Expression Purification and Characterization of SCFV Against Foot and Mouth Disease Virus

Expression, purification and characterization of single
chain fragment variable against foot and mouth

disease virus- type Asia
A Project submitted in partial fulfillment of the requirements for the Degree of
BACHELOR OF TECHNOLOGY IN BIOTECHNOLOGY

Of the
Jawaharlal Nehru Technological University
Kukatpally, Hyderabad, AP. INDIA.
2008-2009
By
Amtul Yasmeen Banu
05H51A2390
DEPARTMENT OF BIOTECHNOLOGY
C.M.R COLLEGE OF ENGINEERING AND TECHNOLOGY
Kanlakoya(V), Medchal Road, Hyderbad-501401
Andhra Pradesh, INDIA
Expression, purification and characterization of single chain fragment

variable against foot and mouth disease virus- type Asia
2008-2009
CERTIFICATION
This is to certify that the work reported in the thesis entitled Expression, Purification and
Characterization of ScFv against FMDV- Type Asia, submitted in the partial fulfillment
of the requirements of the degree of Bachelor of Technology in Biotechnology of the
Jawaharlal Nehru Technological University, Hyderabad is record of bonafide work carried
out by
Name
Regd.No
Amtul Yasmeen Banu
05H51A2390
Our supervision and Guidance
Internal Guide
Department
Head of the
Mrs Kalpana
Reddy
Mrs.T.Rohini
Assistant Professor
Biotechnology
Dept. of
Dept. of Biotechnology
External Examiner
CMR College Of Engineering & Technology
Page 2

ABSTRACT
ABSTRACT
Single chain (ScFv) antibodies represent a new and efficient tool in the research, diagnostics
and therapeutics of infectious diseases like FMDV. In this work presented, efforts were
Page 3

focused on obtaining purified; soluble and active ScFv. Protein expression in Escherichia coli
BL21 strain using T7 expression system is evaluated for its ability to rapidly, efficaciously
and consistently produce active scFv antibodies. Despite the fact that a growing number of
recombinant antibodies have been isolated from phage display libraries, the large number of
established and well-characterized Hybridoma lines still represent a useful source for
recombinant antibody genes. Here, a method is presented to obtain the genetic information
for the antigen binding part of the antibody. An active form of a single-chain antibody (scFv)
from the murine monoclonal antibody (mAb) 1G6, which is specific for type Asia 1 foot and
mouth disease virus (FMDV), was produced in Escherichia coli. This 1G6 culture was taken
for bench/lab scale production, for the expression of soluble ScFv. The protein expressed
predominantly in soluble fractions was purified on Ni-NTA (nickelnitrilotriacetic acid)
Agarose column. The purity of ScFv is qualitatively checked by SDS-PAGE, then the protein
samples are dialyzed and PEG concentrated to get high concentrations of pure ScFv. The
concentration of ScFv antibody yielded from E. coli BL21 strain ranged from 0.7mg1.7mg/150ml of culture. The binding activity of the purified scFv is confirmed and quantified
by enzyme-linked immunosorbent assay (ELISA). The result revealed that the 1G6 ScFv
conserved the same characteristics of specific recognition and binding to type asia1 FMDV
as the parental 1G6 mAb.
DECLARATION
I do hereby declare that the project work entitled Expression,

Purification and Characterization of specific ScFv against FMDV CMR College Of Engineering & Technology
Page 4

type Asia submitted to the Department of Biotechnology, CMR college

of Engineering and Technology, Jawaharlal Nehru Technological University,
Hyderabad, is a bonafied record of work carried out by me, under the
supervision of my project head Mrs. N.V.Sridevi and my in charge Mr.
Shukra Madhaha, Indian immunological ltd Hyderabad.
Amtul Yasmeen
Banu
ACKNOWLEDGEMENT
I hereby thank the following distinguished personalities who stretched their
helping hand in the successful completion of my project.
I thank Dr.Tyaga Rajan the R&D manager, IIL for giving me this opportunity to
carry out this project.
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My sincere thanks to Mrs. Sridevi, PDT lab head and my in charge shukra
madhaha for their cooperation during the project.
It is with gratitude that I record here the very kind and invaluable guidance
suggestions and academic support of Mrs. T.Rohini Reddy, Head of the
Department, CMR college of Engineering and Technology, throughout the
course period.
I would like to thank Mrs. M.kalpalna, my internal guide for extending her
support in completing my project work and Dr.B.Vijaya Lakshmi, Mr.
P.Srinivasa Chary, Mr.S.Ramu, Mr.P.Purushotham Rao, Mr.P.Babji, and
Mrs.V.Vanajakshi Gupta, for their valuable suggestions and guidance to
complete the project successfully.
We would like to thank our Lab-Technicians, Mr.N.Srinivas and Mr.K.Rajendra
Prasad CMR College of Engineering and Technology for their valuable
support and guidance throughout the college education.
I am highly indebted to Dr.M.Ramalinga Reddy, Principle, CMR College of
Engineering and Technology for giving me permission to carry out this
Project.
I express my sincere thanks to Mr. Ch.Gopal Reddy, Secretary, CMR Group
of Institutions for this continuous care towards our achievements.
Last but not least, I thank my parents and friends for their encouragement,
moral support and for their dispensable help.
CONTENTS:
Page 6

Serial No.
TITLE
Page No.
1.
ABBREVATIONS
9-10
2.
INTRODUCTION
12-18
3.
REVIEW OF LITERATURE
20-52
4.
MATERIALS AND METHODS
54-81
5.
RESULTS
83-87
6.
CONCLUSION
88-89
7.
APPENDIX
91-99
8.
REFERENCES
101-112
ABBREVATIONS
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1. ABBREVATIONS
FMD: Foot and Mouth Disease.
FMDV: Foot and Mouth Disease Virus.
E.coli BL21 CELLS: Escherichia coli BL21 CELLS.
RNA: Ribonucleic Acid.
Lac Operon: Lactose Operon.
Ab: Antibody.
Ag: Antigen.
VH-Variable heavy chain
VL: variable light chain.
CDR: Complimentarity Determining Regions.
PMSF: polymethyl sulfonyl fluoride.
Page 8

TB media: Terrific Broth media.

IPTG: Isopropyl Beta Thio D Galactoside.
Ni-NTA Agarose column: Nickel-nitrilotriacetate Agarose Column.
TBS: Tris-Buffer saline.
SDS-PAGE: Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis.
APS: ammonium persulphate.
TEMED: N, N, N, N-Tretramethylenediamine.
NC MEMBRANE: nitrose cellulose membrane.
Anti-HIS: antibody histidine.
PBS: Phosphate Buffered Saline.
PBST: Phosphate Buffered Saline Tween.
DAB: Diamino Benzidine.
TMB: Tetra Methylene Benzamide.
PEG: Poly Ethylene Glycol.
BCA METHOD: Bicinchonic Acid Method.
Mab: Monoclonal Antibody.
ScFv: Single Chain Fragment Variable.
CuSO4: Copper Sulphate.
H2SO4: Sulphuric Acid.
MHz: Mega Hertz.
RPM: Revolutions per Minute.
V: volts
ml: millilitre.
Page 9

L: Litre.
l: microlitre.
INTRODUCTION
Page 10

2. INTRODUCTION
2.1 FOOT AND MOUTH DISEASE (FMD)
FMD is an infectious disease which affects cloven-hoofed animals, especially cattle,

sheep, pigs, goats and deer. The disease is serious for animal health and the economic welfare
of the farming (livestock) industry. Even though FMD is not usually fatal for adult animals,
its effects on loss of productivity can be devastating. FMD can be fatal and on a large scale
for young animals.The after-effects of FMD are serious. Affected animals lose condition and
are especially susceptible to bacterial infections. Animals which recover from FMD are much
more likely to be infertile.
Foot-and-mouth disease virus (FMDV) is the prototype member of the Aphthovirus genus of
the family Picornaviridae.
Picornaviruses are among the most diverse (more than 200
serotypes) and 'oldest' known viruses (temple record from Egypt ca. 1400 B.C.). FMDV was
one of the first viruses to be recognized - Loeffler and Frosch 1898. 'Pico (Greek = very
small) RNA Viruses'. Picornaviridae are nonenveloped viruses with single-stranded RNA
genome of positive polarity and of size 8.5kb (FMDV). In addition, they are highly labile and
rapidly lose infectivity at pH values of less than 7.0.
Foot-and-Mouth Disease (FMD) - a major economic pest world-wide is endemic in parts of
Asia, Africa, the Middle East and South America, with sporadic outbreaks in other
areas.FMD is caused by a virus of which there are 7 main types. These types can only be
differentiated in the laboratory, as their symptoms are identical - fever, followed by blisters
(vesicles) mainly in the foot and mouth. The seven main virus types are: O, A, C, SAT.1,
SAT.2, SAT.3 and Asia 1 - each type has subtypes. The virus is divided into these seven
serotypes based on their cell surface proteins and their geographical location where they are
predominant.
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The average incubation period is between three to eight days - it has been known to be
shorter than and as long as 14 days. An animal that recovers from one virus type is not
protected against infection from any of the other types.
FMDV Type Asia 1: Asia 1 normally only occurs in southern Asia. Antigenically FMD Asia
1 viruses have been considered less diverse than those belonging to types O, A or C. The
genetic variation amongst FMD Asia 1 viruses is much lower than that found within other
serotypes. This may indicate that the serotype has a more recent origin than the others, or that
it has been through severe bottleneck purification with only a single topotype surviving.
The FMD virion has a diameter of about 25 nm. By electron microscopy, the FMD virion
appears to be a round particle with a smooth surface. FMDV is distinguished from other
picornaviruses by the lack of a surface canyon, or pit, which has been shown to be the
receptor binding site for the entero-and cardio viruses. Another feature of the virion is the
presence of a channel at the axis which permits the entry of small molecules, such as CsCl,
into the capsid, resulting in FMDV having the highest buoyant density of the picornaviruses.
FMDV has a relatively short infectious cycle in cultured cells. Depending on the multiplicity
of infection, newly formed infectious virions begin to appear at between 4 and 6 hours after
infection. The virus is cytocidal, and infected cells exhibit morphological alterations,
commonly called cytopathic effects, which include cell rounding and alteration and
redistribution of internal cellular membranes. The virus also causes biochemical alterations,
including inhibition of host translation and transcription.
FMD is characterized by marked salivation, vesicular (blister) lesions and, subsequently,
erosions and ulcers of the epithelium of the lips, gums, soft palate, nostrils, muzzle, and
coronary bands, between the toes and on the teats. Initially, animals may develop a fever.
The lesions in the mouth may make eating painful, so animals refuse to eat, resulting in
weight loss, declined milk and meat production. Foot lesion may cause the animal to be
lame.
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Enormous numbers of the virus are present in the fluid of the blisters, and to a certain extent
in saliva, milk and dung. Animals become infected either as a result of direct contact with a
sick animal or by contact with foodstuffs, dead carcasses, or touching anything a sick animals
has touched.
Unfortunately, presently available (inactivated) vaccines are not entirely effective.
Vaccination blocks disease symptoms (making detection of infection difficult) but does not
always block transmission of the virus to other animals. Sheep can harbors the virus for
several months, cows for up to a year or even longer. Occasional vaccine-linked disease
outbreaks occur as a result. According to the UK Department of Health, human infection of
FMD is extremely rare. The only recorded human case in the UK was in 1966 - symptoms
were similar to influenza (flu), plus some blisters and were fairly mild. There is a human
condition, known as Hand Foot and Mouth disease, which is unrelated to FMD, and does not
affect animals.
2.2 ANTIBODY
Antibodies are the immune systems solution to the problem of molecular recognition.
They are generated and evolved by the immune system to bind with high affinity to
molecules the host has never before encountered. Antibodies are unique in their high affinity
and specificity for a binding partner, a quality that has made them one of the most useful
molecules for biotechnology and biomedical applications. The aspects of antibody structure
permit rapid evolution of high affinity receptors in vivo and also make antibodies excellent
targets for protein engineering. Indeed antibodies offer a paradigmatic case on how protein
engineering can be applied to improve the pharmaceutical properties of a protein drug and to
tailor it for maximum therapeutic efficacy.
2.3 MONOCLONAL ANTIBODIES
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Monoclonal antibodies (mAb) are antibodies that are identical because they are
produced by one type of immune cell that are all clones of a single parent cell. It has now
25years since the advent of monoclonal antibodies.Although the availability of monoclonal
antibodies is considered an essential pre-requisite for any biological study of protein function
and expression, monoclonal antibodies have, to date, only been raised to a few thousand
human proteins. High throughout generation and screening of Hybridoma could theoretically
be used to bridge the gap.
The ability of monoclonal antibodies to bind specifically to antigen and to block
antigen function has been increasingly used in research, diagnostics and therapy. Monoclonal
antibodies can be produced in cell culture or in live animals. Nowadays, bioengineering
allow production of antibodies in heterologous system.
2.4 RECOMBINANT ANTIBODY
Recombinant antibodies are constructed by a process known as Bio-Engineering.

There is alteration of structure and function of antibody for its higher specificity and affinity.
Various formats of antibody fragment can be constructed, such as Fab, Fv and scFv (single
chain variable fragment). Fab fragments consist of the variable domains and the
corresponding first constant domain: VH-CH1 and VL-CL. Recombinant Fab fragments usually
have the same antigen-binding affinities as the intact antibody. But there is a disadvantage
that it has tendency to aggregate. This can be overcome by using short peptide linker to
construct ScFv.
The use of recombinant antibodies is becoming increasingly popular due to their advantages
compared to conventional monoclonal or polyclonal antibodies. In contrast, in the
conventional method, many antigens prove to be non-immunogenic or extremely toxic.
Therefore it can't be used to generate antibodies in animals. Moreover, affinity maturation
(i.e. increasing the affinity and specificity) of recombinant antibodies is very simple and
Page 14

relatively fast. Finally, large numbers of different antibodies against a specific antigen can be
generated in one selection procedure
Using recombinant antibody has significant advantages compared with the
conventional antibody and there for its use becoming more popular now days. The fact that
no animals are needed in the manufacturing procedure of the recombinant antibodies, in
addition, the manufacturing time is relatively short compared with the conventional method
Moreover, the quality of the final product is higher that these of the non recombinant method.
ScFv represent a promising class of therapeutics, which allows one to overcome
problems of the whole antibody molecule. It is made of single peptide chain and its different
domains do not need to reassemble in the host in order to generate tertiary structure. The
immune response against Fc fragment (constant domains) is overcome (Pluckthun and
Skerra). ScFv exhibit faster systemic clearance. ScFv has an improved tissue penetration and
folds more readily into a functional conformation in a range of cellular compartment,
including cytoplasm (Knappik.A, et al). ScFvs expressed in E.coli can be used in biomedical
applications. The peptide linker in scFv can damage its binding conformation and binding
kinetics as it may obscure the antigen-binding site.
2.5 HYDRIDOMA TECHNOLOGY AND ITS APPLICATIONS
Increased research and accumulation of knowledge in the fields of somatic cell

genetics, tissue culture technology and immunology resulted in the development of a
technique for in vitro production of antibodies.
Page 15

MAbs will find use chiefly as research reagents, diagnostics in vitro and in vivo, as the
therapeutic agents and in the purification process and vaccine production. Hybridoma
technology is a form of genetic engineering resulting in the production of specific antibodies
by specialized tissue culture lines. MAb is an antibody directed against one antigenic
determinant or epitope of an antigen, it is single isotype.
Hybridomas are cells that have been engineered to produce a desired antibody in large
amounts. To produce monoclonal antibodies, B-cells are removed from the spleen of an
animal that has been challenged with the relevant antigen. These B-cells are then fused with
myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This
fusion is performed by making the cell membranes more permeable. The fused hybrid cells
(called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will
produce large amounts of the desired antibodies. The production of monoclonal anti-bodies
was first invented by Cesar Milstein, Georges J. F. Khler and Niels Kaj Jerne in 1975.
Selection occurs via culturing the newly fused primary hybridoma cells in selective-media
(HAT). After using HAT it is often desirable to use HT containing media. Cloning occurs
after identification of positive primary hybridoma cells.
Current strategies for the production of therapeutic mAbs include the use of mammalian cell
system store combinantly produce Abs derived from mice bearing human Ig transgenes,
humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human
mAbs has been previously reported; however, this approach has not been fully exploited for
immunotherapy development.
Ever all disease-associated antigens are currently being targeted using therapeutic mAbs
because of their unique pharmacological land safety profiles. Current strategies for the
production of the therapeutic mAbs include the use of mammalian cell systems (i.e.,
CHOorNS0transfectomas) to recombinant produce mAbs derived from immunization of
Page 16

transgenic mice bearing human Ig genes (xenomice) ,humanization of rodent mAbs ,or
through screening of human mAb phagelibraries.
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REVIEW OF LITERATURE
3. REVIEW OF LITERATURE
3.1 FOOT AND MOUTH DISEASE (FMD)
FMD is an infectious disease which affects cloven-hoofed animals, especially cattle,

sheep, pigs, goats and deer. The disease is serious for animal health and the economic welfare
of the farming (livestock) industry. Even though FMD is not usually fatal for adult animals,
Page 18

its effects on loss of productivity can be devastating. FMD can be fatal and on a large scale
for young animals. The after-effects of FMD are serious. Affected animals lose condition and
are especially susceptible to bacterial infections. Animals which recover from FMD are much
more likely to be infertile.
3.2 FMD AND ITS AGENT
The first written description of FMD probably occurred in 1514, when Fracastorius
described a similar disease of cattle in Italy Almost 400 years later, in 1897, Loeffler and
Frosch demonstrated that a filterable agent caused FMD. This was the first demonstration that
a disease of animals was caused by a filterable agent and ushered in the era of virology.
Subsequently it was shown that the agent, FMD virus (FMDV), consists of a single-stranded,
plus-sense RNA genome of approximately 8,500 bases surrounded by four structural proteins
to form an icosahedral capsid (Rueckert et al). FMDV is the type species of the Aphthovirus
genus of the Picornaviridae family. Seven serotypes A, O, C, Asia 1, and South African
Territories 1, 2 and 3 have been identified serologically based on their cell surface proteins
and multiple subtypes occur within each serotype.
Foot-and-mouth disease is an acute and highly contagious febrile disease affecting clovenfooted animals. Identification of the foot-and-mouth disease virus (FMDV), the causative
agent of the disease, posed problems because of the occurrence of many types and subtypes
of the virus. A molecular approach based on oligonucleotide mapping of FMDV RNA has
been used for the identification and characterization of virus isolates obtained in a disease
outbreak (King et al., 1981). One-dimensional oligonucleotide mapping was used for rapid
analysis of FMDV RNA (LaTorre et al., 1982). FMDV types and Asia 1 of Indian origin are
being routinely used for vaccine production in India. This report presents the differences
between FMDV types and Asia 1 at molecular level based on one-dimensional
oligonucleotide mapping of virus-induced poly (A) RNA.
3.3 FMDV TYPE ASIA 1
Page 19

The first reported detection of FMD virus type Asia 1 was in Hong Kong Special
Administrative Region of the Peoples Republic of China (Hong Kong SAR) in March 2005.
The virus was then reported for the first time from eastern Russia close to the border with
China in June 2005.China reported new outbreaks in two other provinces in July 2005. In all
outbreaks, the disease was detected in large and small ruminants (cattle, sheep, and
goats).Although virus appear to be present in China and Myanmar, the first reports of this
type of virus in east Russia and Mongolia raised concerns that the disease was being spread
rapidly through the region.
The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep,
and goats, as well as more than 70 species of wild animals, including deer (Fenner F. J et al.,
1993), and is characterized by fever, lameness, and vesicular on the tongue, feet, snout. In
sheep and goats the disease is generally mild and can be difficult to distinguish from other
common conditions (Donaldson A. I. et al., 2000, Geering W. A. 1967). The disease has
debilitating effects, including weight loss, decrease in milk production, and loss of draught
power, resulting in a loss in productivity for a considerable time.
FMD is one of the most highly contagious diseases of animals or humans, and FMDV rapidly
replicates and spreads within the infected animal, among in-contact susceptible animals, and
by aerosol. FMD is on the list of infectious diseases of animals of the Office International des
Epizooties (OIE) and has been recognized as the most important constraint to international
trade in animals and animal products (Leforban Y 1999). The Smoot-Hawley Tariff Act of
1930, which was passed after the last outbreak of FMD in the United States in 1929,
contained restrictions on importation of susceptible livestock, fresh meat, and animal
products from countries where FMD was present (Bachrach H. L 1965).
Page 20

Fig1Representation of FMDV
Page 21

A) A space-filling representation of the virus (structure determined in the presence of DTT). The
surface is shaded according to radius using Bobscript (Esnouf, 1997) and RASTER3D (Merritt and
Murphy, 1994). The FMDV VP1 GH loop (residues 134157) is highlighted in cyan, with the RGD
integrin-binding tripeptide (residues 145147) picked out in a brighter orange. The heparin motif is
shown in white. The distance from one heparin attachment site to both its 5-fold neighbour and its 3fold neighbour is a little less than 70 , whilst the nearest 2-fold-related neighbour is 90 away.
B) Ribbon cartoon of the virus biological protomer with proteins. The heparin coordinates for five
sugars (CONF1), running left to right, are shown in white ball-and-stick and transparent CPK
representation. The RGD integrin-binding motif is highlighted in blue ball-and-stick and transparent
CPK representation.
(C) A Grasp (Nicholls et al., 1991) accessible surface representation (grey) showing the binding
depression occupied by the five sugar rings with bonds drawn as rods and colored using the standard
convention.
(D) A ball-and-stick representation of the central three heparin sugars (with conventional atom
colors). The protein backbones are drawn with the side chains interacting with the heparin are in balland-stick and colored as the protein backbone; His195 of VP1, Lys134, Arg135 and Tyr138 of VP2,
and Arg56, Ser87 and Asn88 of VP3. Bridging water molecules have been excluded for clarity.
Yellow spheres mark the positions at which sulfates bind in the native virus.
(E) Sugarprotein interactions depicted using LIGPLOT (Wallace et al., 1995) for each of the bound
conformations, CONF1 (left) and CONF2 (right). Note that only the protein side chains that interact
directly are shown and that these are rearranged to clarify the hydrogen-bonding pattern. They are
labeled with the residue number and the chain ID as the least significant digit, e.g. 563 is residue 56
of VP3. Ligand bonds are drawn in black and non-Ligand bonds in green. Hydrogen bonds are
depicted by olive green dashed lines. Non-Ligand residues involved in hydrophobic contacts are
shown as a red, fringed semi-circle. Water molecules are colored red.
3.4 ANTIGENIC VARIATION

Page 22

Antigenic heterogeneity plays an important role in disease control, since immunity

acquired through infection or the use of vaccines is strictly serotype specific. The presence of
7 serotypes and multiple subtypes and variants has added to difficulty of laboratory diagnosis
and control of FMDV. The rise of new variants has inevitably caused by continued
circulation of virus in the field and quasispecies nature of the RNA genome Virus, FMDV in
particular have very high mutation rates in the range of 10 -3 to 10-5 per nucleotide site for
genome replication, due to lack of error correction mechanisms during RNA replication. This
high error rate leads to different serotypes of FMDV. The quasispecies concept was
developed to explain the effects of errors in replication of RNA molecules. The Variability of
FMDV population is manifested when mutation lead to codon changes resulting in a change
in viral phenotype.
Antigenic variation leading to the different serotypes of FMDV occurs mostly within
the capsid coding region of the genome (the p1 region). Antigenic sites on the surface of the
FMD virion have been identified for five of the seven serotypes of virus (SAT1 and SAT3
being the only exceptions) At least four antigenic sites have been identified involving one or
more of the capsid proteins, VP1, VP2 and VP3.However each serotype may not contain all
four sites. Interestingly three of the sites have elements located within the flexible loop which
connects the sheets of the viral proteins, and at least two sited include the C terminus of the
VP1.The RGD receptor binding recognition sequence, major antigenic site to which most of
the immune response is directed, common to all the serotypes, is located within the G-H loop
of the VP1. FMDV antigenic variation, associated with mutation leading to amino acids
replacements within all of the known antigenic sites contributed by the RNA recombination
leads to the genetic diversities in FMDV.
3.5 DESCRIPTION OF THE AGENT
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3.5.1GENOME ORGANISATION
The virion is a 140S particle consisting of a single-stranded RNA genome and
60 copies each of four structural proteins (VP1 [1D], VP2 [1B], VP3 [1C], and VP4 [1A]).
Within the virion, there are small amounts of a cleavage precursor of VP2 and VP4, called
VP0 (1AB) (Araujo J. P. et al., 2002), and one copy of a 23 to 24 amino-acid genome-linked
protein, 3B (VPg), covalently bound to the 5terminus of the RNA (Argos P. et al., 1984,
Arnold E.M et al., 1987). The RNA is translated as a single long open reading frame (ORF)
into a polyprotein, followed by a series of posttranslational proteolytic cleavages to generate
both the intermediate and mature structural and NS viral proteins (Bablanian G. M et al.,
1993 and Bachrach H. L 1968). Based on the initial cleavage products, the genome ORF is
divided into four regions. The 5end, the L region, encodes the N-terminal component of the
polyprotein, contains two in-frame AUG initiation codons that result in the generation of two
L proteins, Lab and Lb (Bahnemann 1975). While both forms of L are synthesized during in
vitro translation of viral RNA (Banerjee R.A. et al., 1997 and Baranowski E. et al., 2001).
The L protein, a papain-like protease (Lpro) plays a role in inhibition of host protein synthesis
and has been identified as a viral virulence factor.
Directly downstream of the L region is the P1 region of the genome encoding the four viral
structural proteins VP4, VP2, VP3, and VP1. Following the P1 region is the P2region
encoding three viral NS proteins, 2A, 2B, and2C, and the P3 region, encoding NS proteins
3A, three copies of VPg, 3Cpro, and 3Dpol. Historically the 2A region was considered part of
the P2 region; however, genetic and biochemical evidence has shown that the FMDV 2A
peptide is cleaved as a P1-2A precursor (Baranowski E. et al., 2001). 3Cpro was identified as
a viral protease (Barclay W. et al., 1998) and is involved in processing the viral polyprotein,
while 3Dpol is the viral RNA-dependent RNA polymerase (Barnett P. V et al., 2002,
Barteling et al., 1991, Vreeswijk et al., 1991, E. P. Black et al, J. ODonnell et al, J. M. Hogle
et al, H. J. Bertschinger et al).
The FMDV genome also contains untranslated RNA found upstream 5untranslated region
5UTR and downstream 3UTR of the ORF. The 5UTR of FMDV contains about 1,300
Page 24

bases (Bautista E. M. et al., 2003, Baxt B. 1995) and can be divided into five functional
elements which play roles in virus translation and RNA replication. The most 5segment, the
S fragment, encompasses about 360 bases and folds into a long stem-loop (Y. Becker and
Baxt B. 1990). The function of the S fragment is in maintaining genome stability in infected
cells and may also be involved in the binding of proteins involved in genome replication
(Baxt, B. et. al., 1990, Bazan, J. F. et.al., 1988 and R. J. Fletterick, Beard C et al., 1999).
Following the S fragment, there is a poly(C) tract comprising over 90% C residues with a
small number of U and A residues. This segment is over 100 bases in length. Just 3 of the
poly(C) tract there is a series of RNA pseudo knot structures of unknown function (Belnap D.
M. et al., 2000).
Fig 2 Genome Organization of FMDV

Downstream of the pseudo knots there is a short hairpin loop structure, the cis-acting
replicative element (cre) (Belsham G. J. et al., 1991). The cre, which has been identified in
the genomes of human rhinoviruses (Benvenisti et al., 2001), poliovirus (Bergmann I.E et.al
1993 and P. A. de Mello 1992) and cardio viruses (Roivainen and T. Hovi 1991, has a stemloop with a conserved AAACA sequence in the loop region. In contrast to the case for other
picornaviruses, where the cre is located within different regions of the ORF, the cre of
FMDV is located within the 5UTR (Mason, P. W. et al., 1993). The cre is essential for RNA
genome replication.
Page 25

The region between the cre and ORF contains a series of highly conserved stem-loop
structures which together constitute the internal ribosome entry site (IRES).All
picornaviruses mRNAs lack the 7-methyl-G cap structure present at the 5 ends of most
eukaryotic mRNAs. An IRES element was subsequently identified within the FMDV genome
(Bienz. K et. al., 1987 and D. Egger 2002). The IRES elements of picornaviruses contain a
high degree of secondary and tertiary structure. They have been divided into three groups,
based on conserved structure as opposed to primary sequence (M. F. Rosas et a., 1995l). The
IRES element for aphthoviruses, which is similar in structure to the cardio virus IRES (group
2 IRES), is about 450 nucleotides in length (Bishop N. E. et al., 1997 and Bittle J. L. et al.,
1982), and has been modeled into a five-domain structure (Blyn L. B. et al., 1997). IRES
elements contain a pyrimidine-rich region at their 3ends immediately preceding the AUG
translation initiation codon, and FMDV contains pyrimidine-rich regions directly upstream of
each of the alternative AUG initiation codons (Bolten, et al., 1998).
The 3 UTR, which follows the ORF termination codon, contains a short stretch of RNA
which folds into a specific stem-loop structure (Pilipenko E. V. et al., 1992) followed by a
poly(A) tract of variable length carried on the genome (Dorsch-Hasler et al., 1975). The 3
UTR also appears to be important for genome replication (Pilipenko. et al., 1992 and
Melchers W. J. et al., 1997).This is supported by studies showing that the 3UTR can bind a
number of picornaviral proteins that are involved in RNA replication (Cui T. and A. G.
Porter, 1995). In contrast, more recent studies have demonstrated that deletion of the FMDV
3UTR reduced the efficiency of in vitro translation (Lopez de Quinto et al., 2002) and
blocked the ability to recover viable virus from transfected cells (Saiz et al., 2001). The poly
(A) tract probably functions in FMDV translation and may also play a role in picornaviruses
RNA replication (Herold J. et al., 2001).
3.5.2 VIRUS STRUCTURE
Page 26

By electron microscopy, the FMD virion appears to be a round particle with a

smooth surface and a diameter of about 25 nm (Bachrach H. L. et al., 1975) The fine
structure of the viral capsid has been determined for a number of serotypes by using X-ray
crystallographic techniques (Lea S., R. et al., 1995, Jackson T. et al., 2003 and Curry S. et al.,
1996).The structural proteins, VP1 to VP3, fold into an eight-stranded wedge-shaped -barrel
which fit together to form the majority of the capsid structure. The VP4 protein is buried
within the capsid and has a myristyl group covalently attached to its N terminus (Chow M.J
et al., 1987). The strands of the -barrels of VP1 to VP3 are connected by loops which form
the outer surface of the virion. FMDV is distinguished from other picornaviruses by the lack
of a surface canyon, or pit, which has been shown to be the receptor binding site for the
entero and cardio viruses (Muckelbauer J. K et al., 1995 Rossmann M. G et al., 1985, Luo et
al., 1987, He Y. et al., 2000). Another feature of the virion is the presence of a channel at the
fivefold axis which permits the entry of small molecules, such as CsCl, into the capsid,
resulting in FMDV having the highest buoyant density of the picornaviruses (Jackson T. et
al., 2003).
Unlike those of other picornaviruses, the FMDV capsid is dissociated at pH of
below 6.5 into 12S pentameric subunits (Brown F. and B. Cartwright 1961). The reason for
this instability is thought to be a cluster of His residues at the interface between VP2 and VP3
which become protonated at low pH, weakening the capsid through electrostatic repulsion
(Ellard F. M et al., 1999 and Curry S. et al., 1996). This low-pH-induced instability of FMDV
leads to differences in the mechanism of its uncoating upon infection of cells compared to
that for other and also probably plays a role in the targeting of the virus to specific tissues
and organs in susceptible hosts.
3.5.3 INFECTIOUS CYCLE
FMDV, like other members of the Picornaviridae, has a relatively short

infectious cycle in cultured cells. Depending on the multiplicity of infection, newly formed
Page 27

infectious virions begin to appear at between 4 and 6 h after infection. The virus is cytocidal,
and infected cells exhibit morphological alterations, commonly called cytopathic effects,
which include cell rounding and alteration and redistribution of internal cellular membranes.
The virus also causes biochemical alterations, including inhibition of host translation and
transcription (Rueckert R. R 1996)
3.5.3.1 EARLY INTERACTION: Adsorption, Penetration, and uncoating
It is generally accepted that FMDV receptors play a role in tissue and organ
tropism which leads to disease pathogenesis (Wimmer E. 1994 and Schneider-Schaulies J
2000). FMDV binds rapidly to cells in culture at both 4 and 37C by using a limited number
of receptor sites, which has measured at between 103 and 104 per cell. Analysis of trypsintreated virus revealed a single cleavage of the VP1 protein at Arg144 (Robertson B. H. et al.,
1985), which was later shown to be located within a surface loop connecting the G and H
strands of the protein (G-H loop) (Logan .D et al., 1993). These results suggested that this
region of the VP1 protein interacted with the cell surface receptor.
In 1984 Pierschbacher and Ruoslahti (Pierschbacher M. D. and E. Ruoslahti, 1984), studying
the binding of fibronectin to cells, reported that the tripeptide sequence Arg-Gly-Asp (RGD)
was a cellular recognition site on the molecule and that this sequence was also found in the
FMDV VP1 protein. In FMDV, while sequences surrounding the RGD sequence within the
G-H loop are variable, the RGD sequence itself is highly conserved (Pfaff E. H et al., 1988).
The first identification of the integrin receptor for FMDV was made based on comparison of
the receptor specificity of the virus with that of a human enterovirus coxsackievirus A9
(CAV9). This virus contains an extension in its VP1 protein that includes an RGD sequence
(Chang K. H. et al., 1989), which was shown to be involved in virus binding to cells in
culture. The receptor utilized by CAV9 was demonstrated to be the integrin V3 (Roivainen
M. et al., 1991), and competition binding studies revealed that CAV9 and antibodies to the
V3 integrin were able to inhibit the binding of FMDV to monkey kidney cells.
Page 28

A number of alternative receptors have been shown to mediate FMDV infection in vitro.
Antibody complexed virus can infect cells via Fc receptor-mediated adsorption (Mason P. W.
et al., 1993). In addition, an artificial receptor which consists of a single chain anti-FMDV
monoclonal antibody fused to intercellular adhesion molecule 1 (ICAM-1) has been
engineered, and this receptor was also able to mediate infection with RGD-deleted virus
(Rieder E. et al., 1996).
After adsorption to the cell surface, the 140S virion breaks down into 12S pentameric
subunits, releasing the RNA (Cavanaugh D. et al., 1978). Recently it has been shown that a
genetically engineered FMDV, which is unable to perform the maturation cleavage of VP0 to
VP2 and VP4 is noninfectious, can adsorb to cells in culture, and is acid sensitive (Knipe T.
et al). Results indicate that the viral receptor is responsible only for docking the virus to the
membrane of the susceptible cell and plays no role in viral uncoating, which is consistent
with the ability of FMDV to utilize multiple receptors for infection in cell culture.
3.5.3.2 VIRAL TRANSCRIPTION & GENOME REPLICATION
Picornaviruses RNA replication presents a number of unique challenges. The

5end of the genome RNA is covalently linked to VPg, and the 3end has a genetically coded
poly (A) tail. Thus, the viral RNA dependent RNA polymerase (3D pol) must distinguish
between viral RNAs and cellular mRNAs, which also contain 3terminal poly (A) tracts. In
addition, since the mRNA and the genome RNA are the same molecule, with the exception of
the genome-linked VPg, there must be a mechanism to distinguish RNAs which are bound
for the ribosome and those which will be packaged into virion particles.
The first step in picornaviruses RNA replication is the synthesis of a minus-strand RNA
molecule. It is thought that translation of the plus-strand RNA must cease before minusstrand synthesis begins (Gamarnik A. V. and R. Andino 1998). One model proposes that
Page 29

initiation begins after circularization of the genome facilitated by the interactions of poly (A)
binding protein (PABP) with the 3 poly (A) tail and the PCBP-3CD 5cloverleaf structure
(Herold J. and R. Andino 2001). In the FMDV genome, the 5interactions probably take
place within the S fragment and may also involve the poly(C) tract. Picornaviruses must have
developed mechanisms enabling the polymerase to recognize viral RN
In picornaviruses-infected cells, both plus- and minus strand RNAs are linked to VPg
(Nomoto A. et al., 1977), and the presence of a small protein-linked dinucleotide, VPgpUpU
in infected cells (Crawford N. M. and D. Baltimore 1983) suggested that VPg might be a
primer for the RNA polymerase. The discovery of the core provided a rational mechanism for
both the uridylylation of VPg and the ability of the polymerase to discriminate viral RNA
from cellular mRNAs. The core is a stem-loop RNA structure, found within the 5UTR in the
FMDV genome with a conserved AAACA motif within the loop. Mutations in this motif
within the FMDV core severely reduced viral replication in cell culture
Following the initiation reaction, elongation of the minus strand begins,
catalyzed by 3Dpol. For this to occur, the initiation complex must translocate to the 3end of
the plus-strand template. The elongation of the nascent strands results in the formation of a
double-stranded molecule, the replicative form (RF). After formation of the RF, new plusstrand synthesis can begin.
Page 30

Fig 3Transcription and genome replication of FMDV
For plus-strand synthesis to proceed, the RF must be unwound. The picornaviruses 2C

protein both has ATPase activity (Klein et al., 1999, Pfister T. et al., 2000) and contains
helicase motifs (Gorbalenya A. E. and E. V. Koonin 1989, Dmitrieva T. M et al., 1991). It has
been shown that 2C and a cellular protein (p38) bind to the minus-strand 3stem-loop (Roehl
H. and Rohll J. B. et al., 1995), and this may act to destabilize the RF molecule.
RNA synthesis occurs within a membranous replication complex, which is derived from
membranes of the endoplasmic reticulum and Golgi and contains viral NS proteins encoded
by both the P2 (2B, 2BC, and 2C) and P3 (3A and its precursors, 3C pro, and 3Dpol) regions
(Egger D. et al., 2002, van Kuppeveld F. J, Teterina N. L, Jecht M. et al., 1997 and Towner J.
S. et al., 1996). Replication complexes containing RNA and 3Dpol have been detected in
Page 31

FMDV-infected cells (Polatnick J. and S. H. Wool 1983). Protein 2C has been found in
membranous aggregates along the periphery of FMDV-infected cells (Tesar M. et al., 1989)
and has been directly implicated in FMDV RNA synthesis by using the picornaviruses RNA
synthesis inhibitor guanidine hydrochloride (Caliguiri L. A. and I. Tamm. 1968).
For FMDV infected cells it has been shown that histone H3 is cleaved by3Cpro
(Grigera P. R. and S. G. Tisminetzky 1984).
3.5.3.3 VIRAL TRANSLATION
The genome-linked protein VPg is cleaved by a cellular enzyme prior to

translation of the incoming RNA. Actively translated viral mRNA does not contain a 7methyl-G cap structure at its 5end and initiates protein synthesis internally at the IRES by a
cap-independent mechanism (Pelletier J. and N. Sonenberg, Kuhn R. et al., 1988). Capdependent mRNA translation is inhibited in infected cells as the result of the cleavage of the
protein synthesis initiation factor eIF4G by Lpro (Kirchweger R. et al., 1994). Intact eIF4G
acts as a bridge connecting the mRNA cap to the 40S ribosomal subunit. Initiation of FMDV
RNA translation requires only the Lpro generated C-terminal eIF4G cleavage product, which
binds to the FMDV IRES and interacts with 40S ribosomal subunit-bound eIF4A and eIF3.
In addition, eIF4B is bound to the FMDV IRES and has been identified in both 48S
preinitiation complexes and 80S ribosomes (Meyer K and Ochs K 1995).
The FMDV IRES interacts with a number of cellular proteins, including initiation factors
important for normal cellular mRNA translation. A host factor of 57 kDa, subsequently
identified as the nuclear polypyrimidine tract binding protein (PTBP) (Kaminski A. et al,
Hellen C. U, Niepmann M. et al), was shown to interact with at least two regions of the IRES
(Pilipenko E. V et al). Deletion of these two sites inhibited both the binding of the protein
and in vitro translation (Luz N. et al., 1991).A second host factor, 45-kDa protein, IRESspecific trans-acting factor (ITAF45), along with PTBP, is required for the formation of the
Page 32

48S translation-initiation complex (Martinez-Salas E. et al., 2001). The presence of the

poly(C) tract upstream of the IRES suggests that it may also play a role in FMDV translation,
genome replication, or both.
Fig4Translation of FMDV
Interestingly, the 3end of the genome may also be required for FMDV translation, since
deletion of either the poly (A) tract or the 3stem loop and the poly (A) tract generated
noninfectious FMDV RNAs which had a lowered translation efficiency in vitro translation
reactions.
Following initiation, translation results in the production of a single polypeptide which
undergoes a series of cleavages leading to the production of both structural and NS proteins
Lpro auto catalytically cleaves itself from polyprotein. 2A, (an 18-amino-acidpeptide), auto
catalytically removes itself from the P2 polyprotein, and remains associated with the P1
precursor (Grubman and Ryan M. D. et al., 2002). This cleavage is a modification of the
translational machinery by the 2A peptide which allows the release of P1-2A from the
ribosome while permitting the synthesis of the downstream proteins to proceed (Donnelly M.
L et al., 2001). 3Cpro is related to the trypsin family of serine proteases (Gorbalenya A. E. et
al., 1989), and Grubman and coworkers have mapped the active site of FMDV 3C pro to
Page 33

Cys163, His46, and Asp84. The protease cleaves at a number of different dipeptide
sequences, including Gln-Gly, Glu-Gly, Gln-Leu, and Glu-Ser (Palmenberg A. C. 1990 and
Robertson B. H. et al 1983).
3.5.3.4 ENCAPSIDATION & MATURATION
The final steps in the replication cycle are the encapsidation of the plus-strand
viral RNA and maturation cleavage of VP0 to VP2 and VP4 to form the mature virion. In
broad terms, the 3Cpro cleavage products of the P1 region are assembled into a protomer
structure containing one copy of each of the proteins VP0, VP1, and VP3. Five protomers can
assemble into a pentamer and 12 pentamers assemble into the final capsid structure.
Following encapsidation of the RNA, the maturation cleavage reaction (VP0 P2 and VP4)
takes place.
Picornaviruses encapsidate only plus-strand RNA, linked to VPg, to the exclusion of all other
viral and cellular RNAs (Nomoto A. et al., 1977, Novak J. E. et al., 1991). In addition, only
newly synthesized plus-strand RNAs are encapsidated, indicating that there is a link between
active RNA replication and encapsidation (Nugent C. I. et al., 1999). Thus, there may be cisacting packaging signals present within the plusstrand RNA to facilitate encapsidation.
Structural proteins of a heterologous picornaviruses are provided in Trans, packaging of the
replicon RNA does not occur. These data provide additional evidence for a packaging
element, specific for each individual picornaviruses, located within the genome outside the
P1 region.
The final step in virion assembly is the maturation cleavage of VP0 into VP4 and VP2,
requiring the presence of viral RNA (Hogle J. M. et al., 1985, Jacobson M. F. et al., 1968 and
Rossmann M. G. et al., 1985). Cleavage is thought to be autocatalytic and results from a
conserved His residue in VP2 which activates local water molecules, leading to a
Page 34

nucleophilic attack on the scissile bond and cleavage (Curry S. et al., 1996 and Hindiyeh M.
et al 1999).
3.6 PATHOGENESIS
While FMD affects a wide variety of cloven-hoofed animals, pathogenesis has

been studied mainly in cattle and pigs. Infection of cattle generally occurs via the respiratory
route by aerosolized virus (Donaldson A. I 1987).Virus is excreted into the milk of dairy
cattle (Burrows R. 1968 and Hyde J. L. et al., 1975 ) as well as in semen, urine, and feces
(Kitching P. 1992), and calves can become infected by inhaling milk droplets. A number of
studies have suggested that the lung or pharyngeal areas are the sites of initial virus
replication (Sutmoller P. et al., 1968), with rapid dissemination of the virus to oral and pedal
epithelial areas (J. W. McVicar et al., 1976), possibly mediated by cells of
monocyte/macrophage origin. In cattle experimentally infected via aerosol, it was found, by
in situ hybridization (ISH), that within the first 24 h, virus was present in respiratory
bronchiolar epithelium, sub epithelium, and interstitial areas of the lung. By 72 h, signal was
Page 35

detected in epithelial cells of the tongue, soft palate, feet, tonsils, and tracheobronchial lymph
nodes (M. E. Piccone et al., 1996).
Pigs usually become infected either by eating FMDV-contaminated food, by direct contact
with infected animals. Animals develop fever, viremia, and lesions on the feet and tongue.
Foot lesions are the most common finding in pigs, while lesions at other sites occur less
frequently tongue lesions are usually small and less noticeable than those in cattle (Kitching
P. and S. Alexandersen 2002.). In young piglets, the infection may be fatal due to
Myocarditis.
Sheep are highly susceptible to virus infection via aerosol and can excrete airborne virus;
however, during outbreaks they are most likely infected by contact with infected animals
(Kitching, P. and G. H. Hughes). Clinical disease in sheep is characterized by lesions on the
feet and mouth, fever, and viremia.
3.7 VIRULENCE FACTORS
In theory, any of the viral structural and NS proteins, elements of the viral RNA,
and host proteins and membranes that participate in the viral replication cycle can be
considered a virulence factor, since defects in the factor or its absence in the cell may lead to
the virus inability to replicate and cause disease in the host species. Viral receptors play a
role in tissue tropism and disease pathogenesis (Evans D. J. et al., 1998, Schneider-Schaulies
J.2000 and Crowell R. L. et al. 1981). FMDV should need three integrin receptors to cause
disease.
Lpro is considered to be a virulence determinant in FMDV. The role of the 3A protein in viral
virulence was demonstrated during studies of the FMDV isolate responsible for an outbreak
in Taiwan in 1997 (designated O/Taw/97).Molecular characterization of the virus revealed
that it contained a 10-codon deletion in the C-terminal half of the 3A protein (Beard C. W.
Page 36

and P. W. Mason 1999). The location of this deletion was similar to that of a 19 to 20 codon
deletion found in FMDV passaged in chicken embryos.
It has been suggested that IRES elements, and possibly the host factors that bind to them,
affect the pathogenicity and virulence of other picornaviruses (Evans D. M. et al., 1985
Kawamura N. et al., 1989 Moss E. et al., 1989, Malnou C. E. et al., 2002). Some recent
preliminary results suggest that the FMDV-specific IRES-binding protein ITAF45 may play a
role in virus virulence by controlling virus tropism within tissues of susceptible species. (V.
ODonnell et al., 2002)
3.8 HOST RESPONSE
The virus elicits a rapid humoral response in either infected or vaccinated

animals. Virus-specific antibodies protect animals in a serotype-specific manner against
reinfection, or against infection in the case of vaccination, and protection is generally
correlated with high levels of neutralizing antibodies (Salt J. S. 1993).The response is
directed to epitopes on the three external structural proteins. In cattle, the immunoglobulin
G1 (IgG1) response predominates over IgG2 (Mulcahy G. et al., 1990, Capozzo A. V. et al.,
1997), and antibody, including IgA, can be detected in upper respiratory secretions early in
infection (McCullough K. C. et al., 1992).
Specific T-cell antiviral responses, involving CD4 + and CD8+ cells, have been
observed in cattle and swine following either infection or vaccination (Gailiunas P et al,
Bautista E. M. et al, Childerstone A. J. et al, Collen T et al).The induction of anti-FMDV
antibody correlates with a lymph proliferative response in cattle and swine (Saiz J. C. et al).
These results suggest that T cells play a role in virus protection and that the reduction of both
Page 37

T-cell numbers and function enhances viral pathogenesis by allowing the virus to spread
within the host, leading to increased viral shedding into the environment
In addition to the IFNs, other cytokines may also play a role in the host
response. In studies of swine which were immunized with a conventional FMD vaccine, it
was shown that vaccinated pigs did not appear to exhibit a systemic inflammatory response,
but chemo tactic activity of plasma on peripheral blood leukocytes increased within the first
week after immunization (Rigden R. C. et al., 2003). Furthermore, in pigs that either were
only vaccinated or were vaccinated and challenged, levels of interleukin-6 (IL-6), IL-8, and
IL-12
in
plasma
increased
after
vaccination
and/or
challenge,
suggesting
monocyte/macrophage activation. Although the levels of IL-6 and IL-8 did not appear to be
related to protection of pigs upon challenge, IL-12 levels were higher in vaccinated pigs,
which were protected from contact challenge, suggesting a role for cytokine-induced
monocytic cell activity in protection from acute-phase disease (Barnett et al., 2002).
3.9 CARRIER STATE
Van Bekkum and colleagues first demonstrated that live FMDV could be
recovered from esophageal-pharyngeal fluids of cattle during the convalescent phase of FMD
(van Bekkum J. G. et al., 1959). Currently, carrier animals are defined as those from which
live virus can be isolated at 28 days, or later, after infection (Sutmoller P. et al., 1968).The
recovered virus probably originates in the pharynx, which appears to be the target region for
persistent infection in cattle (Zhang Z.D. et al., 2001)
Alexandersen and colleagues have proposed two mechanisms for the
development of persistence in the pharynx. FMDV can infect immune system cells, such as
macrophages, or other immunologically privileged sites, leading to evasion of the immune
response. Baxt and Mason examined viral replication in porcine peripheral blood
Page 38

macrophages and found that virus can infect such cells only when presented as an immune
complex, presumably by Fc receptor mediated adsorption.
3.10 DISEASE CONTROL
3.10.1CURRENT VACCINE
The current FMD vaccine is an inactivated whole-virus preparation that is
formulated with adjuvant prior to use in the field. A number of countries have established
vaccine banks which contain concentrated antigen stored in the gaseous phase of liquid
nitrogen (Doel T. R, 2003). The introduction of the killed FMD vaccine has been extremely
successful in reducing the number of disease outbreaks in many parts of the world where the
disease is enzootic. Most virus preparations used for vaccines are concentrated cell culture
supernatants from FMDV-infected cells and, depending on the manufacturer, contain various
amounts of contaminating viral NS proteins. The vaccine does not induce rapid protection
against challenge by direct inoculation or direct contact.
3.10.2 ALTERNATIVE VACCINE STRATEGIES
3.10.2.1 PROTIENS & PEPTIDES

Based on information concerning the FMDV capsid structure, including the
prominent surface exposure of VP1 and the immunologically important VP1 G-H surface
loop, a number of strategies have been employed. Initially these included use of VP1, either
isolated from purified virus or produced by recombinant DNA techniques (Kleid D. G. et al.,
1981); the use of VP1-derived peptides (Strohmaier K. et al., 1982) or chemically
synthesized VP1 peptides (Francis M. J. et al.,1991, Nargi F. et al., 1999); the use of live
Page 39

vectors expressing VP1 fusion protein; inoculation with DNA expressing VP1 epitopes alone
(Wong H. T. et al., 2000) or co administered with DNA encoding IL-2; and use of transgenic
plants expressing the entire VP1-coding region or plants infected with a recombinant tobacco
mosaic virus expressing VP1 (Wigdorovitz A. et al., 1999). All of these strategies present a
limited subset of viral immunogens to the vaccinated animal. The immunogenicity of these
subunit vaccines appears to be due to their ability to present sequential epitopes from the
immunologically important VP1 G-H surface loop.
3.10.2.2 LIVE ATTENUTED VACCINES

Reviews by Bachrach and Brooksby describe, in depth, the attempts to develop
live attenuated FMD vaccines by the classical procedures of serial passage in nonpermissive
animals or in cell culture. Attenuated strains were produced by passage in nonsusceptible
species, such as mice, rabbits, and embryonated eggs, until their virulence for cattle was
weakened. More recently, attempts to develop attenuated vaccines have utilized genetic
engineering to mutate regions of the genome or delete a protein-coding region. By utilizing
recombinant DNA techniques, a virus was created in which the RGD receptor binding site on
VP1. The advantage of this approach, compared to the classical method of attenuation, which
generally introduces mutations at a limited number of sites, is that the risk of reversion to
virulence is significantly reduced.
3.10.2.3 EMPTY VIRAL CAPSIDS
Other experimental FMD vaccines have targeted immunogens that contain the entire
repertoire of immunogenic sites present on intact virus but lack infectious nucleic acid
(Grubman M. J et al., 2002, Lewis S. A. et al., 1991). This strategy involves the molecular
cloning of the regions of the viral genome necessary for the synthesis, processing, and
assembly of the viral structural proteins into empty viral capsids (P1-2A- and 3C pro coding
regions).These structures are naturally produced in FMDV-infected cell culture systems, are
Page 40

antigenic ally similar to virus particles, and are as immunogenic as virions in animals
(Rowlands D. J. et al., 1974 Rweyemamu M. et al., 1979). Animals inoculated with this type
of vaccine could be easily distinguished from infected or convalescent animals by using
currently approved technology, since the regions of the genome coding for the NS proteins
used in the diagnostic assays to detect infection are not present in the empty capsid cDNA
construct. In initial studies, FMDV capsid structures were expressed in Escherichia coli or in
recombinant baculovirus-infected cells and inoculated into animals. An alternative, and very
efficient, antigen delivery system utilizes a live virus vector.
3.10.2.4 ANTI-VIRAL APPROACH
Cells pretreated with IFN-/ productive replication of FMDV are inhibited by

the IFN-induced gene products, double-stranded RNA-dependent protein kinase and 2- 5A
synthetase/RNase L (Chinsangaram J. et al., 1999, Sellers R. F. et al., 1963). IFN is one of
the first lines of host cell defense against viral infection (Vilcek J. and G. C. Sen 1996) and
can rapidly induce a nonspecific protective response against all FMDV serotypes thus far
tested (Sellers R. F. 1963). Furthermore, since effective protection requires the vaccine to be
matched to the outbreak strain and induce a rapid immune response, antiviral therapy could
circumvent these inherent problems of FMD vaccination. Thus, IFN-/ may be useful as an
anti-FMD agent for prophylactic treatment in susceptible animals.
3.11 DIAGONISTICS
An essential component of any disease control strategy includes diagnostic assays to

rapidly confirm the initial clinical determination of infection. In addition, FMDV infection of
sheep and goats can be difficult to detect clinically (Donaldson A. I et al., 2000 and Geering
W. A., et al 1967). Sensitive diagnostic assays are also necessary to distinguish vaccinated
Page 41

from infected or convalescent animals. In addition, these assays can be used for
epidemiological surveillance to confirm the naive status of animals in field situations.
3.12 ScFv FOR FMDV TYPE ASIA
Foot and Mouth Disease (FMD, Latin binomial Aphtae epizooticae), sometimes
called Hoof and Mouth Disease is a highly contagious disease. The etiological agent of this
disease is foot and mouth disease virus (FMDV). Antigenic heterogeneity plays an important
role in disease control, since immunity acquired through infection or the use of vaccines is
strictly serotype specific. For FMDV, it is widely accepted that antibodies have an important
role to play in the protection and recovery from the disease.
The availability of quality diagnostic tests to differentiate infected from vaccinated animals is
crucial to prove freedom from this disease Antibodies against FMDV are found to be useful
in developing specific serological tests to detect the antigen. All current FMDV serological
tests rely on polyclonal or monoclonal hybridoma derived antibody reagents, which can be
difficult to prepare and maintain in a quality-assured manner and in the quantities required.
Comparatively recombinant antibody fragment scFv has high reproducibility and selectivity,
can be over expressed in bacteria and produced in large quantities at low cost to guarantee
the supply of a consistent and well-characterized reagent. The production of recombinant
antibody scFv is the most appropriate method as it does not rely on animal immunization and
does not require the maintenance of viable hybridoma cell lines.
Page 42

The ability of ScFv to express antibody fragments offers several advantages over hybridoma
technology, allowing quick, economical production and generation of antibodies with
increasing affinity of binding and specificity for in vitro and in vivo diagnosis or for
immunotherapy of the disease.
Antibody engineering relates to altering antibody structure and functional properties using
recombinant-DNA tools. These may include altering amino acid sequence to improve their
affinity and specificity, their stability in various environmental conditions, therapeutic
efficacy and improving production yields. These applied molecular techniques allow the
engineering of genes that encode antibodies rather than the manipulation of intact antibodies.
These approaches are expected to make antibodies available for widespread diagnostic or
therapeutic applications. The field of antibody engineering has changed rapidly in the past 10
years, fueled by novel technologies such as phage display technology for the in vitro
isolation of antibodies from combinatorial libraries and their functional expression in
bacteria.
With the growing understanding of immunology and structure-function relationship of
antibody genes, it is possible to engineer antibodies for various applications. An antibody can
be converted into various formats, such as Fab, Fv, single chain variable fragment (scFv) and
bivalent (diabodies) and bispecific antibody fragment. The smallest fragment of the parent
antibody is the Fv fragments that consist of only the variable domains of the heavy and light
chains. Recombinant VH and VL polypeptides that associate to become Fv fragments are
unstable in the physiological pH range. However, stable recombinant Fvs can also be
expressed as a single polypeptide in which the VH and VL domains are joined by a peptide
linker of 14-18 amino acids to create a single chain antibody or scFv. The length and
composition of the linker may affect the binding affinity and stability of the scFv.
ScFv STRUCTURE:
Page 43

Fig ScFv structure

3.13 ANTIBODY
Antibodies are immune system-related proteins called immunoglobulin. Each

antibody consists of four polypeptides two heavy chains and two light chains joined to form
a "Y" shaped molecule. Each light chain is composed N-terminal variable(V) domain(V L)
and a constant (C) domain(C L).Each heavy chain is composed of an N-terminal V domain,
three or four C domains and a hinge region.
Page 44

Fig 1: Structure of an Antibody

The VH and VL domains consists of four regions of relatively conserved sequence
called framework regions (FR1, FR2, FR3 and FR4), which form a scaffold for three regions
of hyper variable sequence (complimentary determining regions, CDRs).The CDRs contain
most of the antigen binding residues.
The heavy chains contain an extended peptide sequence between C H1 and CH2 domains that
has no homology with other domains called the hinge region. It is rich in proline and
cysteine, however , heavy chains lack a hinge region, so they have an additional 110
amino acid domain (CH2) which has hinge like features. Large number of proline residues in
the hinge region confers an extended polypeptide conformation on it, making hinge region
vulnerable to cleavage by proteolytic enzymes like pepsin and papain. Proteolytic digestion
of antibodies releases different fragments termed Fab, Fv and Fc.
The smallest antigen binding fragment is the Fv, which consists of the VH and VL domains.
The Fab fragment consists of the VH-CH1 and VL-CL domains covalently linked by a
disulphide bond.
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Fig 2: Regional activities of an antibody

All the six CDR loops in Fab fragment contact the antigen and three of the six
CDRs account on an average for 73% of the interaction between an antigen and antibody out
of which the heavy chain CDR3 contributes 29% of binding. CDR1 and CDR2 are positioned
at the ends of antigen binding sites with heavy and light chain CDR3 at the center of the site.
Because of their central position CDR3 make majority of the contacts with the epitope
.CDR3 are the most variable of al the CDRs and therefore contribute most of the specificity
of an antibody for its antigen
The base of the Y plays a role in modulating immune cell activity. This region is called the Fc
(Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or
three constant domains depending on the class of the antibody. Based on the Fc portion of the
Antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based on their
constant region structure and immune function
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3.14 RECOMBINANT ANTIBODY
Recombinant antibodies are constructed by a process known as Bio-Engineering.

There is alteration of structure and function of antibody for its higher specificity and affinity.
Various formats of antibody fragment can be constructed, such as Fab, Fv and scFv (single
chain variable fragment). Fab fragments consist of the variable domains and the
corresponding first constant domain: VH-CH1 and VL-CL. Recombinant Fab fragments usually
have the same antigen-binding affinities as the intact antibody. Recombinant Fab fragments
can be as thermostable as the intact antibody. The constant domains of Fabs provide
conserved antigenic determinants, which make it easy to select Fabs with specific antibodies.
But there is a disadvantage that it has tendency to aggregate. The Fv fragment consists of V H
and VL but held by non-covalent hydrophobic, that is prone to dissociate. This can be
overcome by using short peptide linker to construct ScFv.
Fig 3: Fab and Fc regions of an antibody

The use of recombinant antibodies is becoming increasingly popular due to their advantages
compared to conventional monoclonal or polyclonal antibodies. For instance, regulations for
animal usage have become increasingly stringent, whereas, for the production of recombinant
antibodies no animals are needed. Also the production of recombinant antibodies is much
Page 47

faster compared to conventional antibody production and they can be generated against an
enormous number of antigens. In contrast, in the conventional method, many antigens prove
to be non-immunogenic or extremely toxic. Therefore it can't be used to generate antibodies
in animals. Moreover, affinity maturation (i.e. increasing the affinity and specificity) of
recombinant antibodies is very simple and relatively fast. Finally, large numbers of different
antibodies against a specific antigen can be generated in one selection procedure
Using recombinant antibody has significant advantages compared with the conventional
antibody and there for its use becoming more popular now days The fact that no animals are
needed in the manufacturing procedure of the recombinant antibodies, in addition, the
manufacturing time is relatively short compared with the conventional method Moreover, the
quality of the final product is higher that these of the non recombinant method.
3.15 ADVANTAGES OF SCFV OVER FULL LENGTH ANTIBODIES
ScFv represent a promising class of therapeutics, which allows one to overcome

problems of the whole antibody molecule. It is made of single peptide chain and its different
domains do not need to reassemble in the host in order to generate tertiary structure. The
immune response against Fc fragment (constant domains) is overcome (Pluckthun and
Skerra). ScFv exhibit faster systemic clearance. ScFv has an improved tissue penetration and
folds more readily into a functional conformation in a range of cellular compartment,
including cytoplasm (Knappik.A, et al). ScFvs expressed in E.coli can be used in biomedical
applications.
3.16 DISADVANTAGES OF ScFv
The peptide linker in scFv can damage its binding conformation and binding
kinetics as it may obscure the antigen-binding site.
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MATERIALS & METHODS
Page 49

4. MATERIALS & METHODS
The following are the solutions required for the Expression, purification and
characterization of 1G6 ScFv.
4.1 FOR EXPRESSION OF ScFv
4.1.1 pET EXPRESSION SYSTEM

PRINCIPLE
Expression systems are
designed to produce
many copies of a desired
protein within a host cell.
In order to accomplish
this, an expression vector
is inserted into a host
Page 50

cell. This vector contains all of the genetic coding necessary to produce the protein, including
a promoter appropriate to the host cell, a sequence which terminates transcription, and a
sequence which codes for Ribosome binding. The gene for T7 RNA polymerase was inserted
into the host chromosome. This expression system has become known as the pET Expression
System, and is now widely used because of its ability to mass-produce proteins.
Figure 1: The pET Vector. This plasmid contains a drug resistant marker for ampicillin
resistance (green), the lacI gene (blue), the T7 transcription promoter (red), the lac operator
region (pale green) 3' to the T7 promoter, and a polylinker region (black).Also, there are two
origins of replication - one is the f1 origin which enables the production of a single stranded
vector under appropriate conditions, and the other is the conventional origin of replication.
Figure 2: The Host Chromosome. The lac promoter is in red, the lac operator is green, the
gene which encodes the T7 RNA polymerase is pink, and the lac inducer is blue. The host
cell genome is represented by the black line, and the brown line represents the cell
membrane.
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4.1.2 CONTROL OF pET EXPRESSION SYSTEM
Control of the pET expression system is accomplished through the lac promoter
and operator. Before ScFv gene can be transcribed, T7 polymerase must be present. The gene
on the host cell Chromosome usually has an inducible promoter which is activated by IPTG.
This molecule, IPTG displaces the repressor from the lac operator. Since there are lac
operators on both the gene encoding T7 polymerase and ScFv gene, IPTG activates both
genes. Therefore, when IPTG is added to the cell, the T7 polymerase is expressed, and
quickly begins to transcribe ScFv gene which is then translated as ScFv protein. IPTG works
to displace a lac repressor since IPTG is an analogue of lactose .The lac genes express
enzymes which are involved in the breaking down of lactose, and therefore, the presence of
lactose (or it's analogue) would trigger the initiation of transcription of lac genes.
Page 52

MATERIALS: Four 2lt flasks, 2lts Terrific broth, Starter culture, Kanamycine, IPTG, PBS,
Pipettes, Shaker cum Incubator, pET 28a vector, BL21 E.coli cells.
METHOD:
Take stock from -70 and inoculate it in 20ml TB media with kanamycin and put it
overnight in a shaker cum incubator to get a starter culture.
Prepare 2lts TB media and add 50g/ml kanamycin to it.
Subculture the overnight starter culture in 2lts kanamycin containing TB media.
Put it for incubation at 37oC and check the OD till it reaches 0.6.
Induce the culture with IPTG to get final concentration of 1mM IPTG.
Harvest the cells by centrifugation at 8000 rpm for 30min at 4oC.
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Remove the media and resuspend the pellet in TBS.
4.2 FOR CELL LYSIS
4.2.1 SONICATION
PRINCIPLE
Sonication treatment destroys parts of the cells of the biomass liquefying them. In the
following compression phase these bubbles implode under extreme conditions in micro scale
(cavitation). This generates pressures of over 500 bar and enormous shear forces at
temperatures of up to 5,200 Kelvin. These processes tear up the walls of organic cells,
bacteria, fungi etc.
ULTRA SONICATION & CAVITATION PRINCIPLES

In the lower frequency range (20 kHz to 100 kHz) big cavitation bubbles are
generated which excite extremely high shear forces and strong effects. In the middle
frequency range (100 kHz to 1 MHz) the bubbles are smaller but the cavitation is more
effective, generating radical sono-chemical reactions in the water. Sonicating such a liquid
with a frequency of over 1 MHz, the liquid begins to flow on molecular level. The
Sonotronic system works with the frequency of 20 kHz which gives the best reactions and
effects in the biomass.
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Fig Change of the biomass structure through sonication
4.2.2 LYSOZYME ACTIVITY
PRINCIPLE
Lysozyme (also called Muramidase) is a polypeptide of 129 amino acid residues with
molecular weight of 14,400 dalton. It is a basic protein (positively charged) with isoelectric
point of 10.7~11.0. Lysozyme is an enzyme which has the ability to lyses certain Grampositive bacteria by hydrolyzing the b-linkage between N-acetylmuramic acid (NAM) and Nacetyl glucosamine (NAG) of the peptidoglycan layer in the bacterial cell wall.
Page 55

MATERIALS: Lysozyme, PMSF, Sonicator, Centrifuge, TBS.

Before sonication:
Take the pellet in different tubes and add lysozyme (10ng/ml) and PMSF (0.1mM)
and put it in -20oC for over night.
Just before purification the frozen samples were thawed on ice. This thawed culture is
subjected to sonication to reduce its viscosity and make it clear.
METHOD OF CELL DISRUTION BY SONICATION
Take the cell culture (highly viscous) in a beaker within an ice bucket.
Put the cell culture in a sonicator such that its probe should be 60% immersed into the
cell culture, to avoid foaming during sonication.
Sonicate for 15min by using the following parameters a.sonication power of 750
watts; b.pulse duration 9 seconds with 5 seconds gap between the pulses.
Usually Sonication is done for 15-30 mins depending on the viscosity of the culture.
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After sonication the viscous cell culture changes to water like liquid.
Centrifugation to separate cell mass:
The sonicated culture solution is centrifuged at 8000 rpm for 30 min at 4oC.
The supernatant is then separated from the pellet.
This supernatant is taken for purification.
4.3 AFFINITY PURIFICATION OF PROTEINS
PRINCIPLE
Various methods are used to enrich or purify a protein of interest from other proteins
and components in a crude cell lysate or other sample. The most powerful of these methods is
affinity purification, also called affinity chromatography, whereby the protein of interest is
purified by virtue of its specific binding properties to an immobilized ligand.
1. Affinity medium is equilibrated in binding buffer.
2.
Sample is applied under conditions that favour specific binding
of the target molecule(s) to a complementary binding substance (the ligand). Target

substances bind specifically, but reversibly, to the ligand and unbound material washes
through the column.
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3.
Target protein is recovered by changing conditions to favor
elution of the bound molecules. Elution is performed specifically, using a competitive ligand,
or non-specifically, by changing the pH, ionic strength or polarity. Target protein is collected
in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.

FOR PURIFICATION OF ScFv
MATERIALS: Ni-NTA (nickel-nitrilotriacetate) Agarose column, TBS (Tris-buffered saline,

Elution Buffer, 10mM Immdazole wash, 50mM Immdazole wash, and 300mM Immidazole.
METHOD
Make the Ni-NTA bed column by washing the column with TBS thrice and adding
5ml of Ni-NTA Agarose to the purification column.
This column bed must be washed with 20 volumes of the bed volume with 1x TBS.
Start loading the supernatant and collect the flow through.
The column is first washed with 10 mM Immidazole (IZ) to remove the unspecific
bound proteins.
The second wash is done using 50mM immidazole.
The elutions are collected using 300mM immidazole.
4.4 FOR CHARACTERISATION OF ScFv

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4.4.1 SDS-PAGE
PRINCIPLE
A simple, effective and very high resolution method to fractionate and analyze protein
mixtures is the sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE).
SDS-PAGE separates proteins on the basis of their molecular size. SDS is an anionic
detergent, which binds and reacts with the proteins in the solution, and destroys the tertiary
structure of the protein, leading to partial unfolding of the polypeptide chain. In addition,
SDS binds to both the hydrophilic and hydrophobic regions of the polypeptide chain, giving
the protein an excessively net negative charge, which diminishes any intrinsic amino acid
charge. The protein also adopts a cylindrical shape, which is coated along its' entire surface
with negatively charged sulfonate ions. A general rule when using SDS is that the amount of
SDS bound per gram of protein is found to be constant at a SDS: protein ratio of about 1.4
gram/SDS gram protein. Through the use of a supporting medium called polyacrylamide,
with the application of an electric field through this medium, the SDS negatively charged
protein complexes in a protein mixture can be separated. Polyacrylamide is a synthetic
polymer, which is formed by the polymerization of acrylamide monomer with additional
bifunctional cross linking agents (aided by a catalyst). This polymerized polyacrylamide
matrix is a three-dimensional network of pores whose size is determined by the percentage
degree of acrylamide monomer and cross-linker concentration utilized in the mixture (pore
size decreases with higher acrylamide gel concentrations) and is often referred to as a
separating or running gel.. Upon electrophoresis, which applies an electric field through the
pores in the gel matrix, the proteins are sieved through the pores of the gel with the larger
proteins having a slower migration rate than the smaller proteins. The negative charges flow
from the negative cathode terminal into the upper buffer chamber, through the gel, and into
the lower buffer chamber, which is connected to the positive terminal. Therefore, the
negatively charged SDS coated proteins migrate towards the anode. The combination of gel
pore size and protein charge, size and shape determines the migration ability of the protein.
Page 59

SDS PAGE apparatus:
Page 60

SDS-PAGE (Sodium dodecyl Sulphate Polyacrylamide gel Electrophoresis)

Page 61

MATERIALS: Distilled water, acrylamide, Tris buffer ph 8.3, Tris buffer ph 6.8, 10%SDS,
APS, TEMED, 5xSDS dye, 1x running buffer, SDS PAGE apparatus, and scoop.
METHOD:
Preparation of resolving gel/separating gel: The resolving gel is prepared using the given
Composition. APS and TEMED are added just before pouring the gel.
Preparation of Stacking Gel:
The composition of stacking gel is same for all percentages .The stacking gel is
prepared according to given composition.
Sample Preparation:
Samples are prepared in microfuge vials by adding 6ul of 5x SDS dye to 30ul protein
sample and heating at 100oC for 10 mins.
Sample loading: Load 15ul of the protein samples into each well respectively using a
micropipette following the below procedure.

Running of gel:
Remove the gel plates from the set-up clamps and properly set the gels into the
vertical PAGE apparatus.
Fill the PAGE apparatus with 1x SDS buffer. And check for any leakage.
Gently remove the combs without damaging the wells.
Load 10-15ul of each sample into the respective wells
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When the PAGE is done for western blotting use prestain marker and if its done for
Coomassie staining use bench marker.
Adjust the voltage to 100mV and run the gel for 1 hr or till the samples reach the
bottom of the gel.
Remove the gels from the casting plates, carefully using a scoop and 1x SDS buffer.
The gel can either be used for coomassie staining or western blotting.
4.4.2 STAINING PROCEDURES
COOMASSIE STAINING:
MATERIALS: Coomassie Brilliant blue, destaining solution, water, Plastic box, Rocker.
METHOD:
Staining:
Place the Polyacrylamide gel in a plastic box and cover the gel with coomassie brilliant
blue solution and place it on the rocker for half an hour.
Destaining:
Pour out the Coomassie Brilliant blue solution.
Add destaining solution to the gel and destain it for half an hour. Change the
destainer if needed.
Discard the destaining solution and add water, to store the gel.
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4.4.3 WESTERN BLOTTING
PRINCIPLE
The term blotting refers to the transfer of biological samples from a gel to a
membrane and their subsequent detection on the surface of the membrane. Western blotting
(also called immunoblotting because an antibody is used to specifically detect its antigen)
was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis.
The specificity of the antibody-antigen interaction enables a target protein to be identified in
the midst of a complex protein mixture. Western blotting can produce qualitative and semi
quantitative data about that protein.
The first step in a Western blotting procedure is to separate the macromolecules using gel
electrophoresis. After electrophoresis the separated molecules are transferred or blotted onto
a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.
Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the
surface of the membrane. The transferred protein is complexed with an enzyme-labeled
antibody as a probe. An appropriate substrate (DAB-daimios benzidine) is then added to the
enzyme and together they produce a detectable product such as a chromogenic precipitate on
the membrane for colorimetric detection.
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MATERIALS & EQUIPMENT : Electro blotting apparatus, Plastic tray, Whatmann 3 mm

filter paper, fibre pads, 0.45m
Nitrocellulose membrane or PolyVinylidene difloride
membrane, Transfer buffer, Blocking buffer, Washing buffer, blocking solution, Gel rocker,
Measuring cylinders, 50ml centrifuge tubes
Secondary antibody: Anti-Histidine
Substrate: DAB tablet+H2O2 in PBS.
METHOD:
After SDS PAGE, the gels are transferred to protein transfer buffer.
To prepare Trans blot, the fibre pads and the NC (nitrose cellulose) membrane are
soaked into protein transfer buffer.
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The soaked fibre pad is put on the Trans blot apparatus then the NC membrane is
carefully laid on to the fibre pad (do not let the membrane dry).the gel is gently
placed on the NC membrane, which is topped by a fibre pad .thus the gel on NC
membrane is sandwiched between fibre pads in this fashion-(fibre pad, NC
membrane, gel, fibre pad).
After each layer roll out any air bubbles which may have formed.
The Trans blot apparatus is closed and run for 1hr at 20V.
After the protein is transferred onto the NC membrane, block the blot using 30ml of
2% skimmed milk in PBS, for 1hr on a rocker.
Wash the blot thrice with PBS.
Incubate the blocked blot in 25ml Anti-his solution (1:5000 dilution) for 1hr on a
rocker
Wash the blot thrice with PBS and 5 times with PBST.
The blot is developed by incubating it in enzyme substrate DAB (diamino
benzidine) in 50 ml PBS+ 0.6ml H2O2 for 5mins.
The result is observed.
The reaction is stopped by transferring the blot into distilled water.
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4.5 CONCENTRATION OF ScFv
4.5.1 DIALYSIS:
PRINCIPLE
Dialysis works on the principles of the diffusion of solutes and ultrafiltration of fluid
across a semi-permeable membrane under a concentration gradient.
Dialysis is an old established procedure for reducing the salt concentration in
samples. The process utilizes selective permeable membrane to separate the components of
solutions and impurities based on their molecular size and the concentration gradient across
the membrane.
Diffusion, Ultrafiltration and Osmosis are the basic physical principles of dialysis.
Diffusion is the net directional movement of molecules occurring from a solution of higher
concentration to a solution of low concentration. Ultrafiltration is the movement of solvent
across a semipermeable membrane in response to a pressure difference applied across the
membrane. If the solutes dissolved in the solvent are small enough to permeate the
membrane, they are dragged along with the solvent and cross over to the other side, and this
called Convection. Osmosis the movement of the solvent (e.g. water) from the side of low
concentration to the side of higher concentration.
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Dialysis is quite slow, taking as much as several days for almost complete salt removal.
MATERIALS: Snakes skin dialysis membrane, Small beaker, Clamps, 5lt Beaker, PBS.
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METHOD;
Dialysis is a common laboratory technique for removing low molecular weight
impurities, and operates on the same principle as medical dialysis. The affinity purified
protein samples (1G6 ScFv) are placed into a semipermeable dialysis bag, such as a snakes
skin membrane with pore size cut off 3.5 KD and the bag is sealed. The sealed dialysis bag is
placed in a container of PBS for 1-2 days. The buffer can be changed if needed. Molecules
small enough to pass through the tubing (often water, salts, imidazole and other small
molecules) tend to move into or out of the dialysis bag, in the direction of decreasing
concentration. Larger molecules (often proteins, DNA, or polysaccharides) that have
dimensions significantly greater than the pore diameter are retained inside the dialysis bag.
One common reason for using this technique would be to remove the salt from a protein
solution.
4.5.2 PEG CONCENTRATION
PRINCIPLE
Polyethylene glycols are polymers of ethylene oxide typically ranging in size from 200
Da to 20 kDa. PEG is very soluble in water due to the ether oxygen spread along the length
of the polymer, which are strong Lewis bases and form hydrogen bonds with water
molecules. In addition, the formation and equilibration of precipitates take significantly less
time with PEG as the precipitating agent than with ammonium sulfate or ethanol.
PRECIPITATION
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Polyethylene glycols can be used for protein precipitation as PEG is a high molecular
polymer, that aids in the removal of non ionic detergent and other low molecular impurities
dissolved in water by removing the shell of hydrating water around the protein. Water
diffuses out from low concentration (protein solution) to higher concentration (PEG) by a
process called osmosis. As PEG is a higher molecular weight substance it cannot cross the
membrane barrier. Thus, concentrating the protein solution.
MATERIALS: Dialysis tubing, PEG (polyethylene glycol), Plastic box.
METHOD:
Concentration with Polyethylene Glycol is used to concentrate the protein (1G6
ScFv) samples. This is done by placing the protein samples in dialysis tubing (snake-skin
membrane )of 3.5 kDa cutoff, onto crystalline PEG(10 kDa or greater)and completely
covering the tubing with crystalline PEG, for a period of 30 - 60 minutes, depending on the
degree of concentration desired and removing the remaining PEG by rinsing in buffer or
distilled water. Typically, prior to PEG concentration, dialysis step is done.
4.5.3 BCA METHOD (measuring the concentration of protein)
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PRINCIPLE
Bicinchonic acid and copper sulphate solution when added to a protein sample (of
unknown concentration), the alkaline Cu++ reacts with the protein and produces cu+ ions
which reacts with the BCA to produce a intense purple colored complex whose absorbance
can be measured at 562 nm. The color produced is stable and increases in a linear fashion
over a broad working range of increasing protein concentration. Since BCA is stable, it is
incorporated in the reagent formulation at the start of the reaction. Thus, the method offers
mechanical simplification over the method described by Lowry et al.
MATERIALS: Microtitre plate, BSA, E.coli lysate, PBS, BCA and Copper Sulphate,
Spectrophotometer.
METHOD:
Take a micro titer plate and put 50l of PBS in all the wells but the first column.
In the first column, add 50l of positive of known concentration , BSA (bovine serum
albumin) in 1:10 dilution in the 1st well, 50l of 1G6 ScFv in the 2nd well, 50l of
E.coli lysate in 1:20 dilutions in the 3rd well and the 4th well is just 50l of PBS.
Take 25l from the 1st column and Serial dilute all the columns.
Add 100l of BCA+cuppor sulphate solution in 50:1 dilutions to all the wells.
Observe the absorbance at 452nm using a spectrophotometer.
Compare the absorbance of the BSA of known concentration with the absorbance of
1G6 ScFv to determine its concentration.
The concentration of the 1G6 ScFv protein solution is obtained.
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4.6 ELISA- ENZYME LINKED IMMUNOSORBENT ASSAY
PRINCIPLE
Enzyme-Linked Immunosorbent Assay, also called ELISA, Enzyme
Immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample. The ELISA has been used as
a diagnostic tool in medicine and plant pathology, as well as a quality control check in
various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a
surface, and then a specific antibody is washed over the surface so that it can bind to the
antigen. This antibody is linked to an enzyme, and in the final step a substance is added that
the enzyme can convert to a visible signal, which indicates the quantity of antigen in the
sample.
4.6.1 INDIRECT ELISA
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MATERIALS:
Microtitre plate, FMDV antigen, Skimmed milk powder, FMDV Mab,
E.coli lysate, FMDV ScFv, anti-his, anti-mouse, Citrate buffer, TMB (tetramethylene
benzamide), PBS and PBST.
METHOD:
The steps involved in, "indirect," ELISA for determining the affinity of the 1G6 ScFv
towards FMDV type Asia antigen.
Apply 100l of FMDV antigen of concentration 100ng/ml to a surface of each well of
a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple
adsorption of the protein to the plastic surface is usually sufficient. These samples of
known antigen concentrations will constitute a standard curve used to calculate
antigen concentrations of unknown samples. Note that the antigen itself may be an
antibody. Put this antigen coated plate overnight at 4oC.
Add 200l of 2% solution of non-interacting protein, such as skimmed milk powder
to all plate wells. This step is known as blocking, because these proteins block nonspecific adsorption of other proteins to the plate. Put for 1hr incubation at 37oC.
After washing the plate thrice with PBSA, add 100l of PBS into each well except
the 1st the first column of the plate. In the 1 st column, add 100l of a positive of
known concentration in the 1st well, 100l of 1G6 purified ScFv(1.7mg/ml) in the 2 nd
well and 100l of E.coli Lysate(1.7mg/ml) (5l of E.colilysate+95l of PBSA) in
the 3rd well respectively.
Take 100l from the 1st column and serial dilute all the columns and incubate for 1hr
at 37oC.
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Since antigen immobilization in this step is due to non-specific adsorption, it is

important for the total protein concentration to be similar to that of the antigen
standards.
The plate is washed, and 100l of secondary antibody specific (anti-His) in 1:5000
dilution to the ScFv (His tagged) of interest is applied to all plate wells but the first
lane (containing FMDV Mab). To the first lane 100ul of anti-mouse in 1:5000
dilutions is added.
These secondary antibodies are conjugated to the substrate-specific enzyme and the
antibody will only bind to immobilized ScFvs and Mabs on the well surface, not to
the blocking proteins. Incubate for 1hr at 37oC.
Wash the plate thoroughly with PBST and PBS, so that excess unbound enzymeantibody conjugates are removed.
Apply a 100l substrate (TMB-tetramethelene benzamide in 10ml Citrate Buffer+
0.6ml H2O2) which is converted by the enzyme to elicit a chromogenic signal.
View/quantify the result using a spectrophotometer or other optical/electrochemical
device at 450 nm.
Stop the reaction by applying 100l of H2SO4 to each well.
4.6.2 SANDWICH ELISA

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MATERIALS: Microtitre plate, FMDV antigen, Skimmed milk powder, FMDV Mab, E.coli
lysate, FMDV ScFv, anti-his, anti-mouse, Citrate buffer, TMB (tetramethylene benzamide),
PBS, and PBST.
METHOD:
Steps involved in sandwich Elisa for checking the affinity of the FMDV type Asia antigen
towards the 1G6 ScFv
A sandwich ELISA. (1) Plate is coated with a monoclonal antibody; specific to fmdv
type Asia antigen (2) sample containing fmdv antigen is applied, antigen binds to
capture antibody; (3) FMDV ScFv (His-tagged) is added which binds to antigen; (4)
enzyme-linked secondary antibody (anti-HIS) is added which binds to ScFv (His-tagged);
(5) substrate TMB (tetramethyl benzidine) is added, which is converted by enzyme to
detectable form.
The steps are as follows:

Page 75

Apply 100l of FMDV type Asia Mab of concentration 100ng/ml to a surface of each
well of a microtiter plate. The antibody is fixed to the surface to render it immobile.
Simple adsorption of the protein to the plastic surface is usually sufficient. Put this
antibody coated plate overnight at 4oC.
Add 200l of 2% solution of non-interacting protein, such as skimmed milk powder

to all plate wells. This step is known as blocking, because these proteins block nonspecific adsorption of other proteins to the plate. Put for 1hr incubation at 37oC.
After washing with PBS, apply 100l of FMDV antigen of concentration 100ng/ml to
the Surface of each well of a microtiter plate. Incubate at 37oC for 1hr.
Wash the plate, so that unbound antigen is removed.
After washing the plate thrice with PBSA, add 100l of PBS into each well of the
plate. In the 1st column, add 100l of a positive of known concentration in the 1st well,
100l of 1G6 purified ScFv(1.7mg/ml) in the 2 nd well and 100ul of E.coli
Lysate(1.7mg/ml) (5l of E.colilysate+95l of PBSA) in the 3rd well respectively.
Take 100l from the 1st column and serial dilute all the columns and incubate for 1hr
at 37oC.
The plate is washed, and 100l of secondary antibody specific (anti-His) in 1:5000
dilution to the ScFv (His tagged) of interest is applied to all plate wells but the first
lane (containing FMDV Mab). To the first lane 100l of anti-mouse in 1:5000
dilutions is added.
These secondary antibodies are conjugated to the substrate-specific enzyme and the
antibody will only bind to immobilized ScFvs and Mabs on the well surface, not to
the blocking proteins. Incubate for 1hr at 37oC.
Wash the plate thoroughly with PBST and PBS, so that excess unbound enzymeantibody conjugates are removed.
Page 76

Apply 100l of substrate (TMB-tetra methylene benzamide in 10ml Citrate Buffer+

0.6ml H2O2) which is converted by the enzyme to elicit a chromogenic signal.
View/quantify the result using a spectrophotometer or other optical/electrochemical

device at 450nm.
Stop the reaction by applying 100l of H2SO4 to each well.
Page 77

RESULT
Page 78

5. RESULT
5.1 SDS - SDS-PAGE (Sodium dodecyl Sulphate Polyacrylamide gel Electrophoresis)
After lab scale expression of 1G6 culture and its purification on Ni-NTA agarose
column, elution fractions were collected using 300mM immidazole elution buffer.
Presence of purified 1G6 ScFv in the elution fractions is qualitatively checked by SDSPAGE.
1G6 Coomassie Blue stained SDS PAGE gel showing the elution fractions
from E1 to E9 and protein marker (Bench marker).
5.2 WESTERN BLOTTING
Page 79

The result obtained by coomassie staining is confirmed by western blotting of the

purified 1G6 elutions. A band of 32KDa (with respect to marker-30KDa) is observed on
the immunoblot and its confirmed as 1G6 ScFv against FMDV-Type Asia
50KDa
30 KDa
32KDa
1G6 Western blot analysis was performed for pellet, flow-through, wash fractions,
Elution fractions 2,3,4,5 along with protein marker and negative control (E. coli cell lysate).
And a positive control.
Lane1
: Pellet
Lane 2-5 : Elution fractions

Lane 6
: Flow through
Lane7
: Wash
Lane8
: VE E.coli lysate
Lane 9
: +VE E.tenella Mic 2.
Page 80

5.3 PROTIEN ESTIMATION USING BCA METHOD
OD at 562nm
1G6 Protein Estimation

1.4
1.2
1
0.8
BSA
1:10
1:20
1:40
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1.2
Dilution
The concentration of the purified 1G6 ScFv against FMDV type Asia obtained after
affinity purification, dialysis and PEG concentration, using BCA method is 1.7mg/ml.
5.4 ELISA Enzyme Linked ImmunoSorbent Assay

Page 81

5.4.1 INDIRECT ELISA
The binding activity of the purified 1G6 ScFv is confirmed by indirect ELISA.
5.4.2 SANDWICH ELISA
Page 82

The FMDV type Asia antigen affinity towards purified 1G6 ScFv is confirmed by the
sandwich ELISA.
Page 83

CONCLUSION
6. CONCLUSION
In our present study, we sought to apply essential tenets of protein engineering

research for the expression of ScFv constructed from a hybridoma cell line against FMDV
Page 84

Type Asia. Genetically engineered ScFv has a number of advantages over Mabs in clinical
applications.
With rapid progress in antibody gene manipulations and antibody selection methods,
the ability to produce large quantities of functional protein reliably and cost effectively has
increased. ScFv being relatively easily available, smaller in size, less immunogenic, have the
potential for greater tissue penetration and more readily infiltered form nonspecific tissues.
The ability of ScFv to be directed and expressed in different cellular compartments and to
specifically bind to various epitopes of the target molecule have made them suitable for
genetic therapy of viral diseases. Antibody fragments show less possibility of developing anti
immunogenic response in comparison within intact antibodies when applied invivo.
In this study, the ScFv was successfully produced by rDNA technology which offers
the advantages of antibody engineering.
In view of our results, an active scFv could be produced in E.coli with high yield.
Primary characterizations of biological activity indicated that 1G6 ScFv showed same
potential binding activity against FMDV type Asia antigen as its parental Mab.
Hereby, it can be concluded that 1G6 scFv has potential to be a useful agent for
immunodiagnostics and immunotherapeutic applications. Thus, the obtained purified soluble
scFv against FMDV Type Asia will serve for future investigations.
Page 85

APPENDIX
7. APPENDIX
FOR EXPRESSION
Terrific broth (TB medium)
Yeast extracts
Tryptone
5gms
10.0gms
Page 86

K2HPO4
KH2PO4
Glycine
Sodium Chloride
Distilled water
10.0gms
1000ml
CULTURE USED: E.coli BL21 CELLS.

ANTIBIOTIC KANAMYCIN:
Stock: 50mg/ml.
Working: 50g/ml
IPTG:
IPTG is Isopropyl beta-D-thiogalactoside.
stock :1M
working is 1mM..
FOR LYSIS
Lysis reagent:
Lysozyme
10ng/ml
PMSF(polymethylsulfonyl)-protease inhibitor
0.1mM/ml
Page 87

FOR PURIFICATION:
Ni-NTA (nickel-nitrilotriacetate) AGAROSE COLUMN.

TBS (Tris-buffered saline)-1lt -25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4
1x TBS
10xTBS
8 gm NaCl
80 gm NaCl
0.2gm KCl
2 gm KCl
3gm Tris Base

800ml sterile water
30 gm Tris Base
800 ml sterile water
ELUTION BUFFER-IMIDAZOLE:
WASH-1:
Add 0.5 ml of 1M immidazole to 44.5ml of distilled water + 5ml of 10x TBS to make 50ml
of 10mM immidazole in 1x TBS.
WASH-2:
Add 2.5ml of 1M immidazole to 43.5ml of distilled water+5ml of 10x TBS to
Page 88

make 50 ml of 50mM immidazole in 1xTBS,300mm immidazole:
Add 15ml of 1M immidazole to 30ml of distilled water+5ml of 10x TBS to
make 50ml of 300mM immidazole in 1xTBS.

FOR CHARECTERISATION
FOR SDS PAGE

Acrylamide:
Dissolve 290g of acrylamide and 10g of N,N'-methylbisacrylamide in 600ml of H2O.
Heating may be necessary to dissolve the acrylamide.
Adjust the volume to 1L with H2O.
Sterilize the solution by filtration (0.45 micron pore size).
Check the pH (should be 7.0 or less).
Store in dark bottles at room temperature
Tris buffers:
It is essential that these buffers be prepared with Tris base. After the Tris base
has been dissolved in deionized water, adjust the pH of the solution with HCl. If Tris-Cl of
Trizma is used to prepare buffers, the concentration of salt will be too high and polypeptides
will migrate anomalously through the gel, yielding extremely diffuse bands.
Preparation of Tris pH-6.8(1.0M) buffer:
Page 89

Dissolve 12.1g of Tris base in 100ml of distilled water.
Adjust the pH to 6.8 with the help of HCl.
Preparation of Tris pH-8.8(1.5M) buffer:
Dissolve 18.6g of Tris base in 100ml of distilled water.
ii) Adjust the pH to 8.8 with the help of HCl.
APS:
10% APS (Ammonium Per sulfate) can be prepared by dissolving initially 1g of APS
in 9ml of distilled water.
Make the volume to 10ml with distilled water.
TEMED:
N, N, N, N-Tretramethylenediamine accelerates the polymerization of
acrylamide and bisacrylamide by catalyzing the formation of free radicals from APS.
Because this works only as a free base, polymerization is inhibited at low pH.
1x Running Buffer: PH 8.3
25 mM
Tris-Hcl
200 mM
Glycine
0.1% (w/v)
SDS
COMPOSITION FOR SDS-PAGE RESOLVING GEL 12% FOR 5ml

Page 90

Distilled water
1.6ml
30%Acrylamide mix
2.0ml
1.5M Tris (pH 8.8)
1.3ml
10% SDS
0.05ml
10%APS
0.05ml
TEMED
0.002ml
COMPOSITION FOR SDS-PAGE STACKING GEL 5% FOR 2ml
Distilled water
1.4ml
30%Acrylamide mix
0.33ml
1.0M Tris (pH 6.8)
0.25ml
10% SDS
0.02ml
10% APS
0.02ml
TEMED
0.002ml
SDS SAMPLE BUFFER (5x)
Tris-HCl
0.225M (pH 6.8)
SDS
10% (W/V)
Bromophenolblue
0.5%
Page 91

Glycerol
50%
DTT
-mercaptoethanol
0.25M
(10-20l)
SDS RUNNING BUFFER (TRIS GLYCINE BUFFER), PH 8.3 (5X)

Dissolve 15.1gms of Tris base, 94gms of glycine in 800ml of distilled water. Add 50ml of
10% SDS and make up the volume to 1000ml with distilled water.
COOMASSIE STAINING SOLUTION
Methanol
50%
Acetic acid
10%
Distilled water
40%
0.1g of brillant blue is added to 50ml of staining solution

DESTAINING SOLUTION
Methanol
50%
Acetic acid
10%
Distilled water
40%
WESTERN BLOT
Page 92

PROTEIN TRANSFER BUFFER FOR WESTERN BLOT (1x)
Tris base
3.03gms
Glycine
14.4gms
Methanol
200ml
Make up the volume to 1000ml with distilled water.
BLOCKING SOLUTION FOR WESTERN BLOT

2% Skimmed milk powder in PBST solution
ELISA
PHOSPHATE BUFFERED SALINE (PBS, 1x)
NaCl
80gms
KCl
2.0gms
Na2HPO4
14.4gms
KH2PO4
2.4gms
Distilled water
1000ml
Page 93

pH
7.4
PBST
0.05%Tween20 added to 1x PBS solution
SUBSTRATE BUFFER
Citric acid
5.11gms
Na2HPO4
7.3gms
Distilled water
1000ml
pH
5.0
CARBONATE BUFFER - 1 litre

Na2Co3 -1.59 gms
NaHCO3-2.93 gms in 1 litre
STOPPING SOLUTION
1.25M H2SO4
Make up 68ml of Conc. H2SO4 to 1 litre.
Page 94

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Expression Purification and Characterization of SCFV Against Foot and Mouth Disease Virus

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Expression, purification and characterization of single

chain fragment variable against foot and mouth

BACHELOR OF TECHNOLOGY IN BIOTECHNOLOGY

Expression, purification and characterization of single chain fragment

Amtul Yasmeen Banu

Our supervision and Guidance

Expression, purification and characterization of single chain fragment

Expression, purification and characterization of single chain fragment

I do hereby declare that the project work entitled Expression,

Expression, purification and characterization of single chain fragment

type Asia submitted to the Department of Biotechnology, CMR college

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

MATERIALS AND METHODS

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

TB media: Terrific Broth media.

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

FMD is an infectious disease which affects cloven-hoofed animals, especially cattle,

Picornaviruses are among the most diverse (more than 200

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

Recombinant antibodies are constructed by a process known as Bio-Engineering.

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

2.5 HYDRIDOMA TECHNOLOGY AND ITS APPLICATIONS

Increased research and accumulation of knowledge in the fields of somatic cell

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

FMD is an infectious disease which affects cloven-hoofed animals, especially cattle,

Expression, purification and characterization of single chain fragment

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

3.4 ANTIGENIC VARIATION

Expression, purification and characterization of single chain fragment

Antigenic heterogeneity plays an important role in disease control, since immunity

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

Expression, purification and characterization of single chain fragment

Fig 2 Genome Organization of FMDV

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

CMR College Of Engineering & Technology

Expression, purification and characterization of single chain fragment

By electron microscopy, the FMD virion appears to be a round particle with a

FMDV, like other members of the Picornaviridae, has a relatively short

Expression, purification and characterization of single chain fragment

Expression, purification and characterization of single chain fragment