DEVELOPMENT AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINAT FILARIAL THIOREDOXIN (rWb TRX-1)

M.Tech- Biopharmaceutical Technology

P ADHI SESHAN 2011401001

Phase 1

Filariasis
A

neglected tropical disease caused by thread like parasitic nematodes transmitted by bite of infected mosquito.

Eight filarial nematodes are known and are Classified into three groups

Lymphatic filariasis Subcutaneous filariasis Serous cavity filariasis

Life cycle Diagnostic strategies Treatment strategies

LYMPHATIC FILARIASIS

MOLECULAR RESEARCH OF FILARIASIS

PROTEIN LEVEL

GENOMIC LEVEL

Filarial genome project (FGP) established 1994 with funding from from WHO-Tropical Disease s Research. the construction of cDNA and genomic libraries for Brugia, Wuchereria, and Onchocerca B.malayi- complete genome sequenced-100 million bp (Elodie Ghedin et al.,2007) W.bancrofti- mitochandrial genome(Ramesh et al., 2012)

PROTEIN LEVEL

DIAGNOSIS: Wb SXP-1 (Rapid Flow

through filarial diagnostic kits)

TREATMENT/ PROPHYLAXIS:

Bm SXP-1(Brugia Rapid, Pan LF-ELISA) mf SHP

1. Filarial Immunoprophylaxis initiated with mf ES Ag(Harinath et al., 1991) 2. B.malayi based vaccine candidates ALT-2 # r ALT-2 conferred 73% & 64% protection TPX-2 against a challenge in jird and mouse resp. (kaliraj et al., 2007). VAH # BmTPXshowed78%protection in a cocktail vaccine study COX-2 (Kaliraj et al., 2008) COL-4 GST-filarial glutathione-S-transferase TRX-1 a small redox protein(Thioredoxin) TGA

OBJECTIVE

DEVELOPMENT AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST RECOMBINANT Wuchereria bancrofti THIOREDOXIN PROTEIN (rWB TRX-1).

The present study is planned to be carried out in two phases as, PHASE -1: Introduction of Cloned recombinant Wb TRX-1 cloned on expression vector pRSETB into osmatically inducible E. coli strain GJ1158 for over production of rWb TRX-1 by salt (NaCl) induction. Expression and purification of rWb-TRX-1 by introducing pRSET B clone. Characterization of purified rWb-TRX-1. PHASE-2: Immunization of mice with purified recombinant Wb-TRX-1. Periodic analysis of immune response elucidated against immunization of rWb-TRX-1 Isolation of splenocytes from mice and fused with myeloma cell line for production of Hybridoma cells. Screening and characterization of Monoclonal antibody produced against protective filarial protein rWb TRX-1.

Small redox protein (16 kDa-nematodes & 12 kDa-most of the species). Crucial for redox regulation of proteins function and signaling . Two major roles of TRX (Kaliraj et al., 2003):
1.

THIOREDOXIN

2.

3.

Electron carriers necessary for catalytic cycles of biosynthetic and antioxidant enzymes such as ribonucleotide reductases, methionine sulfoxide reductases and peroxidoxins. Secondly they protect cytosolic proteins from aggregation or inactivation via oxidant-mediated formation of inter/intra-molecular disulfides. In addition there are growing evidences that TRX has regulatory effect on immune responses like

4.

Bm-TRX-1 : Immune evasion mechanism (Kunchithapautham et al., 2003).

Cytokine release Enhance cytotoxicity Block the activation of chemokines Control DNA binding activity of immunologically active transcription factors, including NF-kB & AP-1. its ability to inhibit tumor necrosis factor alpha (TNF-) induced activation of p58 mitogen activated protein (MAP) kinase shows that Bm-TRX-1 may function as parasites immune evasion mechanism.

recombinant Wb-TRX-1

osmotically inducible E. coli GJ1158 host with salt (NaCl) as inducer was developed for over production of recombinant Bm TRX-1 cloned in expression vector pRSETB. Induction for over production is facilitated by addition of 1oomM NaCl. Over a period of time induced cells are harvested and lysis by sonication for protein extraction. TRX is purified by ion exchange chromatography. Qsepharose /DEAE matrix used for purification. The bound proteins are eluted by using increasing conc. of Sodium chloride (5mM, 10mM, 15mM, 50mM & 100mM) in TRX binding buffer (pH 8). Purity of eluted samples are checked on SDS-PAGE gel electrophoresis and western blotting. Concentration of purified sample is determined by Bradford method.

Scope of developing monoclonal antibody against rWb-TRX-1

The significance of antibodies as diagnostic and analytical reagents has been known and exploited for almost a century. In recent years, antibodies have become increasingly accepted as therapeutic reagents, particularly for cancer but also for numerous other disorders. Magic bullet- target specific interaction Induction of passive immunity Antibody directed drug delivery system. Immuno-toxins- mAb conjugated toxins.

RESULTS & DISCUSSION

THE AGAROSE ELECTROPHORESIS OF ISOLATED PLASMID SAMPLE AND RESTRICTION DIGESTION OF THE PLASMID SAMPLE.

Lane 1-DNA marker Lane 2-unretricted plasmid sample -1 Lane 3-restricted plasmid sample taken after 2hours incubation Lane 4-restricted plasmid sample taken after 3hours incubation Lane 5-unrestricted plasmid sample- 2 Lane 6-restricted plasmid sample taken after 2hour incubation Lane 7-restricted plasmid sample taken after 3hour incubation

Conformation of purified rWb TRX-1 by SDS-PAGE & Western Blotting

FIG 3: SDS PAGE ANALYSIS OF rWb-TRX FIG 3: WESTERN BLOTTING OF PURIFIED PROTEIN PURIFICATION rWb TRX-1.

Future direction of the study

Immunization of mice with purified recombinant WbTRX-1. Periodic analysis of immune response elucidated against immunization of rWb-TRX-1 Isolation of splenocytes from mice and fused with myeloma cell line for production of Hybridoma cells. Screening and characterization of Monoclonal antibody produced against protective filarial protein rWb TRX-1. Purified Ceruloplasmin (cp) a major plama antioxidant and copper transport protein was incubated with the mAbs, the ferroxidase activity of CP was inhibited up to a maximum 57%. Determination of mAb interference in the activity of rWb-TRX 1 in the assays like:

Insulin reduction assay DNA nicking assay Prothrombin assay P38 activation assay.

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References