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2.
Molecular Cloning:
The identified antimicrobial peptide gene sequence will be cloned in bacterial host and
the cloned antimicrobial peptides will be expressed in expression vectors and purified.
The purified recombinant peptide will be used for further in vitro and in vivo studies
3.
4.
5.
All
groups will be injected intraperitoneally (i.p.) with 100 l PBS containing bacterial
pathogens at 3.6105 cfu ml1. A group of 25 fishes will be maintained separately for
uninfected control (C) fed with basal diet. At weeks 1, 2, and 4 post-infection, six fishes
will be randomly collected from each tank and anesthetize with MS-222 in de-chlorinated
water for 2 min to collect blood samples for hematological and immunological assays.
6.
Hematological Parameters:
i. Haematocrit
ii. Haemoglobin concentration
8.
Immunological Assays:
a. Lysozyme activity Assay
b. Super Oxide Dimutase (SOD) Assay
c. Lipid Peroxidation Assay
d. Measurement of fish Immunoglobulin by ELISA
9.
Invitro antimicrobial activity of AMPs against various bacterial pathogens using minimum
inhibitory concentration technique
The fishes will be fed on four different diets (four different concentrations of recombinant AMPs)
and a control diet for the growth and immunological studies
Each replicate consist of five fishes randomly sampled from a tank at the
beginning, and after 1, 7, 14, and 28 days of AMP feeding
Histological analysis of
Liver, gill and spleen