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Production of purified alpha toxin of C. perfringens is essential for development of diagnostics and vaccines against alpha
toxin mediated clostridial diseases such as gas gangrene. But, purification of native toxins from culture requires a multistep
procedure and the purity is often unsatisfactory. Thus development of recombinant alpha toxin is a promising technique for larger
scale production at low cost with satisfactory qualitative maintenance. Thus in the current study, an attempt was made to clone
and express plc gene encoding alpha toxin. For this, the particular gene (1115bp in size) was amplified from genomic DNA of
C. perfringens obtained from Division of Biological Standardization, I.V.R.I, Izatnagar, using self designed primers. The forward
and reverse primers contained EcoRI and XhoI site respectively. Further the amplicon was cloned into prokaryotic expression
vector pPROEXHTb. The positive clones were confirmed by PCR, double restriction digestion and sequence analysis. Induction
of recombinant clones with 1mM IPTG expressed rAlpha having 46.171 kDa molecular weight. This was subsequently purified
from bacterial lysate using metal chelate affinity chromatography. Removal of urea from elute by stepwise dialysis yielded the
biological active rAlpha as denoted by its hemolytic activity on murine RBC. The concentration of purified protein was 0.13mg/
ml. Antigenicity of protein was proved by immune-blotting using whole cell anti-sera raised in rabbits. Now further studies are
required to test its suitability as a vaccine and diagnostic candidate in various C. perfringens diseases.
Ajay Kumar Rai completed B.V.Sc & A.H degree in 2010 from College of Veterinary Science and Animal Husbandry, MHOW. He got 10th rank in
all India entrance exam for ICAR?JRF. He is presently pursuing his post graduation in Veterinary Bacteriology and Mycology at Indian Veterinary Research Institute, Deemed University, Izatnagar.
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