الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
The current study was under taken to clone and express some non-structural HCV subtype 4a genes which prevalent in Egypt into pure antigens. The purified antigens are suggested to be benefit tool in the vaccine candidate and diagnosis fields in Egypt. In the present study, forward and reverse primers which contain the restriction sites for HCV NS2 and NS4b genes were designed. Both NS2 and NS4b were amplified from HCV genome using PCR. pIVEX vector was purified from agarose gel electrophoresis. NS2 and NS4b were cloned into pIVEX separately using T4 ligase. Both recombinant pIVEX – NS2 and pIVEX – NS4b were used for expression in rapid translation system; RTS 100 E.coli HY kit. Previously prepared recombinant vector; pQE30-NS2 was extracted from E. coli M15 bacteria.
Using T4 ligase, NS2 was ligated into pET21. Transformation of pET21-NS2 into DH5α bacteria was done to produce large quantities of recombinant pET21-NS2. The recombinant pET21-NS2 was extracted from DH5α bacteria to transform into BL21 bacteria. NS2 and NS3 were expressed in E.coli with induction using IPTG. The NS2 and NS3 proteins in this study were soluble when expressed in E. coli with treating under native or denature conditions. Both M15-pQE30-NS3 and BL21-pET21-NS2 production was scaled up. About 25 gram bacterial cells were produced from each strain separately.NS2 and NS3 antigens were purified by affinity chromatography and electroelution under native conditions.The resulted NS2 and NS3 proteins were concentrated using centricon and lyophlized by freezing dry system.These antigens appeared high antigenicity against the sera of HCV patients.