Studies On Cloning And Expression Of Fusion Gene Combining HCV Epitopes Antigen With HBV Core Antigen

C hepatitis infection is a kind of disease which is caused by hepatitis C virus has been distributed all over the world.Hepatitis C virus (HCV) belongs to flaviviridae,and it can be identified to six different genotypes. According to the WHO statistics,170 million people has been infected and 35 thousand new patiens take part into this group every year.In China,there are 40 million HCV carriers .The affection of HCV virus is a chronical symptom,such as hepar chronic inflammation thanatosis,hepar fibrosis,20%～30% of the patients will degenerate to hepatic cirrhosis, 1%～7% of the hepatic cirrhosis patients could be under the danger of hepatic cellular cancer(HCC).HCV vaccine development is still a probablem nowadays,the first reason is HCV is a kind of hight variation virus,the other one is lacking of ideal animal model. Since gene vaccine have been proved to have a effective immunogenicity,some scientists start studing the way of combination between HCV epitopes and immunogenicity intensify vector.Some of the HCV epitopes have been demonstrated that have ideal immunogenicity and the capacity causing CTL. In the past research HCV epitopes are ususlly combined with chemical adjuvant,in this study ,we combined HCV epitopes with HBcAg wich has natural protein structure.HBcAg is a new antigen presentation vector,it can make the extrogensis epitope gene express on the surface of HBcAg. The HCV epitopes we choose come from core 132-140aa;NS3 1073～1081aa;NS4b 1809～1816 aa,core 21～40aa,between every epitope there is a flaxibal peptide to help the complete peptide folding in right way after expression.By using overlapping extention PCR(OE PCR),we construct sequence MCe(231bp),and the sequence from HBcAg 1-77aa:HBc5';the sequence from 84-150aa:HBc3',and complete HBcAg sequence (C).After the following OE PCR we splicing the three sequence as CMCe(630bp).Conbined CMCe and C with pMD18-T vector and inverted pMD18-T-CMCe and pMD18-T-C into E.coli DH5αand sent the sample for sequence testing.The result is completely coinside as we expected .After that we combined CMCe and C with pET-28a(+) using two restrict enzyme: EcoRⅠ,XhoⅠ,and inverted pET-28a-CMCe,pET-28a-C into E.coli BL21.CMCe and C expression are based on 1mmol/L IPTG derivation.We used HBV and HCV infected patients'serum to do the western blot test , CMCe can both combined with HBV an HCV antibodys meanwhile C can only combined with HBV antibody.In the study we conbined fusing gene of HCV multiepitopes and HBcAg,and the sequence test result denmonstrated the sequence we constructed is completely coinside as we designed.Western blot result tells the protein expressed by pET-28a-CMCe has HBV and HCV immunogenicity meanwhile the protein expressed by pET-28a-C has only HBV immunogenicity.The whole study provide a new basic theory for HCV vaccine development.