Cloning, Expression Of A Gene Fragment Encoding HCV Core Antigen And Purification, Antigenicity Analysis Of The Recombinant Protein

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Over 0.17 billions, which accounts for about 3% of the world population, have been affected by HCV. Till now, there is no effective vaccine and therapeutic drugs for the prophylaxis and treatment of HCV infection, so blood donors scanning and early clinical diagnosis become very important in controlling the spreading of HCV.The detection of serum HCV RNA, anti-HCV antibody and HCV antigen are the major assays for the diagnosis of HCV infection. Though HCV RNA amplification by PCR is the most sensitive and specific method for the detection of HCV, the complicated manipulation, easy contamination and high cost prevented PCR from application in a large scale. The low level of serum HCV antigens detected by common immunological and virological methods supports that serum HCV antigen detection can't be a common clinical method for HCV diagnosis.Anti-HCV antibody detection is a most commonly used assay in clinical practice. Enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) are the two chief methods for the detection of anti-HCV antibody. Some uncertain ELISA results should be confirmed by RIBA. ELISA is a high sensitive and specific method suitable for the diagnosis of HCV infection in a large scale. The sensitivity and specificity of ELISA depend on different antigens and epitopes. It is important to optimize different HCV antigens combination to lower the false positive and false negative rates for the satisfied sensitivity and specificity of anti-HCV antibody detection.Core antigen, which induced high level of humoral and cellular immunity, became an essential agent for anti-HCV antibody detection of three generations of anti-HCV antibody ELISA kits. The hydrophilicity profile of HCV core antigen was analyzed and the l~119aa fragment was confirmed as the hydrophilicity region, which contains almost all of the conserved B cell epitopes of core antigen. The research intends to raise the sensitivity and specificity of anti-core antibody using the 119aa fragment as antigen.A 357bp C119 gene was amplified from HCV genome by PCR and the gene was cloned into pET-32a expression vector. Cl 19 gene was fused with Trx gene from pET-32a expression and expressed in E. coli BL21(DE3). The fusion protein C119 was purified using inclusion body purification procedures and SP-Sepharose HP chromatography. The antigenicity of C119 fusion protein was analyzed by Western blot and ELISA. Serial sera were used to confirm the sensitivity, specificity and reproducibility of the purified C119 fusion antigen.The results showed that the nucleic acid and amino acid homologies of C119 gene were over 83% and 93% compared with other HCV genotypes or isolates, respectively. C119 protein chiefly expressed as the form of inclusion body, whose molecular weight was 33kDa and the fusion protein accounted for about 20% of total bacterial proteins. The result of Western blot showed that it was Cl 19 fusion protein but not Trx protein recognized by the serum anti-HCV antibodies, indicating the excellent specificity and antigenicity of the Cl19 protein.When ELISA was used to detect anti-C119 antibodies in human sera, the sensitivity and specificity were 91.5% and 100%, respectively. The results could be well-repeated by reproducible test., whose CVs of intra-assay and inter-assay were 7.49% and 7.81%, respectively.