Retroviral Vector Mediated Hepatitis C Virus Envelope Protein Gene Expression And Study Of Anti-HCV Neutralizing Antibody

Hepatitis C virus (HCV) is a major causative agent of post-transfusion and community-acquired hepatitis in the world. The majority of HCV-infected individuals develop chronic hepatitis progressing eventually to liver cirrhosis and hepatocellular carcinoma. Neither an effective treatment for chronic HCV infection nor a vaccine to prevent HCV infection is available at the present time. The only available treatment, a combination of alpha interferon and ribavirin, is efficacious in only a minority of patients. The development of such therapies will be aided greatly by a more complete picture of the structure-function features of HCV proteins. HCV is an enveloped plus-strand RNA virus of the Flaviviridae family. Its genome is 9.4 kb in length with one open reading frame that is translated as a single polyprotein, which is processed by host and virus proteases into at least three structural and seven non-structual proteins with various enzymatic activities. Two glycoproteins are probably virion envelope proteins, which are obvious candidates for inclusion in a subunit vaccine because of its potential role in HCV attachment. Recently, scholars from all over the world studied structure and function of HCV deeply by means of gene recombination technique .They want to understand infectious chronic mechanism and reciprocity of HCV and host and search effective vaccine and curative medicine. In 1994, researchers of Chiron company report experiment result of HCV vaccine using gorillas. They found gorillas immuned by mammiferous cells express HCV E1E2 protein could induce neutralizing antibody and resist HCV attack. They thinked HCV E2 region especially E2 hypervariable region (HVR, aa386~411) have important action for inducing neutralizing antibody. On the other side, E2 region has high variance and different types and strains have not neutralization. Some scholars considered E2 antibody have no neutralization. Because we have no HCV in vitro reproducing system and concentrated patients serum HCV difficultly, we studied E2 recombinant protein expressed by all kinds of expression systems. Evidences showed that mammiferous cells expressing HCV E1E2 protein can embody structure and character of natural HCV membrane protein. But recently, it is very difficult problem in the world to express and concentrate E1E2 protein highly in mammiferous cells. This effect seriously study and application of E1E2 membrane protein. All of these show study of HCV neutralizing antibody faces a lot of difficulties. The paper has three parts. The first part includes HCV E1E2 protein was expressed in SP2/0 cells'envelope successfully using retroviral express system. Balb/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells and induced specific anti-E1E2 antibody in mouse serum. Virus neutralization antibody was detected .These established bases for studying HCV neutralizing antibody .The second part includes HCV E1E2 gene vaccine was constructed and Balb/c mice were injected intramuscularly with the nucleic acid vaccine. Anti-HCV E1E2 neutralization antibody was screened .The third part is expression of the major antigen region of E2 gene of HCV in E.coli , the fused E2 protein was highly expressed in E.coli prokaryotic expression system in the experiments and provided the basic materials for the HCV diagnose. Main research contents include: 1. The recombinant retroviral vector pBABE-puro-E1E2 was constructed by inserting full-length HCV E1E2 gene of H77 strain into pBABE-puro. Both the recombinant retroviral vector and pVSVg plasmid were transfected into eukaryotic cells 293GP by calcium phosphate transfection method. And then, the pseudovirus was produced. The pseudovirus infected eukaryotic cells SP2/0 and E1E2 protein was expressed. E1E2 protein was detected by puromycin-resistant and FACS analysis. Balb/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells. Anti-HCV E1E2 antibody was screened by FACS. The results showed that HCV E1E2 protein was expressed in SP2/0 cells'envelope successfully. FACS could detect specific anti-E1E2 antibody in SP2/0 cells immune mouse serum. Virus neutralization antibody was detected by HCV pseudovirus .The result showed anti-HCV E1E2 antibody has no neutralization activity. Mouse spleen cells were fused with myeloma SP2/0. Culture supernatants of hybridoms were screened by FACS and four cell lines which could secrete against E1E2 protein of HCV McAbs stably were obtained. The McAbs could react with HCV E1E2 protein specially. The McAbs was detected by HCV pseudovirus .The result showed McAbs has no neutralization activity too. 2. Hepatitis C Virus(HCV) H77 strain E1E2 gene vaccine was constructed by inserting full-length cDNA of HCV E1E2 into an eukaryotic expression vector pcDNA4.0. The recombinant plasmid was transfected into eukaryotic cells 293T bycalcium phosphate transfection method and transient expressive E1E2 envelope protein was analyzed by FACS. Balb/c mice were injected intramuscularly with the recombinant plasmid. Anti-HCV E1E2 antibody was screened by FACS and antigen was SP2/0 cells which expressed HCV E1E2 protein. The results showed that HCV E1E2 protein was expressed transiently in 293T cells. Specific anti-E1E2 antibody could be detected in DNA immune mouse serum by FACS and the antibody could react specially to SP2/0 cells which expressed HCV E1E2 protein. Virus neutralization antibody was detected by HCV pseudovirus .The result showed anti-HCV E1E2 antibody has no neutralization activity. 3. The major antigen region E1and E2 gene of HCV H77 strain were cloned into the expression vector pET32a, the recombinant plasmids pET32a-E1 and pET32a-E2 were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the recipient germs translated and induced by the recombinant plasmid pET32a-E2 could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of HCV.