Construction And Characterization Of Transgenic Mice Model Harboring Hepatitis C Virus Type 1b Full Genome

Hepatitis C virus(HCV) is a positive-strand RNA virus of the Flaviviridae family and has an 9.6 kb genome, which encodes for an 3000 amino acid polyprotein that is posttranslationally cleaved into functional components. Hepatitis C virus(HCV) consist of a single open reading frame(ORF),which is flanked by untranslated regions ( UTRs) at the 5'and 3'ends. Approximately 200 million people in the world are chronically infected with hepatitis C virus (HCV),which occupys about 2～3 per cent in the world of people. HCV infections is severe in China and being infected people occupys about 3.2 per cent.Chronic infection of HCV may lead to cirrhosis and hepatocellular carcinoma (HCC), thereby being a worldwide problem both in medical and socioeconomical aspects. The reseach of HCV in pathogenic mechanism, vaccine and specific antiviral drug is serously limited because of being short of valid small animal model.The first transgenic animal harboring HCV gene generated in the world in 1995 . Many laboratorys in the worldwide prepared the transgenic anminals containing different length HCV gene fragment to study the pathogenic mechanism of HCV. Matsuda J constructed the transgenic mice harboring full genome of HCV in 1998 Who detected the expression of mRNA and the core protein of HCV. There has been no report about transgenic animals of HCV which can express not only the structure but also non-structure proteins in China until now.The HCV type 1b full genome was cloned into the pIRESneo vector with the CMV promotor and retrovirus vector(pLNCX )with the regulatory sequence of long terminal repeat(LTR) to construct the recombinant plasmids of pIRESneo-HCV and pLNCX- HCV.The recombinant plamids were transfected on the cells of BHK and pA317 on which were well expressed by the detecting of ELISA and IFA.The supernatant of pA317 cell could infect the sensitive NIH-3T3 cell and harvested packaging virus which titre achieve to 10-4.67TCID50/0.1ml after being pured by ultracentrifugationThe two pathways was adopted to prepare the tansgenic mice.In one pathway the recombinant plasmid of pIRESneo-HCV was linearized by restriction endonucleases (MluI and Psp1406I) to prepare the transgenic mice by the method of microinjection. Eighty-two founder mice were obtained from the total 992 microinjected eggs. Two founder mice were positive by detecting of PCR.The other pathway was to utilize the method of retroviral-mediated transfection.Forty-one founder mice were generated from the total 846 infected eggs. Five founder mice which three ones were dead were positive by detecting of PCR. Southern Bloting analysis has shown that 8252bp gene of HCV was integrated which length was close to our expectation.The experimental results manifested that founder transgenic mice harboring HCV genotype 1b full genome were obtained.Four founder mice mated with the normal ones to propagate F1 generations which were nine positive from twenty-seven offrings by the detection of PCR.A series of detections were done such as RT-PCR, immunohistochemical method, detection of enzyme (ALT and AST) and Western-Bloting to detect the expression of transgenic mice. The result of RT-PCR and immunohistochemical method has shown that the exogenous gene was expressed mRNA and viral proteins of HCV. The result about detection of enzyme (ALT and AST) displayed that the liver cells of transgenic mice were damaged in different level. Western-Bloting analysis disclosed that not only the structure proteins of core (22kD) and E2(72 kD) but also non-structure ones of NS5 were expressed. In a word the transgenic mice model harboring HCV type 1b full genome are obtained first time in China.The succeeding construction of transgenic mice model of HCV establish the foundation to the study of pathogenic mechanism, vaccine development and screening of antiviral drug.