Clone, Expression, Purification Of HCV Ns5b And Preparation Polyclonal Antibody Against NS5B

In 1989, Hepatitis C Virus (HCV) was first confirmed to be a major pathogeny transmitted by blood transfusion, which is different form HAV and HBV but can cause acute hepatitis, HCV, about 70 percent of whom will end up suffering chronic hepatic diseases, indicating that HCV infection is still one of the problems threatening human health.HCV is a positive-strande RNA virus, whose genome length is 9600nt, encoding some 3000 amino acid residues. In HCV genome 5 ' conversed region, there have internal ribosome entry site, while 3' region determine the genotype of HCV. NS5B is non-structural protein of HCV genome coding, located at C region of HCV polyprotein, with 68kD as its molecular weight. Many experiments show that NS5B is the RNA-dependent RNA polymerace, the key player in RNA replication. In proper conditions of reaction, NS5B can initate many RNA molecules to promote RNA reproduction. Particularly it can effectively reproduce HCV (+) RNA. In this process, NS5B starts RNA synthesisby pattern recognition 3' and conforme the compound by polymerase, template and primer at the 3' region. The bases of the template and primer then strictly pair at the activated site.This study amplified the protein coding gene of HCV NS5B with PCR, used the technique of gene recombination to construct a plasmid pRESTA-ns5b and induced its expression in E.coli. The expression rate amounted to 25% of the total somatic protein, indicating NS5B was effectively expressed. Western-blot analysis showed that the activity to combine with the positive serium from HCV patients was remained fairly well in the recombined NS5B protein. Meanwhile experiments on best induced concentration and best induced time of IPTG showed that the best concentration was Immol/L and the best cultured time was 3 hours. Then chromatography was used to purify the expressed NS5B protein, UV-spectrophotometer showed purfied protein is 0.25mg/ml. In the preparation of anti-NS5B polyclonal antibody, the recombined NS5B was inoculated to New Zealand rabbit. After fundamental immunity and strengthening immunity, the serum of rabbit was isolated. Western-blot showed that its serium antibody still had combination activity at the titer of 1:500.