Expression Of HCV NS3 Protein,Preparation Of Its MAbs And Determination Of Their Binding Region

Hepatitis C Virus (HCV) is the causal agent of Hepatitis c and is transmitted primarily through blood-borne routes. Chronic infection is its distinct characteristic. About 80% infection would develop to chronic hepatitis that can result in liver cirrhosis, hepatocellular carcinoma hereafter. It accounts for more than 170 million cases worldwide. In China, the number of infected people is over 40 million. HCV has been a global severe social public health problem. The present treatment for HCV infection is α-interferon in combination with ribavirin. The efficacy of this therapy shows significant variation depending upon different genotypes. Sustained effect amounted up to about 40%. The two drugs have significant side effects and the patients can't be tolerable. HCV genome also has a high degree of sequence variation which leads to the production of viral quasispecies. It is difficult to prepare the preventive and therapeutic vaccine because thegenome is variable and the existence of quasispecies, especially envelope glycoprotein's great variation. Since the protease inhibitor has a good effect on treating the AIDS, it is a focus on antivirus research on HCV to develop its protease and RNA ploymerase pharmaceutic compounds.HCV belongs to a positive stranded RNA virus of the Flaviviridiae family, hepacivirus genus. The 9.6 kb RNA genome is organized to contain a single translation open-reading frame (ORF). It encodes a ployprotein precursor of 3011~3033 amino acids that is processed into mature proteins and enzymes by cellular signalase and virally-encoded protease. The nonstructure protein NS3 consisted of 631 amino acids located at the nucleotide 3420-5312. NS3 protein has a serine protease activity at one third of the N-terminus, while its C-terminal domain has NTPase and helicase activity. The protease directs proteolytic cleavages activity requiring NS4A protein as the cofactor. It cleavages the nonstructure proteins of polyprotein into single protein. Furthermore, NS3 protein is important for the replication of the genome. Study on NS3 protein has been the focus for researchers at home and abroad since it was the ideal target for anti-HCV.The objective of this study is to amplify ,clone full length and truncated ns3 gene encoding HCV NS3 protein. Then the corresponding prokaryotic and eukaryotic expression plasmid were constructed. When full length NS3 protein was expressed in the E.coli, the BALB/c mice were immunized with the purified protein. The specific monoclonal antibodys (MAbs) binding different region were prepared via the cell fusion technique .1. The primers for amplifying the full length ns3 gene, serine protease and helicase gene were designed according to the ns3 gene sequence from theliterature. The prokaryotic and eukaryotic expression vector of the full length ns3 gene were constructed successfully, named ProEXHTb-ns3full, pcDNA3.1(-)-ns3full, respectively. At the same time, the eukaryotic expression vector containing serine protease and helicase gene were constructed, named pcDNA3.1 (-)-ns3tp, pcDNA3.1 (-)-ns3th, respectively.2. The pProEXHTb-ns3full was transfected into the competent cell E.coli DH5 α and selected in the agarose plate containing the ampicilin. The engineered bacteria were induced by IPTG . SDS-PAGE analysis showed the expression protein was mainly in the form of inclusion. Then recombinant NS3 protein was purified on Ni2+-NTA column under denature condition . The purified protein showed the specificity and immunogen activity by Western-blot assay which was performed with the HCV positive serum as the first antibody.3. The purified recombinant NS3 protein was inoculated in the groin endermic of the BALB/c mice. The monoclonal antibodys (2G11 and 2B8) anti NS3 protein were acquired after cell fusion and coloning. Their specificity was identified with indirect immunofluresent assay and Western-blot assay. Indirect ELISA was used to detect the titer and Ig isotype.4. COS-7 cells were transfected transiently by pcDNA3.1(-)-ns3tp pcDNA3.1(-)-ns3th via lipofectamine method. IFA was performed using the MAbs as the first antibody from above. The results showed that cells expressing the serine protease were positive and cells expressing the helicase were negative when 2G11 was used as the first antibody . On the contrary , cells expressing the serine protease were negative and cells expressing the helicase were positive when 2B8 was used as the first antibody .Therefore the...