Studies On Expression Of Hepatitis C Virus Structural Gene And HCV-like Particles

Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It has been estimated that about 170 million people worldwide are infected with HCV, of whom 50~ 80% will develop chronic liver disease, leading to cirrhosis in 10 to 20% and hepatocellular carcinoma in 1 to 5% of chronically infected individuals. Because lack of tissue culture systems and small animal models, the development of a prophylactic vaccine is very difficult. The linear, single-stranded, positive-sense HCV RNA genome of 9.5 kb contains a single open reading frame (ORF) encoding a polyprotein which is cleaved into the structural proteins and nonstructural proteins by host- and virus-specific proteinases. Three structural proteins including the core protein and two envelope proteins El and E2 play a pivotal role in inducing cellular and humoral immune responses. In this study, first, we expressed a fused protein including HCV truncated core protein with EGFP by using Bac-to-Bac baculovirus expression system, to explore the transfecting efficiency of recombinant baculovirus expression vector, then, we expressed a fused protein including HCV full-length core protein and truncated E2 protein, to study its antigenicity. finally, we studied the HCV-like particles(HCV-VLP) in a recombinant baculovirus that contains the HCV structural gene(core/E1/E2), investigated the binding of HCV-VLP to different cell lines, and evaluated its antigenicity and immunogenicity.1. Expression of hepatitis C virus core protein and EGFP gene in insect cellsHCV truncated core gene amplified by PCR using pGEM-HCJ4 plasmid as the template which contains full-length cDNA clone of HCV, and was inserted into baculovirus expression vector pFastBacl, constructed a recombinant vector, pFastCt. EGFP gene amplified by PCR method, and subcloned into pFastCt, constructed a fusion expression vector, pFastCt-EGFP. After transposition in DHlOBac E .coli., recombinant baculovirus BacmidCt-EGFP were gained. After the transfection of BacmidCt-EGFP to Sf9 cells , using fluorescence microscope observation, we found that the fluorescenced cells transfected with high molecular Bacmid is less than the efficiency of pEGFP-N1. SDS-PAGE and Western blot showed that expressed fusion protein Ct-EGFP had an expected molecular mass of 40 kDa. ELISA results showed that fusion protein Ct-EGFP was able to react with 15 /20 anti-HCV sera samplesfrom HCV patients. The reactivity frequency was 54%.2. Expression of hepatitis C virus core gene and truncated envelope 2 gene in insect cells and its antigenicityTo study the antigenicity of Sf9 insect cells expressed products of HCV different structural gene and obtain HCV core protein and E2 protein, HCV truncated core gene and truncated envelope E2 gene were amplified with PCR method by using plasmid pGEM-HCJ4 as the template, and cloned into pFastBacHTa vector, constructed a fusion expression vector, pFBC-E2t. After transformed into DHlOBac E xoli., we gained recombinant baculovirus transposon-shuttle vector Bacmid C-E2t, and then transfected into Sf9 cells. The expression product was analyed by SDS-PAGE and Western-blot, showed two bands with different molecular weight: 21 kDa band is HCV core protein ,the 33 kDa band is truncated E2 protein, demonstrated that recombinant proteins were expressed and cleaved into the core protein and E2 protein in Sf9 cells. Purified protein could react strongly with monoclonal antibody (mAb) against HCV core protein. ELISA results showed that, fusion protein C-E2t was able to react with 20 /28 anti-HCV sera samples from HCV patients. The reactivity frequency was 71%. using affinity chromatography purified C protein was able to react with 18 /20 anti-HCV sera samples from HCV patients. The reactivity frequency was 64%, The successful expression of recombinant HCV CE2 protein will be helpful in the study of HCV vaccine.3. Expression of hepatitis C virus structural gene and assembly, purification of HCV-like particlesThree fragments(5'NCR- C, 5'NCR- C-E1,5'NCR- C-E1-E2) produced by long template PCR, were separately subcloned into baculovirus donor plasmid pFastBacl, and three expression vectors( pFastA, pFastB, pFastQwere constructed, respectively. The vectors were verified and used to transform DHlOBac. We can see the existence of abundant baculoviruses in the nuclei of transfected cells , and gained recombinant baculovirus transposon-shuttle vector BacmidA, BacmidB, BacmidC to generate recombinant baculoviruses, yielding rBacA , rBacB ,rBacC, respectively. SDS-PAGE and Western blot analysis of lysates from infected Sf-9 cells with either recombinant baculoviruses demonstrated that rBacA showed one novel band of the size of expected HCV C protein which is 21 kD, rBacB demonstrated 2 novel bands...