Cloning, Expression And Product Purification Of HCV F Gene Fragment And Preliminary Application

Hepatitis C virus is a serious and growing threat to human public health and is estimated to infect over 180 million people worldwide. Hepatitis C, it had become clear that the vast majority of persons who develop acute hepatitis C-perhaps over 80 percent-remain infected. A high proportion of persons with chronic HCV infection will advance in time to cirrhosis and progress eventually to terminal chronic liver disease, in some instances after developing HCC.HCV is a member of the Flaviviridae family, with a positive, single-strand RNA genome of 9.6 kb. The genome encodes a single polyprotein that is proteolytically cleaved to produce four structural (core, E1, E2, and p7) and at least six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. The core protein is located at the N terminus of the polyprotein and is predicted to have a length of 191 amino acids and a molecular mass of 23 kDa (p23). HCV core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases.Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence. F protein is conserved in different HCV genotype. It may play an important role in the viral life cycle.So we carry out our work of assessing the prevalence of serum anti-F in patients with hepatitis C virus (HCV) infection, and the correlation between anti-F and HCV infetion.Sectionâ… : Cloning and Expression of HCV F Gene Fragment in Escherichia ColiThe HCV F gene fragment was cloned by PCR and inserted into plasmid vector pGEX-4T-2 digested with BamH I and EcoR I, then the recombinant plasmid was transformed into E.coli TG1. The sequence ofF gene obtained was identified by PCR and sequencing.The recombinant strain was induced by IPTG for expression. SDS-PAGE analysis indicated that a chimeric protein of HCV F fragment with GST was expressed in E.coli. Relative molecular weight of the expressed HCV-F/GST chimeric protein was about 43kD.The engineering E.coli for the expression of HCV-F gene was established successfully, which lays a foundation of purification of the recombinant antigen with good antigenicity and specificity.Sectionâ…¡: Purification and Preliminary Identification of the Expressed HCV-F ProteinThe recombinant strain expressing HCV-F protein by IPTG induction was harvested by centrifugation and lyzed by ultrasonic wave. The expressed HCV-F protein mainly existed in the form of soluble protein. Then the supernatant was purified by Glutathione Sepharose 4B and eluted with buffer (Glutathione) and was analysed by SDS-PAGE.Plasmid pGEX-4T-2 was transformed into E. coli TG1 and expressed GST protein. The GST protein was purified by Glutathione Sepharose 4B.Using the purified HCV-F protein as antigen, we tested sera of patients with hepatitis C virus infection and normal sera by indirect enzyme-linked immunnosorbent assay (ELISA) and western blot. The results indicated the expressed HCV-F /GST protein was reactive to positive serum and non-reactive to normal serum in ELISA. The results preliminarily confirmed the chimeric protein could be produced and purified easily.We successfully expressed and purified the HCV-F recombinant protein antigen with good antigenicity, and it would be very useful for developing the reagents for immunological diagnosis of HCV infections. The purified recombinant protein (HCV-F /GST) was coated onto microtiter plates as antigen. The secondary antibodies were horse radish peroxidase (HRP)-conjugated goat anti-human IgG. Sera of 120 patients with hepatitis C virus infection and 10 normal sera were tested by indirect ELISA for detecting anti-HCV-F antibodies. The results indicated that the recombinant antigen has a specific reaction with about 68% of 120 patients, whereas no reaction with 10 normal persons. There were no significant correlations between anti-F antibody and sex, HCV RNA serum, types of HCV RNA. Patients of midrange,severe range and hepatic cirrhosis have higher rate than others, the positive rate of anti-F is higher in patients over fifties. All above findings showed the chimeric protein has good antigenicity and specificity.We preliminarily developed an indirect ELISA for detecting antiHCV-F antibodies by optimizing the detection reagents and reaction conditions.Sectionâ…£: Preparaion of HCV-F polyclonal antibodyThe purified recombinant protein (HCV-F /GST) were used as antigen to immune BALB/C mice. When sera from mouse tail were detected with the titration of 1:10000, we booster the immunity by annother injection. One week later, sera of these mice were extracted by picked eyes. Western blot test showed the immuned mice sera has a specific reaction with HCV-F/ GST protein whereas no reaction with normal mice. Those findings showed the good and specific polyclonal antibody was abtained . This lays a foundation of further research of HCV-F protein function during HCV infection and course of disease.