The Expression And Purification Of HCV NS3 Protein And Its Antibody Preparation

Hepatitis C virus(HCV)is a leading cause of liver infection and a leading public health threat all over the world.It estimates that 200 million peoples are chronically infected with HCV,approximately 4 million people infected with HCV virus are living in China.However,more than 70%of those infected will develop chronic infection,30%of them were cleared the infection through their own body immune system,including chronic infection,but 5%of patients progressed to liver cirrhosis(Hepatocellular cirrhosis,HC)and liver cancer(Hepatocellular carcinoma,HCC)annually.The current international standard for anti-viral treatment is pegylated interferon α(PEG-IFN-α)and ribavirin(Ribavirin,RBV),but this treatment has controlled 60%cases,however,after termination of treatment there is chance of relapse.Moreover,there is still no vaccine,even no good prospects progress in the vaccine development.The best way for the hepatitis C prevention and control is earlier and correct diagnosis to control the origin of transmission,and interrupt the route of transmission.The main technique for the detection of HCV nucleic acid testing,antibody and antigen detection,HCV nucleic acid identification accuracy,due to laboratory contamination and expensive cost,it is not used in hospitals,Antibody detection easy operation,good repeatability,but there is a window period,and the virus susceptible to mutation,the presence of multiple genotypes and subtypes,likely to cause missed.HCV NS3 protein antigen immunity is strong,the presence of multiple epitopes,it is easy to detect,but at very low levels in the blood,so the establishment of efficient high-sensitivity detection of antigen,the diagnostic reagent to facilitate the operation is important.The purpose of this study was to create a high sensitivity diagnostic kit for HCV NS3 antigen.Using Bioinformatics software,our analysis showed that HCV NS3 protein molecular weight is about 67kDa,NS3 protein in the presence of a plurality of hydrophilic domains,NS3 protein no typical transmembrane region,NS3 protein is present several epitopes.In this study,the whole protein as HCV NS3 protein of the present experiment,the purpose is to obtain high expression rate of protein and high titer antibody.First to J6/JFH1 2a full-length HCV NS3 gene in the genome of the chuck card tailed primers were designed for full-length NS3 protein gene fragment was amplified by PCR,the full-length NS3 protein gene expression plasmid connected to PET28a success,Construction of a recombinant expression vector PET28a-NS3.The recombinant expression vectors PET28a-NS3 was transformed into E.coli expression strain BL21,the recombinant bacteria in shaking at speed of 250r/min,IPTG concentration of lmmol/L,inducing temperature 37℃,induced 5h,The recombinant protein exists mainly in the form of inclusion bodies by sonication cell,after overnight 8M urea dissolved by Ni2+-NTA affinity chromatography can be purified NS3 protein purposes.Use NS3 protein-coated microtiter plates by indirect ELISA method for detecting the plasma of patients showed that the purified NS3 protein can be identified with PCR positive patients have a good response to the effect of the serum,indicating that the purified NS 3 protein has good antigenicity.Using purified NS3 protein to immunize New Zealand white rabbits,by using indirect ELISA assay,Antibody titers showed that rabbit polyclonal antiserum titer can reach 1:512000.Identified by Western blot using rabbit polyclonal antibody showed that rabbit polyclonal antibody prepared with good specificity.NS3 protein immunization with purified BALB/c mice was detected by indirect ELISA serum titer Fourth reach 1:256000 which Identified by Western blot.By indirect immunofluorescence to HCV(J6/JFH1)virus infection of HCC cells plated for 3 days,to prepare a multi-mouse antibody as the first antibody,the results indicated a good fluorescent effect of the immune response.In current study,HCV NS3 full length protein was successfully expressed,purified single protein,strong immunogenicity of purified protein prepared in high titer polyclonal antibodies,which provide the basis for the diversity of HCV antigen detection and development of high-sensitivity HCV antigen detection kit.The NS3 protein purified specific well,with positive serum HCV patients respond well to lay a good foundation HCV NS3 protein function studies and preparation of monoclonal antibodies to HCV NS3.