Cloning And Prokaryotic Expression X Protein Of Hepatitis B Virus (HBx) And Preparation And Identification Of Monoclonal Antibody Against It

The hepatitis B virus (HBV) is a serious worldwide health problem and with an estimate of 400 million people chronically infected with the HBV worldwide. It has been demonstrated now that HBV can induce immune reaction , change the function and structure of hepatocyte by directly or indirectly , initiate hepatocyte apoptosis,necrosis or cancerization . The HBV nonstructual X protein (HBx) is a multifuctional regulator that modulates transcription , signal transduction , cell cycle progerss , protein degradation pathways , apoptosis and genetic stability . It is a key regulatory protein of the virus that is at the intersection of HBV infection,replication and involved in host cell cycle progress,apoptosis and hepatocaroinogenesis . However , the most mechanism of HBx have not been elucidated and many conclusions are contrary because of the quality of anti-HBx antibodies and the difference of cell lines . So , it can provid an useful tool for profound study in the function of HBx and find a new way to prevent the infection of HBV and the development of HBV-related hepatocarcinogenesis that preparing for high-qulity monoclonal antibodies .The aim of our study is to construct the prokaryotic expression vector with HBV X genes, express it highly in E.coli to get purified HBx fusion protein. Then, we are going to prepare monoclonal antibody (mAb) against HBx by hybridoma technique and identification it .PCR was deployed to amplify HBV X gene segment containing EcoRI and XhoI endoenzyme sites. After double enzyme digestion, prokaryotic expression vectors pGEX4T-2 and PCR products were conjuncted to construct the recombinant plasmids which were transformated into E.coli-BL21(DE3) later. After IPGT induction, the fusion proteins were purified with the glutathione-Sepharose 4B gelatum and then dealt with Western-blot detection. Balb/c mice were immunized with HBx fusion protein and monoclonal antibodies against HBx were prepared with hybridoma method. The immunoglobulin (Ig) subtype, the ascites titers,the binding site, and the affinity of the obtained anti-HBx mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis.Enzyme digestion and sequencing analysis showed that the target gene had been inserted into recombinant vector. SDS-PAGE analysis showed that the target protein had been expressed successfully and had been purified With the affinity chromatography of glutathione-Sepharose 4B. The Western-blot detection showed that inductive expressed fusion proteins have specific antigenicity. From over thirty positive hybridoma which secreting monoclonal antibody to HBx fusion protein, we screening out a pair of hybridomas, named AF5 and DA12 . The Ig subtype of AF5 and DA12 mAb was both IgG1 , and the ascites titers of them were 1: 106 and 1:108 , respectively. Our study also demonstrated that these two mAbs, which recognized the same epitope, could both specifically bind to the HBx protein . The relative affinity constant of AF5 and DA12 mAb was determined as 105 and 106 respectively. Western blot identification suggested that mAb could bind specially to the antigen.In conclusion , we obtained the GST-HBx fusion protein and purified the target protein with high-purity and the correct molecular size through the method of gene recombination and prokaryotic expression . Two monoclonal antibodies against HBx protein were obtained and characterized finally. The results establish the foundation of the profound approaching the biological function of HBx and the role in the infection of HBV. Moreover, it provides us new insights into the preventative and clinical therapy of HBV and HBV-related hepatocarcinogenesis.