The Cloning, Expression, Purification And Immunological Identification Of Wild-Type And Mutant Hepatitis B Virus X Gene

Background and Objectives:The hepatitis B virus (HBV) infection is a worldwide human health problem and is a major risk factor for the formation of human hepatocellular carcinoma. The HBV genome contains four open reading frames (ORF), S, C, P and X Area. Among them, the X area consisting of465bases about, i.e. nucleotide1374-1838, is the smallest open reading frame (ORF) in the HBV genome. The hepatitis B virus X antigen (HBxAg) coded by X gene has a significant correlation with liver cirrhosis and hepatocellular carcinoma. HBV mutates frequently because of the unique HBV replication system, host immune pressure and other factors. It was reported that patients with HBV-related liver cancer had higher viral mutation rate than asymptomatic HBV carriers. The most common type of mutation is A1762T/G1764A double mutant. A1762T/G1764A double mutation stays in the core promoter (CP) coding region. Many scholars believed that A1762T/G1764A double mutant affects the core promoter activity to some extent. However, due to the coding regions in the HBV genome with a high degree of overlapping, A1762T/G1764A double mutant just causes amino acid130and131changes of HBxAg. This change is likely to bring about changes in the HBV protein structure and function, thereby affecting the HBV-related tumorigenesis and disease progression. In order to prove the hypothesis, we constructed prokaryotic expression systems for the wild-type and A1762T/G1764A double mutant hepatitis B virus X genes respectively. After inducing, recombinant HBxAg-wild and HBxAg-mutant were purified and identified immunologically. In addition, we made and purified with an affinity chromatography colum (HiTrap rProtein A FF) rabbit polyclonal HBxAb-wild and HBxAb-mutant against HBxAg-wild and HBxAg-mutant respectively. This study settled a solid foundation for research on implication of A1762T/G1764A mutation in HBV X gene.Method:The hepatitis B virus (HBV) genomes were extracted from the sera of patients who had been infected with HBV. The wild and mutant(A1762T/G1764A) HBV X genes were amplified with polymerase chain reaction (PCR) from HBV genome and were confirmed with gene sequencing. With TA cloning and subcloning techniques, the two HBV X genes were inserted into pGEX-6P-2respectively. Prokaryotic expression vectors pGEX-6P-2-HBV Xw and pGEX-6P-2-HBV Xm (A1762T/G1764A) were constructed and transformed to Trans1-blue; wild and mutant HBxAgs were expressed through IPTG respectively; after refolding of inclusion body, the wild and mutant HBxAgs which owns GST-tag were purified with GSTrap FF; the wild and mutant HBxAgs were analysised by SDS-PAGE, Western blot and ELISA; Make rabbit polyclonal HBxAb-wild and HBxAb-mutation.Identification of rabbit polyclonal HBxAb use ELISA; Purify rabbit polyclonal HBxAb use HiTrap rProtein A FF.Result:1. SDS-PAGE found that the two expression systems were able to express target protein efficiently.2. After refolding and purifying, the concentrations of purified wild HBxAg and mutant HBxAg were4.88mg/ml and5.07mg/ml respectively.3. Western blot certified both the wild HBxAg and the mutant HBxAg could be recognized by the same monoclonal antibody against HBxAg, implying the two kinds of recombinant antigen has a similar antigenic.4. The two expressed fusion antigens coated in microtiter plate were able to react with the sera of HBV infected patients but not with the sera from healthy donors in ELISA. 5. It was confirmed with ELISA method that the rabbit polyclonal HBxAb-wild, and HBxAb-mutation could specifically identify wild type and mutant HBxAg.Conclusions:We established the systems for expression of hepatitis B X gene (wild and mutant) and made rabbit polyclonal HBxAb-wild and HBxAb-mutant successfully and set up the foundation for further research on the role and molecular mechanisms of the mutant HBxAg in the progress of chronic HBV infection and hepatic tumorigenicity.