野生型及突变型乙型肝炎病毒X基因在pGEX-6P-2系统中的克隆、表达、纯化及免疫鉴定

构建野生型和A1762T/G1764A双突变型乙型肝炎病毒(HBV)X基因原核表达系统,为深入研究HBV X基因及其双突变后对HBV慢性感染者病情发展和肿瘤发生的作用奠定基础。从慢性乙型肝炎患者血清中抽提HBV基因组DNA,经PCR扩增和基因测序,证实并获得野生型及A1762T∕G1764A双突变型HBV X基因。应用TA克隆及亚克隆技术将2基因分别插入pGEX-6P-2载体,构建pGEX-6P-2-hbvxw(野生型)及pGEX-6P-2-hb-vxm(A1762T∕G1764A突变型)重组表达载体;转化入宿主菌进行诱导表达;包涵体复性后,GSTrap FF蛋白纯化柱纯化带有GST标签的野生型和A1762T∕G1764A突变型HBV x抗原(HBxAg);应用SDS-PAGE、Westernblot和ELISA方法对表达的野生型和突变型HBxAg进行鉴定。结果SDS-PAGE证实本研究构建的2个表达系统均能高效表达目的蛋白;复性、纯化后野生型和突变型GST-HBxAg分别达到4.88mg/mL和5.07mg/mL;Western blot鉴定证实所纯化的野生型和突变型HBxAg均能被特异性的单克隆抗体所识别;ELISA方法检测显示2种抗原都能成功包被于微量滴定板上并与乙肝患者血清反应。证实本研究成功地构建了野生型和A1762T∕G1764A突变型HBxAg表达系统,为进一步研究A1762T∕G1764A基因突变对肿瘤发生学和慢性HBV感染者疾病进展的作用和分子机制奠定了基础。

The cloning, expression, purification and immunological identification of wild-type and mutant hepatitis B virus X gene in pGEX-6P-2 system

To settle the foundation for the future research on the influence of wild and mutant (A1762T/ G1764A) HBV X gene on the progress of chronic HBV infection and hepatic tumorigenicity, wild and mutant (A1762T/G1764A) HBxAgs expression system was constructed. The wild and mutant (A1762T/ G1764A) HBV X genes were amplified with polymerase chain reaction (PCR) from HBV genome were inserted into pGEX-6P-2 and confirmed by sequencing respectively. Prokaryotic expression vectors pGEX-6P-2-hbvx(w) and pGEX-6P-2-hbvx(m) (A1762T/G1764A) were constructed and transformed to Trans1-blue; wild and mutant HBxAgs were expressed through IPTG induction respectively; after refolding of inclusion body, the wild and mutant HBxAgs were purified with GSTrap FF; and analysised by SDS-PAGE, Western blot and ELISA. SDS-PAGE analysis showed that the expression system was able to express target protein efficiently; the concentrations of purified wild HBxAg and mutant HBxAg were 4.88 mg/mL and 5.07 mg/mL respectively; Western blot analysis certified both the wild HBxAg and the mutant HBxAg could be recognized by the same monoclonal antibody against HBxAg; 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. Results demonstrated that we successfully established a system for expression of hepatitis B x antigen and lay 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.