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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。
To construct a prokaryotic expression system of wild-type and A1762T / G1764A double-mutant hepatitis B virus (HBV) X gene, and to lay a foundation for further study of HBV X gene and its double mutation on the progression of disease and tumorigenesis in patients with chronic HBV infection. HBV genomic DNA was extracted from the serum of patients with chronic hepatitis B, and the wild-type and A1762T / G1764A double mutant HBV X genes were confirmed and obtained by PCR amplification and gene sequencing. The recombinant plasmids pGEX-6P-2-hbvxw (wild type) and pGEX-6P-2-hb-vxm (A1762T / G1764A mutant) were constructed by inserting the two genes into pGEX-6P-2 vector using TA cloning and subcloning techniques The recombinant plasmids were transformed into host bacteria for inducing expression. After renaturation of inclusion bodies, GST-tagged wild-type and A1762T / G1764A mutant HBV x antigen (HBxAg) were purified by GSTrap FF purification column. SDS-PAGE, Western blot and ELISA Methods The expressed wild type and mutant HBxAg were identified. Results SDS-PAGE confirmed that the two expression systems constructed in this study could efficiently express the target protein. The renatured and purified GST-HBxAg were 4.88mg / mL and 5.07mg / mL, respectively. Western blot confirmed that The purified wild-type and mutant HBxAg can all be recognized by specific monoclonal antibodies. The ELISA method showed that both antigens can be successfully coated on the microtiter plate and reacted with the serum of hepatitis B patients. This study confirmed that the wild-type and A1762T / G1764A mutant HBxAg expression system was successfully constructed, which laid the foundation for the further study on the role and molecular mechanism of A1762T / G1764A mutation on the progress of tumorigenesis and chronic HBV infection.