乙型肝炎病毒X基因克隆及其表达

Clone of hepatitis B virus X gene and its protein expression

目的:构建乙型肝炎病毒X基因原核表达载体,并诱导其表达。方法:从乙肝病人血清中提取HBVDNA;以带有EcoRI和HindⅢ酶切位点的引物,用PCR方法扩增X基因;而后定向克隆到原核表达载体pMAL-C2X上,经酶切鉴定和序列分析;以IPTG诱导X融合蛋白的表达。结果:PCR扩增显示有类似HBVX基因片断大小的条带;构建的原核表达重组体PMAL-C2X-HBV-X经酶切有相应大小的的基因插入片断,序列分析证实为正确的有完整读码框的X基因;这一重组体在IPTG诱导下有相应大小的X融合蛋白表达。结论:成功构建了HBVX基因原核表达重组体pMAL-C2X-HBVX;在IPTG的诱导下,该重组体在大肠杆菌中可表达X融合蛋白,为进一步纯化HBVX蛋白和研究其生物学作用奠定了基础。

Objective To construct the hepatitis B virus （HBV） X gene recombinant and to induce the expression of X protein. Methods HBV DNA was extracted from the serum of patient with hepatitis B. The X gene was amplified by PCR using the primers with EcoRI and Hindlll digestion sites, and then cloned into pronucleus expression vector pMAL-C2X, which was detected by EcoRI and Hindm digestion and sequence. Finally, the recombinant was induced by IPTG to express X protein in JM109. Results The band similar to X gene was amplified by PCR. There were fragments similar to X gene when the recombinant was digested by the enzyme digestion. It was tested by DNA sequence that the correct and entire opening reading frame of HBV X gene was inserted. The X protein was expressed by the IPTG induction. Conclusion Pronucleus expression recombinant pMAL-C2X-HBV-X is constructed successfully and with the IPTG induction, the recombinant pMAL-C2X-HBV-X can express the X protein in E. coli JM109, which lays the foundation for the HBV X protein purification and its biological study.