乙型肝炎病毒X基因真核表达载体的构建及其在巴斯德毕赤酵母中的表达

Construction and Expression in Pichia pastoris of an Eukaryotic Vector for Hepatitis B Virus X Gene

目的:在巴斯德毕赤酵母中表达乙型肝炎病毒(HBV)X蛋白,为探讨HBVX蛋白与慢性乙型肝炎及肝细胞癌发生的关系奠定基础。方法:用PCR方法扩增X基因序列,并分别在上下游引入XhoⅠ和XbaⅠ酶切位点,插入pPICZαA载体,转化大肠杆菌TOP10,筛选阳性克隆,对其进行PCR和双酶切及测序鉴定,构建HBVX蛋白毕赤酵母表达质粒pPICZαA-HBx;电击转化毕赤酵母GS115,对阳性克隆进行诱导表达后经SDS-PAGE和Western blotting鉴定目的蛋白。结果:双酶切pPICZαA-HBx后,琼脂糖电泳可分别见到大小约为3.1kb和465bp的片段,表明目的片段已插入载体中,序列测定表明其含有完整的X基因片段,Western blotting结果显示含有pPICZαA-HBx的毕赤酵母GS115能分泌表达X蛋白。结论:构建了毕赤酵母表达载体pPICZαA-HBx,并能在毕赤酵母GS115中分泌表达X蛋白。

Objective： To construct and express a Pichia pastoris expression vector of HBV X protein for exploring the contribution of X protein to the chronic hepatitis and the hepatocellular carcinoma. Methods： X gene with Xho Ⅰ and Xba Ⅰ restriction enzyme sites was obtained by PCR, and subcloned into the pPICZαA vector. After identified by restrictive enzymes digestion and sequencing, reconstructed plasmid was transformed into P.pastoris GS115. Expression of X protein was assayed in P.pastoris GS115 culture supernatant by SDS and Western blotting. Results： The target gene X fragment about 465 bp was intergrated into the genome of P.pastoris GSllS. The recombinant X protein with molecular weight of 17 kDa was secreted into the culture supernatant and was confirmed by Western blotting. Conclusion： P.pastoris expression vector pPICZαA-HBx was constructed successfully and recombinant X protein was expressed in P.pastoris GS115.