乙型肝炎病毒前S2基因克隆及真核表达载体的构建

Cloning of Hepatitis B Virus PreS2 Gene and Construction of Eukaryotic Expression Vector

目的:克隆乙型肝炎病毒前S2基因,构建其真核表达质粒。方法:以乙型肝炎患者血清HBV DNA为模板,设计引物扩增全长前S2基因,再将目的基因插入到真核载体pcDNA3.1(+)中,经PCR、酶切和DNA测序确认。结果:从患者血清抽提的DNA中扩增得到约860bp大小的目的片段,经回收、纯化、酶切后转化大肠杆菌DH5α,阳性重组质粒经PCR扩增得到约860bp产物;重组质粒经BamHⅠ和EcoRⅤ双酶切得到5.4kb和860bp两个片段,与预期一致;经DNA测序与已知序列比对确认前S2基因片段正确插入pcD-NA3.1(+)载体。结论:成功构建了含前S2基因的真核载体,为进一步研究HBV前S2蛋白的生物学功能及其实验诊断意义提供了条件。

Objective:To clone HBV preS2 gene and construct an eukaryotic expressive vector pcDNA-S2.Method:HBV preS2 gene was amplified by PCR from serum of a chronic HBV carrier,and cloned into the eukaryotic vector pcDNA3.1(+),then authenticated by PCR,digestive enzymes and sequencing.Results:A 860bp fragment of the preS2 gene was successfully amplified by PCR from the serum,and inserted into the eukaryotic vector pcDNA3.1(+). The positive clone was identified by PCR,enzyme digestion and DNA sequencing.Conclusion:Eukaryotic expression vector pcDNA-S2 was successfully constructed,which provides a tool for further studies on its biological function and further investigation of its roles in HBV diagnosis.