乙型肝炎病毒X基因真核表达的载体构建及鉴定

目的:构建HBV X基因与pCDNA3.1相融合的高效真核表达载体,为进一步研究HBV X基因的功能奠定基础。方法:设计并合成HBV X基因的引物,以HBV DNA阳性血清PCR扩增得到HBV X基因全序列;将X基因连接到真核表达载体pCDNA3.1上后,酶切图谱分析、PCR检测和扩增产物序列分析等,鉴定所构建的真核表达载体。结果:以重组载体为模板扩增得到的片段大小与已知HBV X基因大小相同,酶切也得到目的基因片段,测序结果也显示与已知X基因序列相同。结论:成功构建了HBV X基因的真核表达载体,为进一步研究HBVX基因及其产物的功能奠定了基础。

Construction and Identification of HBV X Gene Eukaryotic Expression Vector

Objective: To construct a highly effective eukaryotic expression vector pCDNA3.1 with HBV X gene(pCDNA3.1-X).Methods: HBV DNA was extracted from the HBV DNA positive serum.HBV X gene was amplified by PCR,and identified by gelose electrophoresis analysis.The PCR products and the eukaryotic expression vectors pCDNA3.1 were digested by restriction endonucleases,then the digested X gene was inserted into the digested eukaryotic expression vector. After screening,the positive recombinants were picked out.PCR analysis,Kpn Ⅰ and EcoR Ⅰ restriction endonucleases digest analysis and sequence analysis were used to identify the recombinants.Results: The 480 bp DNA fragment was firstly amplified by PCR from the HBV DNA positive serum.The identical DNA fragment was also amplified from the recombinants.The products of double restriction endonucleases digest analysis were X gene and a 5.5 kb DNA fragment.Sequence analysis showed that the HBV X gene was successfully inserted into pCDNA3.1 vector.Conclusion: The eukaryotic expression vector pCDNA3.1-X has been successfully constructed.