HBV X基因真核表达载体的构建研究

The Construction of HBV X Gene Eukaryotic Expression Vector

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,从HBV DNA阳性的血清中,PCR扩增得到HBV X基因全序列,将其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,为下一步研究HBV X基因及其产物的功能奠定了基础。

Object：To construct a highly effective eukaryotic expression vector pCDNA3.1 with HBV X gene （pCDNA3.1 - X） for exploring the contribution of X gene to the chronic hepatitis and the bepatocellular. Methods：Abstract HBV DNA from the HBV DNA positive serum. HBV X gene was amplied by PCR, identified by gelose electrophoresis analysis. The PCRproduct and the eukaryotic expression vector pCDNA3.1 were digested by restruction endonucleases, then the digested X gene was inserted into the digested eukaryotic expressionvector. After screened, the positive recombinants were picked out. PCR analysis, Kpn I and EcoR Irestriction endonucleases digest analysis and sequence analysis were used to identify the recombinants. Results The 480bp DNA fragment was firstly amplified by PCR from the HBV DNA positive serumre. The identical DNA fragment was also amplified from the recombinants. The products of double restriction endonucleases digest analysis were X gene and a 5.5kb DNA fragment. Sequence analysis showed that the the HBV X gene was successfully inserted into pCDNA3, lvector. Conclusion：The eukaryotic expression vector pCDNA3.1 -X has been successfully constructed.