HBV全基因组逆转录病毒载体的构建及鉴定

Construction of A Retrovirus Plasmid Containing the Whole Genome of Hepatitis Virus B

目的构建HBV全基因组逆转录病毒载体,并进行酶切鉴定和测序。方法用合适的引物,通过PCR方法扩增pBlue Script sk(-)-HBV载体上的HBV全基因组,分别用HindⅢ和SalⅠ进行PCR产物和pLEGFP-N1载体的双酶切,用连接酶将HBV DNA和载体进行连接,转化连接产物后,进行质粒提取、酶切和测序鉴定。结果PCR产物凝胶电泳可见3.2 kb条带。pLEGFP-N1-HBV重组质粒酶切结果电泳后,可看见6.9 kb和3.2 kb的条带,测序结果表明重组质粒含有HBV全基因组。结论成功构建了HBV全基因组逆转录病毒载体,为下一步形成稳定的细胞模型奠定基础。

Objective To construct a retrovirus-mediated expression system containing the whole genome of hepatitis virus B, Methods full length hepatitis virus B genome was amplified by PCR from the pBlueScript sk （-）-HBV recombinant plasmid, The PCR amplified HBV DNA was inserted into retroviral vector pLEGFP-N1 and then the recombinant plasmid was identified by restriction endonuclease analysis and sequencing, Results 3.2 kb DNA fragment was PCR amplified. 6.9 kb and 3.2 kb bands can be seen when the recombinant plasmid was double digested. Result of the DNA sequence of the recombinant plasmid showed the whole genome of hepatitis virus B was cloned. Conclusion The recombinant retroviral pLEGFP-HBV plasmid was constructed successfully.