不同启动子驱动HDV核酶的逆转录病毒载体构建

Construction of retrovirus expression vectors with different promoters expressing HDV ribozymes

目的分别构建CMV、H1、tRNA、U2、U3和U6启动子驱动反式丁型肝炎病毒核酶(HDV核酶)的逆转录病毒表达载体。在这些载体中,HDV核酶设计为靶向HBV基因序列PreS2和C区。方法用PCR技术分别扩增CMV、U2和U3启动子,并连入pMD18-T载体。合成靶向PreS2和C区HDV核酶并利用SalⅠ和H indⅢ的酶切位点分别连入逆转录病毒表达载体pLEGFP(pLEGFP-R z)。然后利用BamHⅠ和SalⅠ的酶切位点分别把CMV、H1、tRNA、U2、U3和U6启动子连入重组载体pLEGFP-R z。所有的重组载体经PCR和酶切的方法验证。结果成功地构建了分别含有6种启动子驱动HDV核酶的逆转录病毒载体。结论这些重组载体的构建为筛选高效表达核酶的启动子奠定基础。同时这些重组载体也可用于进一步研究HDV核酶抑制HBV复制的效率。

Objective To construct recombinant retrovirus vector expressing trans-acting HDV ribozymes driven by six different promoters CMV,H1,tRNA,U2,U3 and U6.In these vectors,HDV ribozymes were designed to target PreS2 and C gene of HBV.Methods CMV,U2 and U3 promoters were amplified by PCR and cloned into pMD18-T vector.HDV ribozymes of PreS2 and C were synthesized and ligated into pLEGFP vector by SalⅠand Hind Ⅲ restriction enzyme.Then the CMV,H1,tRNA,U2,U3 and U6 promoter were inserted into recombinant pLEGFP-Rz vector by BamHⅠand SalⅠsites,respectively.All the recombinant vectors were tested by PCR and restriction enzyme digestion.Results All the recombinant retrovirus vectors carrying trans-acting HDV ribozymes driven by six promoters were constructed successfully.Conclusion These constructs are the first step to screen high efficient promoters for ribozyme expression.Meanwhile,these recombinant vectors could be used for the study on the inhibition efficiency of HDV ribozymes against HBV replication.