【摘要】 目的构建1.3拷贝乙型肝炎病毒(HBV)拉米夫定耐药株的重组逆转录病毒载体及其包装细胞系。方法在野毒株1.3拷贝HBV载体的基础上采用定点突变的方法将rtL180M,rtM204V位点突变,构建拉米夫定耐药病毒株,并通过PCR、限制性内切酶酶切,分别以正向和反向将野毒株和耐药株分别连接于逆转录病毒载体pLNCX2的相应酶切位点,构建成重组逆转录病毒载体,经双酶切及PCR扩增鉴定。重组载体转染包装细胞,筛选培养细胞克隆并进行病毒滴定。结果通过双酶切、PCR扩增、测序鉴定证实成功构建了1.3拷贝HBV拉米夫定耐药株的逆转录病毒载体。成功建立包装细胞系并测得病毒滴度均为1×105cuf/ml。结论含正向及反向1.3拷贝HBV拉米夫定耐药的重组逆转录病毒载体及其包装细胞系构建成功,可以用于拉米夫定耐药细胞模型研究。更多还原

【Abstract】 Objective To Construct recombinant retrovirus vector containing 1.3-fold-overlength genome of lamivudine-resistant HBV and its packaging cell line.Methods The 1.3-fold-overlength genome of lamivudine-resistant HBV was obtained by the method of site-directed mutagenesis base on the 1.3-fold-overlength genome of wild strain HBV.The corresponding mutation in the HBV reverse transcriptase domain of the polymerase are L180M and M204V.Then it was inserted into the retrovirus pLNCX2 vector cloning site in the sense or antisense orientation respectively with recombinant technigue. PT67 cells were transfected with the recombinant retrovirus vectors. The virus titers of packaging cell lines were determined by infecting NIH3T3 cells.Results The recombinant retrovirus vector carrying sense or antisense 1.3-fold-overlength genome of lamivudine-resistant HBV have been constructed. It have been identifed by restriction enzyme digestion and electrophoretic analysis fragments of PCR amplification and DNA sequence were checked with automatic sequenator. The virus titers of packaging cell lines were about 1×10~5 cuf/ml.Conclusion The recombinant retrovirus vector carrying sense or antisense 1.3-fold-overlength genome of lamivudine-resistant HBV and its packaging was constructed successfully.It would apply to the study of lamivudine-resistant cell lines.更多还原