携带可调控人前脑啡肽原基因逆转录病毒载体的构建及鉴定

Construction and identification of recombinant retroviral vector with controllable human preproenkephalin gene

目的构建四环素调控的含人前脑啡肽原基因(hPPE)逆转录病毒载体。方法用PCR方法扩增目的基因hPPE,定向克隆入逆转录病毒四环素反应质粒(pRevTRE)中,酶切反应、PCR 及DNA测序鉴定重组逆转录病毒载体pRevTRE/hPPE;在脂质体介导下分别将pRevTRE/hPPE和逆转录病毒四环素调节质粒(pRevTet-on)转入包装细胞PT67,经相应的抗生素稳定筛选得到抗性细胞克隆。RT-PCR鉴定得到阳性产病毒细胞系PT67/Tet-on和PT67/TREhPPE。用NIH3T3细胞测定病毒滴度。结果经限制性酶切分析、RT-PCR及DNA序列分析证实pRevTRE/hPPE中含有hPPE基因。转染有pRevTet-on的FT67细胞病毒滴度为2.6×105 CFU/ml,转染有pRevTRE/hPPE的PT67细胞病毒滴度为3.2×105 CFU/ml。结论成功构建了含有受四环素调控hPPE基因的逆转录病毒载体,建立了能产较高滴度逆转录病毒的包装细胞系,为可调控细胞移植镇痛的研究奠定了实验基础。

Objective To construct a recombinant retroviral vector with human preproenkephalin gene regulated by tetracycline. Methods Human preproenkephalin gene was amplificated by polymerase chain reaction （PCR） and was cloned into retrovirus tetracycline responsive plasmid. Then this recombinant plasmid and regulatory plasmid pRev Tet-On were transferred into packaging cell PT67 respectively. The transfected PT67 / Tet-On and PT 67 / TREhPPE cells were selected by the corresponding antibiotics and identified by RT-PCR. The selected cells were enriched and virus titer was assayed using NIH3T3.Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant RevTRE/hPPE vector was constructed successfully. The virus titer of PT 67/TREhPPE was 3.2 × 10^5 CFU·ml^-1 and the virus titer of PT67/Tet-On was 2.6 × 10^5 CFU· ml^-1 . Conclusion A recombinant retroviral vector with human preproenkephalin gene regulated by trtracycline and stable virus producing lines have been successfully constructed, providing a good basis for further research on regulated cell therapy of chronic pain.