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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 tetracycline-containing retroviral vector containing human proenkephalin gene (hPPE). Methods The hPPE gene was amplified by PCR and cloned into retrovirus tetracycline plasmid pRevTRE. The recombinant retroviral vector pRevTRE / hPPE was identified by restriction endonuclease digestion and DNA sequencing. PRevTRE / hPPE and retroviral tetracycline-regulated plasmid (pRevTet-on) were transferred into packaging cell PT67, and stable cell lines were screened by corresponding antibiotics. Positive virus-producing cell lines PT67 / Tet-on and PT67 / TREhPPE were identified by RT-PCR. The virus titer was determined using NIH3T3 cells. Results Restriction analysis, RT-PCR and DNA sequence analysis confirmed that pRevTRE / hPPE contained hPPE gene. The titers of FT67 cells transfected with pRevTet-on were 2.6 × 10 5 CFU / ml, and the PT67 cell virus titer transfected with pRevTRE / hPPE was 3.2 × 10 5 CFU / ml. Conclusion A retroviral vector containing tetracycline-regulated hPPE gene was successfully constructed and a packaging cell line capable of producing high titer retroviruses was established, which laid the experimental foundation for the study on the regulation of analgesic cell transplantation.