bFGF cDNA重组逆转录病毒表达载体的构建及在哺乳动物细胞中的表达

Construction of a recombinant retroviral vector containing human bFGF cDNA and its expression in mammalian cells

目的 建立逆转录病毒介导的人bFGF体外表达体系。方法 应用DNA重组技术 ,将人bFGFcDNA片段重组到逆转录病毒质粒pLXSN中 ,经PA3 17细胞包装后 ,产生的重组逆转录病毒感染COS 7细胞 ,用RT PCR和WesternBlotting检测bFGFmRNA和蛋白表达。结果 重组pLXSN bFGF质粒 ,经酶切鉴定正确。重组逆转录病毒pLXSN bFGF滴度可达 2 3× 10 4CFU/ml,感染COS 7后能稳定表达。结论 逆转录病毒能介导人bFGF在哺乳动物细胞中的稳定表达 。

Objective To constract a retroviral mediated expression system of human basic fibroblast growth factor (bFGF). Methods Recombinant retroviral vecter pLXSN bFGF was generated by cloning a full length human bFGF cDNA into retroviral vecter pLXSN. COS 7 cells were infected with the viral supernatant from the highest productive PA317 clones. RT PCR was used for the detection of bFGF mRNA and bFGF protein in the COS 7 cells was detected with Western Blotting. Results The restriction enzyme analysis with EcoR Ⅰ and Xho Ⅰ showed that the recombinant retroviral vector had been constructed correctly. The titer assayed on NIH3T3 cells was up to 2.3×10 4 CFU/ml. Using the RT PCR and Western Blotting methods, the bFGF mRNA and bFGF protein were expressed in COS 7 cells after pLXSN bFGF infection. Conclusion The constructed retroviral vector is able to generate effective expression of human bFGF in mammalian cells, with potential utility in the gene therapy for central nervous system diseases such as Parkinsonism dimentia.