周期型马来丝虫基因稳定转染细胞株的建立

摘要:目的　将含周期型马来丝虫半胱氨酸蛋白酶抑制剂（Bm-CPI）基因的重组质粒pcDNA3.1（+）-Bm-CPI转染人宫颈癌细胞（Hela细胞）获得重组质粒稳定转染细胞株，为重组蛋白的获得提供基础。方法　将成功构建的重组质粒pcDNA3.1（+）-Bm-CPI转染Hela细胞，以G418筛选转染细胞，通过RT-PCR和SDS-PAGE鉴定G418筛选后的单克隆抗性细胞株。结果　重组真核表达质粒pcDNA3.1（+）-Bm-CPI转染Hela细胞后，d 14可见G418抗性细胞株开始形成。G418抗性Hela细胞株筛选、扩大培养后RT-PCR扩增出621bp左右的目的条带；SDS-PAGE检测获得了明显的目的条带。结论　实验证实pcDNA3.1（+）-Bm-CPI重组质粒被成功转入Hela细胞，并获得稳定表达；为进一步研究Bm-CPI表达、蛋白纯化和测定其生物学活性奠定了基础。

Building of stably transfected cell strain on periodic brugia malayi gene

Abstract: Objiective To obtain stable transfected cell strain by plasmid pcDNA3.1 (+) -Bm-CPI transfected Hela cell. Methods Recombinant plasmid pcDNA3.1 (+) -Bm-CPI transfected Hela cell by lipofectin, G418 was used for screening, The monoclonal resisting cell strain screened by G418 was confirmed with RT-PCR and SDS-PAGE. Results The monoclonal cell strains with resistance to G418 was formed on 14th day after Hela cell was transfected with pcDNA3.1 (+) -Bm-CPI. A 621bp special fragment was amplified by RT-PCR in the monoclonal cell strains and SDS-PAGE also detected obvious purpose stripe after Hela cell strains of G418-resistant being screened and expanded. Conclusion Plasmid pcDNA3.1 (+) -Bm-CPI expressive recombinant has been transfected successfully into Hela cells and expressed steadily, which sets up a base for further study of Bm-CPI expression, purification and its biological activity.