周期型马来丝虫基因稳定转染细胞株的建立  

Building of stably transfected cell strain on periodic brugia malayi gene

作　　者：陆施娟[1] 徐倩[1] 徐邦生[1] 方政[1] 

机构地区：[1]南通大学医学院形态学实验室,江苏南通226001

出　　处：《现代预防医学》2015年第21期3952-3954,3958,共4页Modern Preventive Medicine

基　　金：南通大学研究生科研创新实践计划项目(YKC14049);南通市应用研究计划项目(BK2012017);南通大学校级自然科学类科研项目(12Z007)

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pc DNA3.1(+)-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pc DNA3.1(+)-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pc DNA3.1(+)-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pc DNA3.1(+)-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。Objiective To obtain stable transfected cell strain by plasmid pc DNA3.1（＋）-Bm-CPI transfected Hela cell. Methods Recombinant plasmid pc DNA3.1（＋）-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 14 th day after Hela cell was transfected with pc DNA3.1（＋）-Bm-CPI. A 621 bp 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 pc DNA3.1（＋）-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.

关 键 词：周期型马来丝虫 半胱氨酸蛋白酶抑制剂 转染 细胞株 

分 类 号：R117[医药卫生—公共卫生与预防医学]