mGITRL真核表达质粒的构建及其在Lewis细胞中的表达  

Construction of eukaryotic expression plasmid containing mGITRL gene and its expression in Lewis cell

作　　者：胡正军[1] 王胜军[1] 朱燕萍[1] 鲍俊峰[1] 仝佳[1] 马洁[1] 成军[2] 孙长贵[2] 许化溪[1] 

机构地区：[1]江苏大学医学技术学院免疫学与免疫检验学系,江苏镇江212013 [2]中国人民解放军117医院,浙江杭州310013

出　　处：《江苏大学学报（医学版）》2008年第2期107-110,共4页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省自然科学基金资助项目(BK2004405);江苏大学高级人才资助项目(05JDG042)

摘　　要：目的:构建含小鼠GITRL基因的真核表达质粒pIRES2-eGFP/mGITRL,体外转染小鼠肺癌细胞株Lewis细胞。方法:利用PCR方法扩增mGITRL基因,克隆至pIRES2-eGFP载体,选择阳性克隆并进行序列测定。以电穿孔法转染Lewis细胞,通过G418筛选,获得稳定表达细胞株。结果:构建了真核表达质粒pIRES2-eGFP/mGITRL,基因测序与GenBank中发表的序列完全一致,体外转染Lewis细胞,经G418筛选,体外传代30代以上,RT-PCR及流式细胞仪检测该细胞株稳定表达mGITRL。结论:成功构建了真核表达质粒pIRES2-eGFP/mGITRL,并在小鼠Lewis细胞中稳定表达,为进一步研究GITRL的生物学功能提供研究基础。Objective： To construct eukaryotic expression vector containing mGITRL gene pIRES2-eG- FP/mGITRL and transfect it into Lewis cell. Methods： The mGITRL gene was amplified from intermediate vector PMD18-T/mGITRL by PCR and inserted into pIRES2-eGFP vector, and the sequence of DNA was analyzed. The mGITRL gene was transfected into Lewis cell by electroporation and the positive clone was selected by G418. Results： Expression plasmid pIRES2-eGFP/mGITRL was successfully constructed, sharing 100% homology with the sequence of mRNA for mGITRL in GenBank. mGITRL gene and the protein could be detected by RT-PCR and FCM in the transfected Lewis cell. Conclusion： pIRES2-eGFP/mGITRL vector has been constructed successfully and was expressed stably in Lewis cell, which brought a foundation for further researching on its biological function.

关 键 词：GITRL 细胞转染 肿瘤 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]