STGC3基因突变载体的构建及其在CNE2细胞中的表达  

Construction of the STGC3 recombinant mutation vector and its expression in CNE2 cells

作　　者：李素云[1] 刘慧晴 刘芳[2] 周小兵[1] 贺修胜[3] Li Suyun;Liu Huiqing;Liu Fang;Zhou Xiaobing;He Xiusheng(Clinical Anatomy&Reproductive Medicine Application Institute,University of South China,Hengyang 421001,China;University of South China,Hengyang 421001,China;Cancer Research Institute,University of South China,Hengyang 421001,China)

机构地区：[1]南华大学应用解剖与生殖医学研究所,衡阳421001 [2]南华大学,衡阳421001 [3]南华大学肿瘤研究所,衡阳421001

出　　处：《解剖学杂志》2019年第5期453-457,共5页Chinese Journal of Anatomy

基　　金：湖南省科技厅资助项目(2012TT2020);南华大学博士启动基金(2016XQD14);湖南省大学生研究性学习与创新性实验资助项目(2018XJXZ154)

摘　　要：目的:构建STGC3基因单碱基位点突变载体并观察CNE2细胞中STGC3蛋白表达情况,为探讨STGC3基因发挥抑瘤作用的功能性位点提供前期基础。方法:针对STGC3基因糖基化、蛋白激酶C磷酸化、酪蛋白激酶Ⅱ磷酸化3个(656C、725C、913T)可能性功能位点,运用Stratagene定点突变技术,对STGC3基因656C、725C、913T位点分别进行单碱基定点突变,使656C突变为G,725C突变为T,913T突变为G,构建突变表达质粒,同时构建野生型质粒为对照。质粒经转化、抽提、酶切及测序鉴定。将重组真核表达质粒,用脂质体转染至鼻咽癌CNE2细胞系,经G418筛选,得到稳定表达STGC3基因3种突变型CNE2细胞系。实验设6组,即空白对照组(Negative control)、空载体组(Vector)、野生型组(His-STGC3)和3个STGC3基因突变型组(His-STGC3-C656G、His-STGC3-C725T、His-STGC3-T913G)。运用RT-PCR、免疫印迹及免疫细胞化学,观察重组质粒突变型STGC3基因是否正常表达。结果:突变质粒经酶切和测序鉴定,酶切结果显示目的片段大小一致,测序结果显示突变序列正确,成功构建带His标签的3个突变型(His-STGC3-C656G、His-STGC3-C725T、His-STGC3-T913G)重组真核表达质粒。RT-PCR、免疫印迹和免疫细胞化学结果显示,突变质粒均正常表达His-tag-STGC3基因及融合蛋白质,融合蛋白定位于细胞质和细胞核。结论:成功构建STGC3基因单碱基位点突变质粒,并在CNE2细胞中表达STGC3融合蛋白。Objective:To construct the recombinant plasmids carrying a single base site mutation of STGC3 gene and detect the expression of STGC3 protein in CNE2 cells,so as to provide preliminary reference for exploring the functional sites of the nasopharyngeal candidate tumour suppressor gene STGC3.Methods:Site-directed mutagenesis of STGC3 plasmid at sites of C656G,C725T and T913G was induced by the Stratagene mutagenesis method and transfected into CNE2 cells.The CNE2 cells line with stable expression of STGC3 gene was obtained after detection by RT-PCR,Western blotting and immunohistochemistry.Results:The three mutants plasmids His-STGC3-C656G,His-STGC3-C725T and His-STGC3-T913G were successfully constructed by restriction enzyme identification and sequence analysis.These mutant plasmids stabely expressed STGC3 gene according to RT-PCR and Western blotting,and immunocytochemistry showed proteins of mutant STGC3 genes were expressed in the cytoplasm and nucleus of CNE2 cells.Conclusion:Recombinant plasmids with point mutations at C656G,C725T and T913G of gene STGC3 are successfully established,and mutant STGC3 genes are stabely expressed in CNE2 cell lines.

关 键 词：STGC3基因 质粒 突变 

分 类 号：R73[医药卫生—肿瘤]