期刊论文

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

中文

解剖学杂志

1001-1633

2019

42

5

453-457

10.3969/j.issn.1001-1633.2019.05.005

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)。

本校为第一机构

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（Neg...

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.Resul...