含突变型p53和LMP1双基因重组质粒的构建与鉴定  

Construction and Identification of Recombinant Plasmid Containing Mutant p53 and LMP1 Genes

作　　者：何迎春[1] 田道法[1] 

机构地区：[1]湖南中医学院,湖南长沙410007

出　　处：《湖南中医学院学报》2005年第3期1-3,共3页Journal of Hunan College of Traditional Chinese Medicine

基　　金：中国博士后科学基金第35批资助项目(2004035644);湖南省杰出青年基金项目资助(03JJY3040);湖南省教育厅资助项目(03C287)。

摘　　要：目的构建一种双基因真核表达载体,使之表达人突变型p53蛋白(p53mt)和EB病毒潜伏膜蛋白1(LMP1),为研究这两个蛋白在鼻咽癌变中的交互作用奠定基础。方法通过基因重组技术,构建含突变型p53基因和EB病毒LMP1基因的真核表达载体pLMP1—p53mt;采用脂质体lipofectAMINETM2000将此载体转入体外培养的HeLa细胞中,转染后48h,采用免疫细胞化学方法分析突变型p53基因和EB病毒LMP1基因的表达。结果对重组载体进行限制性内切酶酶切及PCR鉴定分析,结果与预期相符合;转染试验观察到转染细胞中突变型p53基因和LMP1基因的阳性表达。结论成功构建了表达人突变型p53蛋白和EB病毒LMP1的真核载体pLMP1—p53mt,其研究思路可以为中医体质病机研究提供借鉴。Objective To construct a new eukaryotic expression vector that expresses both human mutation p53 protein and Epstein-Barr virus(EBV) latent membrane protein 1 (LMP1) so that the reciprocity of mutation p53 with LMP1 in the tumorogenesis process of nasopharyngeal carcinoma could be investigated more easily.Methods The recombinant vector pLMP1-p53mt containing mutation p53 gene and Epstein-Barr virus LMP1 gene was constructed by combinant technology. Then, the recombinant vector was transfected into HeLa cells cultured in vitro by lipofectAMINE^(TM) 2000 and the expression of mutation p53 and LMP1 was detected by immunocytochemical measure after 48 hours following transfection. Results Following the recombinant vector digested by restricted endoenzymes and identified by PCR procedures, gel electrophoresis showed that the DNA band distribution was basically the same as that of theoretic expect. The positive expressions of mutation p53 and LMP1 were examined in transfected HeLa cells by transfecting experiments. Conclusions It demonstrates that the experiment is successful in the construction of recombinant eukaryotic expression vector pLMP1-p53mt expressing human mutation p53 protein and Epstein-Barr virus LMP1.

关 键 词：突变型P53基因 LMP1基因 基因重组 鉴定 

分 类 号：Q344.12[生物学—遗传学] Q344.13