肿瘤坏死因子受体2编码基因重组质粒的构建及意义  

Tumor necrosis factor receptor 2 gene mutation eukaryotic expression plasmid: construction and implications

作　　者：卫娜[1] 白瑞[1] 车星星[1] 王泽慧[1] 肖传实 边云飞[1] 

机构地区：[1]山西医科大学第二医院心内科,太原030001 [2]山西省医科大学第一医院

出　　处：《中国药物与临床》2012年第6期712-716,共5页Chinese Remedies & Clinics

基　　金：山西省科技攻关项目(20090311057-4)

摘　　要：目的 构建真核表达质粒pcDNA6.0-TNFR2^196MET和pcDNA6.0-TNFR2^196ARG.方法 人工合成TNFR2196MET目的 片段,与pMD18-T simple 载体连接形成克隆载体,再通过设计突变引物,PCR定点突变扩增出含有突变位点的质粒,转化到肠埃希菌感受态细胞JM109中,通过氨苄青霉素抗药性筛选出成功转化的阳性克隆,并进行扩增.抽提质粒进行测序鉴定,得到pMD18-T simple-TNFR^2196ARG.双酶切pMD18-T simple-TN-FR^2196MET、pMD18-T simple-TNFR^2196ARG及pcDNA6.0,将TNFR^2196MET、TNFR^2196ARG插入pcDNA6.0线性质粒,即构建成pcDNA6.0-TNFR^2196MET和pcDNA6.0-TNFR^2196ARG真核表达质粒,转化到Ecoli感受态中,筛选阳性克隆行菌液PCR和双酶切及测序鉴定.结果 酶切鉴定和测序分析验证TNFR2基因196MET和196ARG表达载体构建成功.结论 成功构建表达载体为下一步心血管疾病研究奠定了实验基础.Objective To construct the eukaryotic expression plasmid, pcDNA6.0-TNFR2^196MET and pcDNA6.0- TNFR2^196MET. Methods TNFR2^196AR Gtarget fragment was artificially synthesized and was linked to pMD18-T simple for forming cloning vectors. This was followed by amplification of plasmids with point mutation for further transformation into JM109, the competent E. coli cells, via polymerase chain reaction on the basis of mutation primers designed previously. The positive clones via transformation were screened based on ampicillin resistance and were subjected to amplification. The pMD18-T simple-TNFR2^196MET was obtained by sequencing of the extracted plasmids. The plasmids, pMD18-T simple-TNFR2^196MET, pMD18-T simple-TNFR2^196ARG and pcDNA6.0, underwent double enzyme digestion. Both TNFR2^196METand TNFR2^196ARG were then inserted into pcDNA6.0, the linear plasmid, for construction of eukaryotic expression vectors, pcDNA6.0-TNFR2^196MET and pcDNA6.0-TNFR2196ABC, which were subsequently transformed into competent E. coli followed by screening the positive clones as confirmed by polymerase chain reaction, double enzyme digestion and sequencing test. Results TNFR2 gene expression plasmids, pcDNA6.0-TNFR2^196MET and pcDNA6.0-TN- FR2^196ABG, were successfully constructed, as confirmed by DNA sequencing and enzyme digestion analysis. Conclusion The successful construction of peDNA6.0-TNFR2^196MET and peDNA6.0-TNFR2^196ARC expression plasmids has provided foundation for further research on cardiovascular diseases.

关 键 词：受体 肿瘤坏死因子 Ⅱ型 突变 遗传载体 心血管疾病 

分 类 号：R363[医药卫生—病理学]