突变型人PRKAG2基因的克隆及其表达载体构建  

Cloning and construction expression vector of mutant human PRKAG2 gene

作　　者：叶忠[1] 张必利[1] 徐荣良[1] 雷军平[1] 石秀英[1] 陈挺[1] 郑兴[1] 

机构地区：[1]第二军医大学附属长海医院心内科,上海200433

出　　处：《生物医学工程与临床》2012年第4期390-394,共5页Biomedical Engineering and Clinical Medicine

基　　金：国家自然科学基金项目(81000038)

摘　　要：目的构建含有定点突变的人PRKAG2基因表达载体。方法首先应用Trizol法抽提健康人全血mRNA,通过实时聚合酶链反应(RT-PCR)克隆得到野生型人PRKAG2 cDNA;继而通过聚合酶链反应(PCR)定点突变的方法获得突变型人PRKAG2基因,命名为G100S,最后应用酶切连接的方法得到重组载体pEGFP-G100S。结果Trizol法提取人细胞mRNA,经RT-PCR合成cDNA第一条链,特异性引物PCR扩增得到PRKAG2基因片段,目的片段条带在1 761 bp左右。分段克隆得到的G100S片段分别为300 bp与1 500 bp左右。转化Top10感受态细菌,PCR筛选阳性菌落5个克隆有3个克隆可见1 800 bp左右的阳性片段,筛选得到pEGFP-G100S重组阳性克隆。测序证实重组载体pEGFP-G100S构建成功。结论构建的含定点突变人PRKAG2基因重组载体pEGFP-G100S为进一步研究基因PRKAG2 G100S突变的功能奠定了基础。Objective To construct human PRKAG2 gene expression vector with containing site--directed mutagenesis. Methods Healthy human whole blood mRNA was extracted by Trizol method. The wild-type human PRKAG2 eDNA was amplified by real- time polymerase chain reaction （RT-PCR） from human whole blood mRNA, and then the site-directed mutagenesis of human PRKAG2 gene（PRKAG2 G100S） was made by polymerase chain reaction（PCR）. Finally, the recombinant vector pEGFP-G100S was constructed by digested connection. Results The human cell mRNA was extracted by Trizol method, and the first strand eDNA was synthesized by RT-PCR, the specific primers PRKAG2 gene fragment was amplified by PCR, and the target fragment bands was about 1 761 bp. The G100S fragments obtained by segment cloned were about 300 bp and 1 500 bp. The Topl0 competent bacteria was transformed, PCR screened 5 clones of positive bacteria, and about 1 800 bp positive fragment were observed in 3 clones. It was confirmed that the recombinant vector pEGFP-G100S was successfully constructed by sequencing. Conclusion It is demonstrated that the construction of PRKAG2 G100S lays the foundation for its further functional study.

关 键 词：PRKAG2基因 定点突变 pEGFP—N1载体 人 

分 类 号：R541[医药卫生—心血管疾病]