人SNCA及其致病突变基因的逆转录病毒pEGZ/MCSHA载体的构建

Construction of Retroviral Vector pEGZ/MCS HA Containing Human SNCA Gene and Its Pathogenic Mutation

目的:克隆人野生型SNCA基因,构建野生型SNCA基因及其致病突变Ala30Pro、Ala53Thr的逆转录病毒表达载体。方法:通过逆转录聚合酶链式反应(RT-PCR)方法从人胎脑扩增SNCA基因,T-A克隆后测序。在此基础上利用单核苷酸差异引物定点诱变法构建其致病突变Ala30Pro、Ala53Thr的SNCA基因的逆转录病毒载体,并用这些重组逆转录病毒载体转染宿主细胞。结果:PCR、酶切及测序证明逆转录病毒表达载体构建成功。目的基因序列在宿主细胞成功表达。结论:人野生型SNCA基因及其Ala30Pro、Ala53Thr突变基因的重组逆转录病毒pEGZ/MCSHA载体的成功构建,为进一步构建表达野生型及Ala30Pro、Ala53Thr突变型SNCA的PD细胞模型奠定基础。

Aim： To clone human wild type SNCA gene and construct retroviral vector pEGZ/MCS HA containing human wild type SNCA gene and its Ala30Pro, Ala53Thr pathogenic mutation. Methods ： Human wild type SNCA gene was cloned from fetus brain by using RT-PCR. By T-A extension cloning, the gene was ligated with T-vector and sequenced. Based on it, the retroviral vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation were constructed by site-directed mutagenesis using primer variance in mononucleotide, and these recombinant retroviral vectors were transfected into host cells respectively. Results： According to the result of PCR, the double digestion and gene sequencing, it was comfirmed that recombinant retroviral vector containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation had been constructed successfully, Objective gene sequence was expressed successfully in host cells, Conclusion： The successful construction of retroviral vector pEGZ/MCS HA containing human wild type SNCA gene or its Ala30Pro, Ala53Thr pathogenic mutation lays the foundation for the further study of PD cell model expressing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation.