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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。
OBJECTIVE: To clone human wild-type SNCA gene and construct a retroviral vector containing the wild-type SNCA gene and its mutations Ala30Pro and Ala53Thr. Methods: The SNCA gene was amplified from human fetal brain by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. On this basis, the retroviral vectors of SNCA gene whose mutations Ala30Pro and Ala53Thr were constructed by site-directed mutagenesis with single nucleotide differential primers were constructed and transfected into host cells with these recombinant retroviral vectors. Results: PCR, restriction enzyme digestion and sequencing proved that the retroviral expression vector was successfully constructed. The target gene sequence is successfully expressed in the host cell. CONCLUSION: The successful construction of recombinant retrovirus pEGZ / MCSHA vector containing human wild-type SNCA gene and its Ala30Pro and Ala53Thr mutants laid the foundation for further construction of PD cell models expressing wild-type and Ala30Pro and Ala53Thr mutant SNCA.