K-Ras^(G12D)基因突变体慢病毒载体的构建及其鉴定  

Construction and Identification of Retrovirus Vector of K-Ras Gene Mutation(G12D)

作　　者：赵善民[1] 汤球[1] 刘志学[1] 余琛琳[1] 孙伟[1] 蔡丽萍[1] 袁子彦[1] 徐晨[1] 崔淑芳[1] 

机构地区：[1]第二军医大学实验动物中心,上海200433

出　　处：《现代生物医学进展》2014年第2期223-225,250,共4页Progress in Modern Biomedicine

基　　金：上海市科技发展基金项目(10140900800)

摘　　要：目的:构建K-RasG12D基因突变体慢病毒载体。方法:从病人组织中提取RNA通过RT-PCR反转录获得cDNA作为K-RasG12D基因模板,通过PCR法扩增出K-RasG12D基因突变体片段。将酶切的片段克隆入真核表达载体pCDH-CMV-MCS-EF1-RFP中,构建K-RasG12D基因突变体逆转录病毒真核表达载体。将连接产物转化至感受态大肠埃希菌DH5α,挑取转化平板上的细菌克隆,在抗生素培养液中培养过夜后进行PCR鉴定。经测序正确后转染293T细胞系,利用重组质粒PCR及串联基因表达的检测等方法对目的基因的转录与表达进行分析与鉴定。结果:所构建的K-RasG12D突变体基因逆转录病毒真核表达载体经PCR鉴定和测序鉴定正确,转染293T细胞后可以观察到可检测到高强度表达的RFP荧光信号。结论:成功构建了重组真核表达载体,为下一步建立稳定转染细胞系及进一步研究K-Ras突变在癌症发病中的作用奠定了基础。Objective： The aim is to construct retrovirus vector of K-Ras Gene Mutation （G12D）. Methods： Total RNA was isolated from the tissues of patients. First-strand complementary DNA （cDNA） was synthesized by reverse transcriptase using the RNA as template. K-Ras gene mutation （G12D） was amplified by PCR from the cDNA. Then the target genes were identified by enzyme digestion and then cloned into the eukaryotic expression vector pCDH-CMV-MCS-EF1-RFP, the recombinant plasmid was transfected into 293T cell line after sequencing. PCR was performed to determine the recombinant plasmid and then transient expresse of the gene was analysised by immunofluorescence to confirm the tandem gene were expressed. Results： The results showed that the recombinant plasmid was constructed successfully. Due to the high fluorescencesignals, 293T cells tmnsfected could easily be detected under a standard fluorescence microscope. Conclusion： The stable transfected cell lines were constructed successfully, which was useful for construction stable transtected cell lines and laid a foundation for further study of the fimction of K-Ras gene mutation in carcer development.

关 键 词：K—Ras G12D突变体 真核表达 

分 类 号：Q75[生物学—分子生物学] Q78