| 赵善民汤球刘志学余琛琳孙伟蔡丽萍袁子彦徐晨崔淑芳△.K-RasG12D基因突变体慢病毒载体的构建及其鉴定*[J].,2014,14(2):223-225 |
| K-RasG12D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 GeneMutation (G12D)* |
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| DOI： |
| 中文关键词: K-Ras G12D突变体 真核表达 |
| 英文关键词: K-Ras Gene mutation Eukaryotic expression |
| 基金项目:上海市科技发展基金项目（10140900800） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| ABSTRACT Objective：The aim is to construct retrovirus vector of K-Ras Gene Mutation (G12D). Methods：Total RNA was
isolated fromthe 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 transfected 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 function of K-Ras gene mutation in carcer
development. |
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