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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: To construct a lentiviral vector with K-RasG12D gene mutation. METHODS: RNA was extracted from patient’s tissues and cDNA was reverse transcribed by RT-PCR to obtain the K-RasG12D gene template. The K-RasG12D mutant was amplified by PCR. The digested fragment was cloned into the eukaryotic expression vector pCDH-CMV-MCS-EF1-RFP to construct a retroviral eukaryotic expression vector with K-RasG12D gene mutation. The ligation product was transformed into competent E. coli DH5α, and bacterial colonies on the transformation plates were picked and cultured in antibiotic culture medium overnight for PCR identification. 293T cell line was transfected into 293T cell line after sequencing correctly, and the transcription and expression of the target gene were analyzed and identified by using recombinant plasmid PCR and tandem gene expression detection. Results: The eukaryotic expression vector of retroviral K-RasG12D gene was identified by PCR and sequenced correctly. After transfected 293T cells, RFP fluorescence signal with high intensity could be detected. Conclusion: The recombinant eukaryotic expression vector was successfully constructed, which laid the foundation for further establishment of stable transfected cell lines and further study on the role of K-Ras mutation in the pathogenesis of cancer.