【摘要】 目的构建法尼基二磷酸法尼基转移酶1(farnesyl diphosphate farnesyl transferase 1,FDFT1)基因重组质粒并在人胚肾上皮细胞(293T)验证质粒表达。方法设计FDFT1基因引物并经两次PCR扩增获取FDFT1目的基因;用相应的限制性内切酶将pCDH-CMV-MCS-EF1-copGFP(pCDH-GFP)质粒线性化;FDFT1基因及线性化载体经纯化后以同源重组反应连接构建重组质粒;重组质粒通过菌落PCR、双酶切验证并测序做进一步验证;在293T细胞中利用三质粒共转染法包装FDFT1目的质粒并收集含病毒的上清液;以病毒液感染293T细胞验证FDFT1基因表达。结果菌落PCR及双酶切说明目的基因成功插入载体质粒,测序结果说明重组质粒中FDFT1基因与NCBI中登记的相应基因同源。构建成功的pCDH-GFP-FDFT1质粒包装为慢病毒后感染293T细胞,可检测到细胞中FDFT1基因表达与对照病毒感染相比升高15.13倍。结论本研究成功构建高表达FDFT1基因的重组慢病毒表达载体,为进一步研究FDFT1基因在肿瘤中的作用机制提供了实验基础。更多还原

【Abstract】 Objective To construct recombinant plasmids of farnesyl diphosphate farnesyl transferase 1(FDFT1)and verify the expression of FDFT1 plasmids in human embryonic kidney epithelial cells(293 T).Methods FDFT1 gene primers were designed and FDFT1 target genes were obtained by PCR amplification twice.The pCDH-CMV-MCS-EF1-copGFP(pCDH-GFP)plasmid was linearized with corresponding restriction enzymes.After being purified,the FDFT1 genes and the linearized vectors were linked by homologous recombination reaction to construct recombinant plasmids.The recombinant plasmids were verified by colony PCR,double enzyme digestion and further verified by sequencing.The FDFT1 target plasmids were packaged in 293 Tcells by three-plasmid co-transfection and the supernatant containing virus was collected.The expression of FDFT1 genes was verified by 293 Tcells infected with supernatant containing virus.Results Colony PCR and double enzyme digestion indicated that the target genes were successfully inserted into the vector plasmids.Sequencing results showed that FDFT1 genes in the recombinant plasmids were homologous to corresponding genes registered in NCBI.The successfully constructed pCDH-GFP-FDFT1 plasmids were packaged into lentivirus and then infected with 293 Tcells.It was detected that the expression of FDFT1 genes increased by 15.13 times in comparison with that of control infected virus.Conclusion In this study,recombinant lentivirus vectors with high expression of FDFT1 gene were successfully constructed,providing an experimental basis for further study on the mechanism of FDFT1 genes in tumors.更多还原