异柠檬酸脱氢酶1与突变体重组慢病毒的制备及表达

Preparation and expression of isocitrate dehydrogenase 1 and mutant recombinant lentivirus

目的:制备异柠檬酸脱氢酶1野生型(IDH1)和RI32H突变型(muIDH1)基因重组慢病毒,感染人胶质瘤来源的细胞系U87细胞并观察目的基因的表达。方法:利用基因克隆技术构建IDH1和mulIDH1基因重组慢病毒载体pLVX-IDH1-mCMV-ZsGreen-PCK Puro和pLVX-mutIDH1-mCMV-ZsGreen-PGK-Puro,转染293T细胞,进行慢病毒包装,获得慢病毒颗粒;慢病毒颗粒感染HEK293细胞,采用逐孔稀释滴度测定法测慢病毒滴度;IDH1和mutIDH1基因重组慢病毒感染U87细胞,观察感染效率,WesternBlot检测IDH1和mutIDH1基因在U87细胞的表达。结果:成功制备IDH1和mutIDH1基因重组慢病毒颗粒,病毒滴度均为1.0×10^8TU/ml;感染U87细胞,Westerm Blot结果显示:/DHI组中IDH1蛋白水平高于muIDH1组和空载体对照组;mutIDH1组中mu-tIDH1蛋白条带清晰,表达量高,IDH1组和对照组均不表达mutIDHl蛋白。结论:成功制备IDH1和mutIDH1基因重组慢病毒,感染U87细胞后能使目的基因表达。

Objective;Preparing isocitrate dehydrogenase 1 wild type(IDH1)and RI32H mutant lype(mulIDH1)lentiviruses to observe the expression of target gene by infecting human glioma cell line U87.Methods:IDH1 and mu-tIDH1 recombinant lentiviral vectors were constructed by gene eloning technology,which were pLVX-IDH1-mCMV-Zs-Green-PGK Puro and pLVX-mutIDH1-mCMV-ZsGreen-PGK Puro.Recombinant lentiviral vectors were transfected into 293T cells to pack for lentiviruses;HEK293 cells were infected with lentiviruses to detect lentiviral titers by hole-by-hole dilution titration.We used these lentiviruses to infect U87 cells to observe the infection efficiency.W estern Blot was used to detect the expression level of IDH1I and mutlDHI genes.Results:The IDH1 and mutIDH1 recombinant lentivirus particles were sucessfully prepared.The titers of lentiviruses were 1.0×10^8TU/ml.U87 cells were infected with lentiviruses,and results of w esterm Blot showed that the IDH1 protein in the IDH1 group was higher than that in mulIDH1 group and Control group.The mutIDH1 protein band in the mutIDH1 group was clear and the expression quan-tity was high.There was no mutIDH1 protein expressed in IDH1 group and Control group.Conclusion:The IDH1 and mulDHI lentiviruses were sucessfully prepared.They can effectively infect U87 cells in ritro and make the target gene express.