腺病毒介导ERK-2基因在生长板软骨细胞中的表达

目的:构建ERK-2基因重组腺病毒载体,检测构建的腺病毒感染原代大鼠生长板软骨细胞的效率以及目的基因的表达。方法:将ERK-2 cDNA亚克隆到腺病毒穿梭载体pAdTrack-CMV中,线性化后与腺病毒骨架质粒pAdEasy-1共同转染E.Col.i B J5183,将筛选、鉴定的重组腺病毒质粒线性化后转染HEK293细胞进行病毒颗粒的包装;流式细胞术检测不同感染复数(MO I)ERK-2重组腺病毒感染原代培养的大鼠肋生长板软骨细胞的效率,W estern b lot检测腺病毒感染的生长板软骨细胞中ERK-2蛋白的表达。结果:成功构建ERK-2重组腺病毒,MO I 50的腺病毒感染原代生长板软骨细胞的效率大于90%,感染的生长板软骨细胞中ERK-2表达显著增加。结论:构建的重组腺病毒可介导ERK-2基因在原代大鼠生长板软骨细胞中高表达。

Expression of ERK- 2 gene in growth plate chondrocyte mediated by adenovirus vector

Aim: To construct recombinant adenovirus vector carrying ERK-2 gene,and detect the infective efficiency of constructed vector on primary cultured rat growth plate chondrocyte(RGC),as well as the target gene expression.Methods: cDNA of ERK-2 was subcloned into shuttle vector pAdTrack-CMV,the linearized resultant plasmid and adenoviral backbone plasmid pAdEasy-1 were cotransfected into E.Coli.BJ5183 for recombination.Recombinant adenoviral plasmids were screened and identified,the linearized recombinant plasmid was transfected into HEK293 for adenovirus package.Infective efficiency of different MOI(multiplicity of infection) recombinant adenovirus on primary cultured RGC were measured by FACS,ERK-2 expression of the infected RGC was detected by western blot.Results: Successfully constructed recombinant adenovirus carrying ERK-2 gene,the infective efficiency of MOI 50 adenovirus on(primary) RGC>90%,ERK-2 expression of the infected RGC increased significantly.Conclusion: Constructed recombinant adenovirus mediates ERK-2 gene overexpression in primary cultured RGC.