人骨生成诱导因子基因慢病毒载体构建及在人主动脉平滑肌细胞中稳定过表达

Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

目的:构建人骨生成诱导因子基因(osteoinductive factor,OIF)慢病毒载体,并在人主动脉平滑肌细胞(human aorticsmooth muscle cell,HASMC)中稳定过表达OIF。方法:PCR扩增OIF-3FLAG,并克隆到慢病毒载体pMSCV PIG中,构建慢病毒表达载体pMSCV PIG-OIF-3FLAG,并测序鉴定。应用脂质体将pMSCV PIG-OIF-3FLAG或pMSCV PIG与VSVG、GAG-POL共转染293T细胞,48 h后收集细胞上清,感染HASMC,48 h后加入嘌呤霉素4 d,即筛选出稳定过表达pMSCV PIG-OIF-3FLAG的HASMC细胞株,采用real-time PCR和Western blot检测OIF mRNA和蛋白表达。结果:构建OIF基因慢病毒表达载体pMSCV PIG-OIF-3FLAG,经酶切及测序后证实目的基因的插入位点和读码框正确;包装病毒感染HASMC,经real-time PCR和Western blot检测证实OIF mRNA和蛋白水平在该细胞株中高表达。结论:成功构建人OIF基因的慢病毒载体,并获得稳定过表达该基因的HASMC。

Objective：To construct human osteoinductive factor（OIF） recombinant lentivirus vector and over-express OIF stably in human aortic smooth muscle cells（HASMCs）. Methods：OIF-3FLAG was amplified by PCR and cloned into vector（pMSCV PIG）.The lentivirus vector pMSCV PIG-OIF-3FLAG was constructed and indentified by sequencing.pMSCV PIG-OIF-3FLAG or pMSCV PIG with helper vectors of VSVG and GAG-POL were co-transfected into 293T cells.Infectious lentivirus was harvested at 48 h post-transfection.The lentivirus was added in HASMCs.After 48 h,puromycin was added for 4 d.When the cells reached 90%,HASMCs over-expressed pMSCV PIG-OIF-3FLAG stably.Human OIF expression was verified by quantitative real-time PCR and western blot analysis. Results：The lentivirus vector pMSCV PIG-OIF-3FLAG was successfully constructed and confirmed by enzyme digestion and sequencing.HASMCs were infected by lentivirus supernatant.Over-expression of OIF in HASMCs was revealed by real-time PCR and western blot. Conclusions：Human OIF gene recombined lentivirus vector and a cell model which over-expessing OIF stably in HASMCs were successfully established.