EIF4A3 shRNA慢病毒载体的构建及其稳定转染细胞系的建立

Construction of EIF4A3 shRNA lentiviral vector and establishment of its stable transfection cell line

目的:构建真核细胞翻译起始因子4A3(EIF4A3)-短发夹RNA(shRNA)慢病毒载体,建立Neuro-2a-EIF4A3-shRNA稳定转染细胞系。方法:通过美国国家生物技术信息中心(NCBI)数据库检索EIF4A3基因序列,设计并合成PCR鉴定引物,并将其连接至经EcoRⅠ和AgeⅠ酶切的慢病毒GV493载体,构建GV493-EIF4A3-shRNA慢病毒质粒,PCR筛选阳性克隆并测序鉴定。将GV493空载质粒和GV493-EIF4A3-shRNA重组质粒分别转染至HEK293T细胞中,分别为GV493对照慢病毒和GV493-EIF4A3-shRNA慢病毒,转染48 h后收集慢病毒进行包装并测定病毒滴度。将Neuro-2a细胞分为空白组、GV493对照组和GV493-EIF4A3 shRNA组,空白组不作处理,GV493对照组和GV493-EIF4A3 shRNA组分别采用相应慢病毒感染Neuro-2a细胞,慢病毒感染复数(MOI)为100,使用10 mg·L-1嘌呤霉素筛选成功感染慢病毒的Neuro-2a细胞,荧光显微镜观察各组Neuro-2a细胞的生长状态和绿色荧光表达情况;实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组Neuro-2a细胞中EIF4A3 mRNA及蛋白表达水平。结果:PCR测序结果显示GV493-EIF4A3-shRNA重组质粒基因序列与设计合成的EIF4A3-shRNA序列一致,成功构建GV493-EIF4A3慢病毒载体。荧光显微镜观察可见HEK293T细胞荧光表达强烈,生长状态良好,慢病毒包装成功。GV493-对照慢病毒和GV493-EIF4A3-shRNA慢病毒的滴度均为2×10~8 TU·mL^(-1),GV493对照组和GV493-EIF4A3 shRNA组Neuro-2a细胞生长状态良好且表达绿色荧光,表明慢病毒感染稳定细胞系构建成功。RT-qPCR法,与空白组和GV493对照组比较,GV493-EIF4A3shRNA组Neuro-2a细胞EIF4A3mRNA表达水平明显降低(P<0.01)。Western blotting法,各组在相对分子质量49000处出现特异性条带,提示Neuro-2a细胞中EIF4A3蛋白表达成功;与空白组和GV493对照组比较,GV493-EIF4A3 shRNA组Neuro-2a细胞中EIF4A3蛋白表达水平明显降低(P<0.01)。结论:成功构建GV493-EIF4A3-shRNA慢病毒载体,建立了Neuro-2a-EIF4A3-shRNA稳定转染细胞系,为EIF4A3在颅内动脉粥样硬化的作用机制研究提供了参考。

Objective:To construct the eukaryotic cell translation initiation factor 4A3(EIF4A3) short hairpin RNA(shRNA) lentiviral vector,and to establish the Neuro-2a-EIF4A3-shRNA stable transfection cell line.Methods:The EIF4A3 gene sequence was retrieved from the National Center for Biotechnology Information(NCBI) database;the PCR identification primers were designed and synthesized,and connected to the lentiviral GV493 vector digested with EcoR Ⅰ and Age Ⅰ enzymes to construct the GV493-EIF4A3-shRNA lentiviral plasmid;PCR method was used to screen the positive clones,which were sequenced for the identification;the GV493 empty plasmid and GV493-EIF4A3-shRNA recombinant plasmid were transfected into the HEK293T cells,regarded as GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus,respectively.After 48 h of transfection,the lentiviruses were collected for packaging and the viral titer was determined.The Neuro-2a cells were divided into blank group,GV493control group,and GV493-EIF4A3 shRNA group.The Neuro-2a cells in blank group were untreated,and the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group were infected with the respective lentiviruses at a multiplicity of infection(MOI) of 100.The infected Neuro-2a cells were selected by 10 mg·L-1 puromycin,and the growth status and green fluorescence expression of the Neuro-2a cells in various groups were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR) and Western blotting methods were used to detect the expression levels of EIF4A3 mRNA and protein in the Neuro-2a cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the GV493-EIF4A3-shRNA recombinant plasmid was consistent with the designed EIF4A3-shRNA sequence,indicating successful construction of the GV493-EIF4A3 lentiviral vector.The fluorescence microscope observation results showed that there was strong fluorescence expression and good growth status in the HEK293T cells,confirming successful lentiviral packaging.The viral titers for GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus both were 2×10~8 TU·mL~(-1).The growth status of the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group was good,and they expressed green fluorescence,indicating successful construction of the stable transfection cell line.The RT-qPCR results showed that compared with blank group and GV493 control group,the expression level of EIF4A3 mRNA in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).The Western blotting results showed that the specific bands was at a relative molecular mass of 49 000,indicating successful EIF4A3 protein expression in the Neuro-2a cells.Compared with blank group and GV493 control group,the expression level of EIF4A3 protein in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).Conclusion:The GV493-EIF4A3-shRNA lentiviral vector is succfssfully constructed,and the Neuro-2a-EIF4A3-shRNA stable transfection cell line is established;the results provide the reference for the study of the effect of EIF4A3 on the intracranial atherosclerosis.