NTE基因ShRNA慢病毒载体的构建与鉴定

【目的】构建神经病靶酯酶(NTE)蛋白低表达的ShRNA重组慢病毒表达系统。【方法】依照ShRNA序列设计原则设计带酶切位点的NTE ShRNA序列,与表达载体pLL3.7连接构建NTE ShRNA重组表达质粒,转染DH5α菌观察重组表达状况,与pCVM-VSV-G包膜质粒和pCMV-dr8.2dvpr表达质粒共转染293T细胞,包装得到重组慢病毒。用病毒稀释法以绿色荧光蛋白(GFP)为标记,确定转染效率及病毒含量。【结果】经酶切及测序鉴定结果显示成功构建pLL3.7-NTE ShRNA慢病毒载体。重组慢病毒质粒与辅助质粒共转染293T细胞后获得高表达的细胞,共转染48h后,收集病毒液检测病毒滴度达4.5×10~4/mL。【结论】成功构建NTE ShRNA慢病毒重组表达系统。

Construction and identification of NTE ShRNA gene Lentivirus vector

Objective]In this study,the SbRNA recombinant slow virus expression system was constructed with low expression of NTE protein.Methods]The sequence of ShRNA-NTE with restriction sites was designed according to the design principles of ShRNA sequence,then connected with vector pLL3.7 toconstruct recombinant expression plasmid of ShRNA NTE,which were transfected in E.coli DHSα to observe the expression.The plasmids of pLL3.7-NTE,pCVM-VSV-G and pCMV-dr8.2 dvpr were co-transfeeted to 293T cells,the recombinant Lentiviral was packaged.Dilution method was applied to measure the transfection efficiency and virus content using green fluorescent protein (GFP) as a marker.Results] Enzyme digestion and sequencing analysis showed that the ShRNA pLL 3.7-NTE vector was successfully constructed.The high expression cells were obtained after co-transfection to 293T cells with the PLL3.7-NTE plasmid and helper plasmids.The titer of Lentivirus was 4.5 × 104 units /ml after 48 h of co-transfected.Conclusion]NTE-ShRNA expression lentiviral vector is successfully constructed.