慢病毒载体介导的APP695过表达SH-SY5Y细胞系的构建和鉴定

Construction and identification of APP695 gene-transfected SH-SY5Y cells by lentiviral vector

目的通过构建pLVX-APP695-PGK-Puro慢病毒载体,用以建立可以稳定过表达APP695蛋白的SH-SY5YAPP695细胞株。方法利用PCR方法扩增目的基因片段APP695,并构建pLVX-APP695-PGK-Puro慢病毒载体,鉴定后将构建好的载体与慢病毒包装系统共转染293T细胞,并以最适感染复数感染神经母细胞瘤SH-SY5Y细胞,筛选稳转SH-SY5Y-APP695细胞株并用PCR和Western blot鉴定稳转株细胞APP695表达。结果对pLVX-APP695-PGK-Puro载体进行酶切鉴定及DNA测序证明构建过表达APP695的重组慢病毒载体成功,PCR和Western blot显示构建的SH-SY5Y细胞稳转株成功。结论成功构建APP695基因的慢病毒载体并成功构建了SH-SY5Y-APP695细胞株,该载体可在SH-SY5Y细胞株中高水平表达APP695。

Objective To establish the SH-SY5Y-APP695 cell lines with stable APP695 overexpression by constructing the recombinant lentiviral vector pLVX-APP695-PGK-Puro. Methods The APP695 gene was amplified by PCR and subcloned into the lentiviral vector to generate the pLVX-APP695-PGK-Puro. The recombinant lentiviral vector and lentivirus packaging system were co-transfected into 293 T cells, followed by the infection of SH-SY5 Y cells using the fittest multiplicity of infection（MOI）. The APP695 expression was detected by PCR and Western blot in the screened SH-SY5Y-APP695 cells. Results The recombinant lentiviral vector pLVX-APP695-PGK-Puro was successfully constructed using restriction enzyme digestion and DNA sequencing, and the SH-SY5 Y cells with stable APP695 overexpression were also established by PCR and Western blot. Conclusion The SH-SY5Y-APP695 cells with APP695 overexpression are successfully constructed by APP695 gene-trasfected lentiviral vector.