携带OPCML基因的慢病毒表达质粒的构建

目的:建立携带和表达OPCML基因的慢病毒(lentiviral viruses,LV)表达质粒。方法:用内切酶将OPCML基因从已构建的表达质粒pcDNA3-OPCML上切下,插入慢病毒载体pWPI-GFP中,构建慢病毒表达质粒pWPI-OPCML,通过酶切、测序和检测验证OPCML蛋白后,将pWPI-OPCML、pCMV-dR8.74和pMDG共转染包装细胞293T,获得重组慢病毒,再感染靶细胞A2780,通过检测标志蛋白-绿色荧光蛋白(GFP)和目的蛋白OPCML进一步验证pWPI-OPCML在细胞中OPCML的表达。结果:(1)pWPI-OPCML中携有正确的OPCML基因,并能在人类细胞中表达;(2)pWPI-OPCML共转染包装细胞293T能产生重组病毒;(3)目的基因OPCML能被重组慢病毒高效地转导入靶细胞A2780,并达到稳定的表达,转导效率几乎达100%,荧光显微镜下能直接观察到GFP,Western blotting能检测到OPCML和GFP蛋白在靶细胞中的表达。结论:pWPI-OPCML能在人细胞中表达OPCML蛋白,共转染包装细胞获得的重组慢病毒能感染人细胞,作为转基因的工具,具有高效性。

Construction of the lentiviral expression plasmid carrying OPCML gene

Objective:To construct the lentiviruses expression plasmid carrying OPCML gene.Methods:Digested the palsmid pcDNA3-OPCML with endoenzyme and subcloned OPCML into the lentiviral vector,pWPI-GFP,to generate the lentiviral expression vector,pWPI-OPCML.The corrected OPCML was confirmed by endoenzym digestion,sequencing and Western blotting.Recombinant lentiviruses were produced by 293T cells following the co-transfection of pWPI-OPCML,with the packaging plasmids pCMV-dR8.74 and pMDG.The resulting recombinant lentiviruses which carrying OPCML were then used to infect A2780 cell lines.GFP and OPCML protein expression in 293T and A2780 were dectected by fluorescent microscope and Western blotting analysis.Results:(1) plasmid pWPI-OPCML carried the OPCML gene,and it could be expressed in human cell line;(2) The recombinant lentiviruses which carrying OPCML could be product by co-transfection pWPI-OPCML to 293T;(3) The GFP and OPCML protein were dectected by fluorescent microscope and Western blotting analysis in 293T and A2780,the recombinant lentiviruses which carrying OPCML could infect and deliver OPCML and GFP genes to A2780 cells,The infection efficiency was almost 100%.Conclusion:The plasmid pWPI-OPCML can express OPCML protein in human cell lines,the recombinant lentiviruses can deliver target gene and have high infection efficiency.