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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。
OBJECTIVE: To construct lentiviral viruses (LV) expression plasmids carrying and expressing OPCML gene. METHODS: The OPCML gene was cut from the constructed expression plasmid pcDNA3-OPCML by endonuclease and inserted into the lentiviral vector pWPI-GFP to construct the lentiviral expression plasmid pWPI-OPCML. The expression of OPCML protein was confirmed by restriction enzyme digestion, sequencing and detection The pWPI-OPCML, pCMV-dR8.74 and pMDG were co-transfected into 293T packaging cells to obtain the recombinant lentivirus. The recombinant lentivirus was then infected with target cell A2780 to further verify pWPI by detecting the green fluorescent protein (GFP) and the target protein OPCML OPCML Expression of OPCML in Cells. Results: (1) pWPI-OPCML carries the correct OPCML gene and can be expressed in human cells; (2) pWPI-OPCML co-transfection of 293T can produce recombinant virus; (3) the target gene OPCML can be recombined The lentivirus transduced efficiently into target cell A2780 and reached stable expression. The transduction efficiency was almost 100%. GFP was directly observed under fluorescence microscope. Western blotting detected the expression of OPCML and GFP protein in target cells. CONCLUSION: pWPI-OPCML can express OPCML protein in human cells. Recombinant lentivirus co-transfected into packaging cells can infect human cells, which is highly efficient as a gene transfer tool.