过表达CD47重组慢病毒的制备及表达

Preparation and Expression of Overexpressing CD47 Recombinant Lentivirus

目的 构建hCD47基因重组慢病毒并观察其在人胶质瘤U87细胞中的表达。方法 采用聚合酶链式反应(PCR)扩增目的基因hCD47,采用双酶切实验鉴定重组慢病毒载体pLVX-hCD47-3flag-ZsGreen-Puro,接着进行慢病毒的包装,将质粒载体导入293T细胞观察转染效率。进一步获取慢病毒转染效率,将慢病毒干预HEK293观察转染效率,最后采用逐孔稀释梯度法检测病毒滴度。采用稳转细胞系的方法将构建好的hCD47重组慢病毒转入胶质瘤U87细胞中,倒置荧光显微镜下观察荧光强度及范围,Western blot检测CD47蛋白表达情况。结果 PCR以及酶切鉴定表明重组慢病毒载体克隆为阳性,病毒包装以及检测转染效率实验结果均显示较高的荧光强度,表明慢病毒被成功构建。病毒滴度检测结果显示为1×10^(8)TU·mL^(-1)。Western blot结果显示,目的基因CD47在稳转hCD47重组慢病毒U87细胞中(hCD47)相比对照组(NC)表达升高。结论 成功构建能够体外有效转染U87细胞的hCD47慢病毒。

Objective To construct the recombinant lentivirus of hCD47 gene and observe its expression in human glioma U87 cells. Methods The target gene hCD47 was amplified by polymerase chain reaction(PCR).Then the recombinant lentiviral vector pLVX-hCD47-3flag-Zs Green-Puro was identified by double digestion assay. Then lentivirus was packaged and the transfection efficiency was observed by introducing the plasmid vector into 293T cells. The transfection efficiency of lentivirus was further obtained. The transfection efficiency was observed by lentivirus intervention HEK293 and finally,the viral titer was detected by the well-by-well dilution gradient method. The hCD47 recombinant lentivirus was transferred into glioma U87 cells by stable cell line transfer method. The fluorescence intensity and range were observed by inverted fluorescence microscope,and the expression of CD47 protein was detected by Western blot. Results PCR and enzyme digestion identification showed that the recombinant lentivirus vector was cloned positively. The results of virus packaging and transfection efficiency test showed high fluorescence intensity,indicating that the lentivirus was successfully constructed. The virus titer was 1×10^(8)TU·mL^(-1).Western blot results showed that the expression of target gene CD47 was increased in hCD47 recombinant lentivirus U87 cells(hCD47)compared with the control group(NC).Conclusion hCD47 lentivirus can be successfully constructed and transfected into U87 cells in vitro.