人色素上皮衍生因子慢病毒表达载体的构建及鉴定

Construction and identification of hPEDF lentiviral expression vector

目的构建人色素上皮衍生因子(human pigment epithelium-derived factor,hPEDF)慢病毒载体,包装过表达PEDF的慢病毒,构建过表达PEDF的人角质形成细胞(human keratinocyte,Ha Ca T)的细胞株。方法将PEDF基因插入慢病毒载体p LVX-IRES-puro,构建p LVX-IRES-puro-PEDF慢病毒重组质粒并转染293T细胞,包装获得病毒上清,进行病毒扩增,收集病毒上清液,测定扩增后的病毒滴度。扩增后的病毒上清液以最佳感染复数感染体外培养的Ha Ca T细胞作为Ha Ca T-PEDF组(实验组),同时以含有空载体的慢病毒感染Ha Ca T细胞株作为Ha Ca T细胞组(对照组),扩增72 h后收集两组细胞及条件培养基,通过实时荧光定量PCR(real-time PCR,RT-PCR)和蛋白质免疫印迹(western blot,WB)技术检测各组细胞PEDF基因m RNA以及蛋白的表达情况,通过酶联免疫吸附试验检测两组细胞PEDF蛋白分泌情况。结果经限制性内切酶酶切鉴定、Sanger测序证实,成功构建了携带PEDF基因的重组慢病毒表达载体,且包装的慢病毒滴度可达1×107pfu/ml。RT-PCR和WB检测结果显示,实验组细胞中PEDF的m RNA和蛋白均显著高于对照组(P<0.05),并能分泌至细胞外。结论成功构建了表达PEDF基因的慢病毒载体,在体外培养条件下可以成功感染Ha Ca T细胞,并高表达PEDF蛋白。

Objective To construct the human pigment epithelium-derived factor(hPEDF)lentiviral vector,package lentivirus overexpressing PEDF and construct the human keratinocyte(Ha Ca T)cell line overexpressing PEDF.Methods The PEDF gene was inserted to lentiviral vector p LVX-IRES-puro to construct p LVX-IRES-puro-PEDF lentivirus recombinant plasmid.The p LVX-IRESpuro-PEDF lentivirus recombinant plasmid was transfected into 293 T cells.The virus was packaged and amplificated.The virus supernatant was collected and the titer after amplification was authenticated.In the experimental group,Ha Ca T cells were infected with the constructed p LVX-IRES-puro-PEDF;whereas in the control group,Ha Ca T cells were infected with lentivirus containing empty vector.After amplification of 72 h,the Ha Ca T cells in both groups and conditioned medium were collected.The expression of PEDF m RNA in Ha Ca T cells of each group was detected by real-time PCR(RT-PCR)and the expression of PEDF protein was detected by western blot(WB).The PEDF protein secretion of Ha Ca T cells in both groups was detected by enzyme-linked immunosorbent assay(ELISA).Results It was confirmed that the recombinant lentivirus vector carrying PEDF gene was successfully constructed by using restriction enzyme digestion and Sanger sequencing.The titer of packaged lentivirus was up to 1×10~7 pfu/ml.The RT-PCR and WB results revealed that the expression of PEDF m RNA and protein in experimental group was significantly higher than that in the control group(P<0.05).Conclusion The recombinant lentiviral vector carrying PEDF gene was successfully constructed,and the Ha Ca T cell line infected with packaged lentivirus could over-express PEDF in vitro.