新型慢病毒载体的构建及其在人骨髓间充质干细胞基因转导中的应用

摘要

背景:构建一个组成性表达某特定基因同时携带荧光报告基因和抗生素筛选基因的慢病毒载体目前未见报道.目的:观察新型慢病毒载体pLVpuro/EF1α-PEDF-IRES-EGFP在人骨髓间充质干细胞基因转导中的表达.方法:通过PCR在PEDF基因的两端加上attB位点,构建表达载体pLVpuro/EF1α-PEDF-IRES-EGFP,将表达载体与包装质粒(ViraPowerTM Lentiviral Packaging Mix)共转染293FT细胞.通过多次感染的方法将慢病毒载体导入人骨髓间充质干细胞,转导后第7天开始使用1~5 mg/L嘌呤霉素筛选5 d,得到表达PEDF和EGFP的人骨髓间充质干细胞,并进行Western、Elisa的鉴定分析.结果与结论:经PCR和测序证实,慢病毒表达载体pLVpuro/EF1α-PEDF-IRES-EGFP构建成功;将其成功导入人骨髓间充质干细胞,经过筛选获得纯化的过表达PEDF基因的绿色荧光细胞群.%BACKGROUND: Construction of a lentiviral vector concurrently containing a specific gene, a fluorescent reporter and an antibiotic selection marker under the control of different constitutive promoters for gene overexpression studies has not been reported before.OBJECTIVE: To construct the lentivector, pLVpuro/EF1α-PEDF-IRES-EGFP, which containing the elements of PEDF and EGFP gene driven by EF1α promoter and puromycin resistant gene driven by PGK promoter through multisite gateway technology and to transduce it into human mesenchymal stem cells (hMSCs) to detect the protein expression of PEDF gene.METHODS: The PEDF cDNA was flanked with attB sites by PCR and then cloned into pDONRTM221 to generate pDown-PEDF by utilizing the BP recombination reaction. The entry clones, pUp-EF1α, pDown-PEDF, pTail-EGFP were then recombined into the destination vector pDEST-puromycin to construct expression lentiviral vector, pLVpuro/EF1α-PEDF-IRES-EGFP by LR recombination reaction. The lentiviral particles were prepared by transient co-transfection of the resulting vector and ViraPower? Lentiviral Packaging Mix into 293FT cells using Lipofectamine 2000. The viral particles were harvested concentrated and used to infect hMSCs. 7 days after transduction, 1-5 mg/L puromycin was added to the culture and lasted for 5 days. Transduced hMSCs were purified and then analyzed by Western Blot and ELISA assay.RESULTS AND CONCLUSION: In this study, the lentivector pLVpuro/EF1α-PEDF- IRES-EGFP was successfully constructed by multisite gateway technology and it was confirmed by PCR and gene sequencing and the hMSCs were successfully transduced by lentivirus. After antibiotic selection, pure infected cell population which expressed PEDF gene and EGFP were attained.