hPDGF-A/hBD_2双基因共表达腺病毒载体的构建及表达

Construction and identification of recombinant adenovirus expressing hPDGF-A and hBD-2

目的制备人血小板源性生长因子-A(human platelet-derived growth factor A,hPDGF-A)和人β防御素2(human beta defensins,hBD2)双基因共表达腺病毒载体并观察其在大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)中的表达。方法将hPDGF-A和hBD2通过内部核糖体插入位点(internal ribosome entry site,IRES)连接后,以同源重组的形式插入腺病毒表达载体,由人胚肾293细胞包装,获得有感染能力的重组腺病毒颗粒。重组病毒感染BMSCs后,用RT-PCR检测重组腺病毒的表达。结果①成功构建穿梭质粒pAdTrack-hPDGF-A-IRES2-hBD2。②成功构建重组腺病毒质粒pAdeasy-hPDGF-A-IRES2-hBD2。③293细胞包装获得有感染能力的重组腺病毒。④转染的BMSCs高表达hPDGF-A和hBD。结论构建了hPDGF-A/hBD双基因共表达腺病毒载体,证实其转染BMSC后的表达。

Objective To further determine their possible synergistic effect on accelerating wound healing, adenovirus vector containing recombinant human hPDGF-A and hBD2 genes was constructed and the expression of exogenous genes in transformed mesenchymal stem cells derived from rat bone marrow was observed. Methods By putting IRES in the middle of hPDGF-A and hBD2, these two genes were expected to be expressed individually. The shuttle vector was named as pAdTrack-hPDGF-A-IRES2-hBD2, which homologously recombinated with Adeasy-1 in BJ5183 cells and formed the mammalian expression vector pAdeasy-hPDGF-AIRES2-hBD2. Furthermore, the recombinant vector was packaged in 293 cells into infectious recombinant adenovirus, which were used to infect BMSCs. The expression of hPDGF-A and hBD2 in BMSCs was detected by RT-PCR. Results We successfully constructed recombinant adenovirus vector that simultaneously expressed hPDGF-A and hBD2. The expressions of hPDGF-A and hBD2 were confirmed by RT-PCR on transformed BMSCs. Conclusion The established BMSCs that overexpressed hPDGF-A and hBD2 provide a new strategy of combining cell therapy and gene therapy to promote wound healing, especially the chronic one.