人β防御素2腺病毒表达载体的构建及其在大鼠真皮多能干细胞中的表达

Construction of human beta-defensin 2 adenovirus expression vector and its expression in dermal multipotent stem cells in rats

目的构建和鉴定人β防御素2(HBD2)的重组腺病毒表达载体,并观察其转染大鼠真皮多能干细胞(dMSCs)后的表达。方法反转录聚合酶链反应(RT-PCR)扩增HBD2全长cDNA,亚克隆至穿梭质粒(pAdTrack-CMV)中,然后转化含骨架质粒(pAdEasy-1)的大肠杆菌B J5183,通过同源重组产生腺病毒载体质粒。PacⅠ酶切阳性的重组质粒后转染293细胞,包装出重组病毒载体。用所得重组腺病毒感染dMSCs细胞,并采用RT-PCR和荧光免疫组织化学检测其在dMSCs中的表达情况。结果经测序、酶切及聚合酶链反应(PCR)鉴定,表明已成功地扩增到HBD2全长cDNA,并将其顺序克隆到穿梭质粒和骨架质粒中,进而组装出腺病毒表达载体。经RT-PCR和免疫组织化学证实构建的病毒载体可感染dMSCs,并有效表达HBD2。结论本实验成功地构建了含HBD2基因的腺病毒表达载体,并证实其可在dMSCs中表达。

Objective To develop a gene therapy vector of human beta-defensin-2 （ HBD2 ） using recombinant adenovirus, and to observe the expression of HBD2 in dermal multipotent stem cells （dMSCs） after transfected with the vector. Methods RT-PCR was performed to get full-length cDNA of HBD2 gene. The gene was then subcloned into the pAdTrack-CMV shuttle vector. The resultant plasmid was transfected into E. coli BJ5183 cells containing backbone plasmid pAdeasy-1 to undergo homologous recombination, by which HBD2 was cloned into pAdeasy-1 （ pAdeasy/ HBD2 ）. Then pAdeasy/ HBD2 was linearized with Pac Ⅰ and transfected into 293 cells to construct recombinant adenovirus vector. Dermal muhipotent stem cells were isolated and transfected with the vector,and the expression of HBD2 in dMSCs was monitored by RT-PCR and fluorescent immunocytochemistry. Results By employed sequencing,restriction endonuclease analysis and PCR, it was confirmed that the full-length cDNA of HBD2 was obtained and cloned into pTrack-CMV and pAdEasy-1, and recombinant adenovirus vector of HBD2 was successfully constructed. The results of RT-PCR and fluorescent immunocytochemistry showed that HBD2 could effectively expressed in dMSCs after being transfected with the recombinant adenovirus vector. Coulusion The recombinant adenovirus vector of HBD2 was successfully constructed, and HBD2 could effectively expressed in dMSCs after transfection.