大鼠骨桥蛋白重组腺病毒的构建及体外表达

Construction and Expression of Recombinant Adenovirus Vector Containing OPN Gene in Vitro

目的:构建表达大鼠骨桥蛋白(osteopontin,OPN)基因的重组腺病毒载体,并观察其感染人胚肾293AD(humanembryo kidney 293AD,HEK293AD)细胞后OPN的表达。方法:提取大鼠骨组织总RNA,利用反转录聚合酶链式反应(reversal transcript polymerase chain reaction,RT-PCR)扩增目的片段,将产物双酶切后连入腺病毒穿梭质粒载体中,构建pacAd5 CMV-IRES-GFP-OPN。PCR及双酶切鉴定正确后,与腺病毒骨架载体pacAd5 9.2-100同时经PacI酶切,利用HEK293AD细胞进行包装、生产,构建出重组腺病毒。通过HEK293AD细胞绿色荧光表达反映重组腺病毒的表达情况,并用RT-PCR检测OPN的表达。结果:成功构建了携带OPN的重组质粒,脂质体介导下转染HEK293AD细胞,7 d后可见明显的荧光表达,12 d后有半数细胞飘起,收集原病毒液感染的293AD细胞后,实验组OPN的表达明显高于对照组细胞,24 h感染效率为15%,36 h感染效率为21%。结论:利用腺病毒载体系统RAPAD成功构建了含OPN基因的重组腺病毒载体,为进一步研究OPN对皮肤创口愈合的影响提供了一定的实验基础。

Objective： The study was designed to construct a recombinant adenovirus vector containing rat osteopontin （OPN） by RAPAD vector, and to study its expression in human embryonic kidney 293AD （HEK293AD） cells. Methods： The OPN cDNA was obtained by RT-PCR amplification from rat bone tissue, and then inserted into pacAd5 CMV-IRES- GFP shuttle vector. The plasmid was checked and verified by digestion and DNA sequence analysis, then it and pacAd5 9.2-100 Vector were linearized by PacI. The recombinant adenovirus was transfected into HER293AD cells for packaging and amplification. Result： The recombinant adenovirus vector containing OPN gene was constructed successfully in vitro and OPN gene expression was increased significantly in HEK293AD cell after infection by the recombinant virus. Conclusion： The recombinant adenovirus vector containing OPN gene was constructed successfully in vitro. It may be a useful tool for the research on the OPN gene.