摘要： 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(human embryo 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。
关键词:
骨桥蛋白,
腺病毒载体,
质粒重组,
基因克隆
Abstract: 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.
Key words:
osteopontin(OPN),
adenovirus vector,
recombinant plasmid；gene clone
中图分类号:
刘安娜，王佐林. 大鼠骨桥蛋白重组腺病毒的构建及体外表达[J]. 《口腔颌面外科杂志》, 2012, 22(4): 233-237.
LIU An-na, WANG Zuo-lin. Construction and Expression of Recombinant Adenovirus Vector Containing OPN Gene in Vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2012, 22(4): 233-237.