摘要
目的探讨构建携带人骨形成蛋白7(human bone morphogenetic protein-7,hBMP-7)基因的重组35型腺病毒(serotype35adenovirus,Ad5F35)载体,为基因治疗研究提供一种新的方法,并观察其在兔骨髓间充质干细胞(MSCs)中的表达。方法以pcDNA1.1/Amp-hBMP7质粒为模板扩增骨形成蛋白(BMP-7)基因(1.3kb),将回收的PCR产物片段克隆入pDC316载体,获得重组质粒pDC316-hBMP7。骨架质粒pBHG-fiber5/35和穿梭质粒pDC316-hBMP7共转染293细胞,同源重组产生重组腺病毒rAd5/F35-hBMP7。同样的方法构建rAd5/F35-EGFP。在体外用不同感染复数(M.O.I)值的rAd5/F35-hBMP7和rAd5/F35-GFP分别转染兔MSCs,并留置空白对照。采用流式细胞仪、RT-PCR方法检测目的基因的转染效率和表达。结果PCR、酶切鉴定以及序列测定与比对分析表明,重组腺病毒rAd5/F35-hBMP7质粒构建正确。转染兔MSCs的最高效率可达99%,hBMP-7基因存在于转染后的MSCs中并表达相应的mRNA。结论本实验成功构建了含hBMP-7基因的重组腺病毒rAd5/F35-hBMP7载体,在体外能高效转染兔MSCs。
Objective To investigate the construction of an Ad5F35 based vector carrying bone morphogenetic protein- 7 （hBMP- 7） and observe their expression in rabbit bone mesenchymal stem cells in order to facilitate gene therapy research using recombinant adenovirus＇ s containing fibers derived from B - group serotype 35 adenovirus （Ad5F35） vector as gene transfer vehicle. Methods The coding sequence （1.3 kb） of BMP- 7 was amplified by PCR from the pcDNA 1.1 （ ＋ ） plasmid containing the human BMP - 7 eDNA. After purified, the gene fragment was cloned into a plasmid pDC316 and termed plasmid pDC316 - hBMP7. Two hundred ninety three cells were transfected with the pDC316 - hBMP7 and pBHG- fiber5/35 plasmid and termed recombinant plasmid rAd5/F35 - hBMP7. rAd5/F35- hBMP7 was determined by polymerase chain reaction （PCR） and digest with restriction enzyme. Following infection with tAd5/F35 - hBMP7 at different multiplicities of infection of vector per cell and subsequent culture, mesenchymal stem cells were assessed qualitatively for BMP - 7 production. Results The recombinant plasmid pDC316 - hBMP7 and rAd5/F35 - hBMP7 was identified by PCR and digest with restriction enzyme. The best transfection showed an efficiency of 99 % in mesenchymal stem cells. BMP- 7 express/on in mesenchymal stem cells was identified by RT - PCR. Conclusion The hBMP- 7 recombinant Ad5F35 vector is successfully constructed and rAd5/F35 - hBMP7 could efficiently infect MSCs.
出处
《中国骨与关节损伤杂志》
2008年第8期655-658,共4页
Chinese Journal of Bone and Joint Injury
基金
广东省科委重点攻关项目(C30307)
关键词
骨形成蛋白
腺病毒
基因
治疗
Bone morphogenetic protein
Adenovirus
Gene
Treatment
