重组人骨形态发生蛋白7腺病毒载体构建及在小鼠成肌细胞中的表达  

Construction of the recombinant adenoviral vector carrying human bone morphogenetic protein-7 cDNA and its expression in myoblasts of rats

作　　者：杨民[1] 蔡善保[1] 马庆军[1] 郭均[1] 党耕町[1] 

机构地区：[1]北京大学第三医院骨科,北京市100083

出　　处：《中国临床康复》2005年第6期56-58,i003,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金课题(30170947)~~

摘　　要：目的:构建重组人骨形态发生蛋白7的腺病毒载体(AdBMP7),并观察其在小鼠成肌细胞C2C12细胞中的表达情况。方法:实验于2004-05/10在北京大学医学部完成。RT-PCR方法从HKE293细胞中克隆出hBMP7cDNA并亚克隆入穿梭质粒pAdtrackcmv中,构建pAdtrackcmv-hBMP7质粒。该质粒和腺病毒骨架质粒pAdEasy-1在BJ5183菌中进行同源重组后挑选阳性重组体pAd-hBMP7质粒。线性化pAd-hBMP7质粒转染293A细胞后检测病毒噬斑形成情况,并于转染后9d收获病毒,大量扩增并制备高纯度、高滴度的AdBMP7。将AdBMP7按MOI=100来感染C2C12细胞,48h后分别应用RT-PCR和免疫组化方法来检测hBMP7在mRNA和蛋白水平的表达。结果:成功克隆出hBMP7cDNA,酶切鉴定pAdtrackcmv-hBMP7和pAd-hBMP7质粒正确重组,获得纯化后滴度达到5×1012vp/mL的AdBMP7。AdBMP7能有效感染C2C12细胞并表达hBMP7蛋白。结论:应用AdEasy-1腺病毒载体系统可在短期内制备高滴度的携带GFP和hBMP7的重组腺病毒AdBMP7,并在为进一步研究BMP7基因治疗促进骨愈合打下基础。AIM: To construct the recombinant adenoviral vector carrying human bone morphogenetic protein 7(AdBMP7) cDNA and observe its expression in C2C12 cells of rat myoblasts.METHODS: The experiment was conducted in Peking University Health Science Center from May to October 2004.Human bone morphogenetic protein 7(hBMP7) cDNA was cloned by RT PCR from HEK293 cells and subcloned into shuttle plasmid pAdtrackcmv.The linearized shuttle plasmid pAdtrackcmv hBMP7 was co transformed into bacteria BJ5183 with adenoviral backbone vector AdEasy 1 to obtain the recombinant adenoviral plasmids pAd hBMP7 by homologous recombination.The linearized pAd hBMP7 was transfected into 293A packaging cells to detect the formation of viral plaque.After the viruses were hanvested at 9 days,the recombinant adenovirus dBMP7 was multiply and purify further.C2C12 cells were infected by AdBMP7 at an MOI of 100,and mRNA and protein expression of hBMP7 were detected by RT PCR and immunehistochemistry 48 hours after infection respectively.RESULTS:The cloned cDNA was confirmed to be hBMP7 cDNA.Recombinant pAdtrackcmv hBMP7 and pAd hBMP7 were correctly constructed and confirmed by restriction endonuclease analysis.The titer of purified AdBMP7 could reach as high as 5× 1012 vp/mL.The AdBMP7 could infect C2C12 cells in vitro effectively and express hBMP7 protein.CONCLUSION:The AdBMP7 carrying high titer GFP and hBMP7 is successfully constructed in a short time by using AdEasy 1 adenoviral vector system,which may lay a foundation for BMP7 gene therapy of bone healing.

关 键 词：骨折愈合 骨形态发生蛋白质类 腺病毒 人 基因治疗 

分 类 号：R318[医药卫生—生物医学工程]