重组人骨形态发生蛋白7的腺相关病毒载体的构建  

Construction of an Adeno-associated Virus Vector Carrying Human Bone Morphogenetic Protein-7 gene

作　　者：杨民[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：《中国骨与关节损伤杂志》2005年第7期458-461,共4页Chinese Journal of Bone and Joint Injury

基　　金：国家自然科学基金(编号:No.30170947)

摘　　要：目的构建重组人骨形态发生蛋白7(hBMP7)的腺相关病毒(AAV)载体。方法通过RT-PCR方法,从HEK293细胞中扩增hBMP7全长cDNA并亚克隆入通用型AAV载体质粒pSNAV。将pSNAV-hBMP7质粒DNA转染BHK-21细胞,筛选出携带hBMP7的AAV载体细胞株—BHK-21细胞。用能表达Rap和Cap的重组1型单纯疱疹病毒HSV1-rc/ΔUL2按MOI=0·1感染BHK-21载体细胞株后收获病毒液,通过氯仿—PEG/NaCl沉淀—氯仿抽提纯化病毒。结果所克隆的hBMP7全长cDNA经测序证实和已知hBMP7序列完全一致。通过用能表达Rap和Cap的重组1型单纯疱疹病毒HSV1-rc/ΔUL2感染携带hBMP7基因的AAV载体细胞株BHK-21细胞的方法获得了滴度为2×1012vg/ml、纯度达99%的重组人骨形态发生蛋白7的腺相关病毒载体(AAV-hBMP7)。结论RT-PCR方法从HEK293细胞中扩增克隆hBMP全长cDNA是一种有效可行的方法。成功地构建了高滴度、高纯度的AAV-hBMP7载体,为进一步体内及体外研究基因治疗促进骨愈合提供了有力的工具。Objective To clone hBMP7 gene and construct an adeno - associated virus vector carrying human bone morphogenetic protein- 7 gene. Methods Human BMP7 full length cDNA was amplified by RT - PCR from HEK293 cells and inserted into a general AAV vector plasmid pSNAV. The AAV vector ceils - BHK - 21 carrying hBMP7 gene was then screened. BHK - 21 cells were subsequent infected with recombinant virus-HSV1 -rc/△UL2, which expresses AAV-2 Rep and Cap, and possessing packaging functions for AAV. The infected BHK - 21 cells were collected after 48 hours and processed by chloroform treatment, PEG8000/Nacl precipitation and chloroform extraction. The titration and purity of the vector were performed by quantitative DNA dot blots and SDS - PAGE respectively. Results The cloned cDNA was confirmed to be hBMP7 cDNA by sequencing. The purified AAV - BMP7 reached to 2 × 10^12vg/ml and more than 99% in purity. Conclusion It is an effective way to clone hBMP7 cDNA from HEK293 ceils by RT- PCR. The high - titer, purified AAV - BMP7 stock provide us a useful tool in the next experiments.

关 键 词：重组人骨 形态发生蛋白7 病毒载体 hBMP7 腺相关病毒 AAV 

分 类 号：R681[医药卫生—骨科学]