重组pcDNA3.1-BMP7真核表达载体的构建与鉴定  

Construction of carrier of recombinant pcDNA3.1- BMP7 eukaryotic express and its identification

作　　者：曲福军[1] 戚良晨[2] 刘丽玲 陈化兰 侯立中[1] 

机构地区：[1]吉林大学再生医学科学研究所哈尔滨医科大学附属二院药学部 [2]吉林大学中日联谊医院 [3]哈尔滨兽医研究所

出　　处：《中国地方病学杂志》2005年第4期442-445,共4页Chinese Jouranl of Endemiology

基　　金：国家自然科学基金资助项目(c03030306)

摘　　要：目的研究将家兔骨形成蛋白-7(BMP7)的cDNA构建于pcDNA3.1,形成重组DNApcDNA3.1-BMP7真核表达载体。方法将自建的pGEM-3Zf(-)-BMP7与pBluescriptSK(M13-)用HindⅢ及KpnI双酶切,电泳回收BMP7及pBluescriptSK(M13-)酶切片段,连接得重组载体pBlue-BMP7,DH5a转化,阳性克隆扩增及提取纯化,并用NotI和KpnI双酶切,PCR及序列分析鉴定。将载体pBlue-BMP7及pcDNA3.1用NotI及KpnI双酶切,电泳回收BMP7及pcDNA3.1酶切片段,连接得真核表达载体pcDNA3.1-BMP7,将该载体用DH5a转化,阳性克隆扩增及提取纯化。再一次用NotI和KpnI双酶切,PCR及序列分析鉴定。结果通过酶切、PCR及序列分析证明pcDNA3.1-BMP7真核表达载体的BMP7目的基因序列约为1.5kb,与报道的鼠、人BMP7全长序列一致,无突变。结论成功构建了pcDNA3.1-BMP7真核表达载体。Objective To reconstruct recombinant DNA pcDNA3.1-BMP7 eukaryotic expressing vector byamplifying rabbit bone morphogenetic protein-7(BMP7) gene and then cloning into pcDNA3.1. Methods pGEM-3Zf(-)-BMP7 and pBluescript SK(M13-) were digested by HindⅢ and KpnI, and then BMP7 gene was cloned into pBluescriptSK (M13-). Recombinant plasmid was identified respectively by PCR analysis, restriction endonuclease analysis andnucleotide sequencing. Positive recombinant pBlue-BMP7 and pcDNA3.1 vector were digested by NotⅠ KpnⅠ andBMP7 gene was cloned into pcDNA3.1. The recombinant was verified by restriction endonuclease, PCR analysis andsequencing. Results The cloned BMP7 gene was 1.5 kb long, having the same length and sequence of the gene that ratsand human possessed. Conclusion Eukaryotic expressing vector of pcDNA3.1-BMP7 has been constructed successfully.

关 键 词：骨形态发生蛋白质类 真核表达载体 基因 

分 类 号：Q78[生物学—分子生物学]