重组pcDNA4 His/Max人骨形态发生蛋白9真核表达载体的构建与鉴定

背景：腺病毒临床应用存在安全隐患,利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。目的：将人骨形态发生蛋白9的cDNA构建于pcDNA4 His/Max,得到重组真核表达载体pcDNA4 His/Max人骨形态发生蛋白9。方法：将已有的Padtrack-cmv-人骨形态发生蛋白9进行PCR扩增,电泳回收人骨形态发生蛋白9片段,将pcDNA4 His/Max用Not Ⅰ和KpnⅠ双酶切,得到pcDNA4 His/Max酶切片段,连接人骨形态发生蛋白9和pcDNA4 His/Max片段得到重组质粒pcDNA4 His/Max人骨形态发生蛋白9,将该载体用DH5α转化,阳性克隆扩增及纯化、序列分析鉴定。结果与结论：通过PCR及序列分析证明pcDNA4 His/Max人骨形态发生蛋白9真核表达载体的人骨形态发生蛋白9基因长度为1.3 kb,与报道的人骨形态发生蛋白9全长序列一致,无突变。结果证实,实验成功构建了pcDNA4 His/Max人骨形态发生蛋白9真核表达载体。

Construction and identification of a recombinant pcDNA4 His/Max human bone morphogenetic protein eukaryotic expression vector

BACKGROUND:Adenovirus mediated method has been used numerously in human bone morphogenetic protein 9 (hBMP-9) induced osteogenesis researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.OBJECTIVE:To reconstruct a recombinant DNA pcDNA4 His/Max hBMP-9 eukaryotic expressing vector by amplifying hBMP9 gene and then cloning into pcDNA4 His/Max.METHODS:Padtrack-cmv-hBMP-9 was amplified by PCR, and hBMP-9 gene was retrieved by electrophoresis. pcDNA4 His/Max was digested by NotⅠ, Kpn Ⅰand then the hBMP-9 gene was cloned into pcDNA4 His/Max. The recombinant plasmid was transformed by DH5α, clonal expansion, and purification, and then verified by sequencing.RESULTS AND CONCLUSION:The cloned hBMP-9 gene was 1.3 kb long, having the same length and sequence of the gene that human possessed. Eukaryotic expressing vector of pcDNA4 His/Max hBMP-9 has been constructed successfully.