人BMP4基因的克隆及其原核表达载体的构建

Cloning of Human Bone Morphogenetic Protein 4 and Construction of Its Prokaryotic Expression Vector

以成熟人胎盘组织为材料来源,克隆人BMP-4基因的全长cDNA,经过PCR扩增后与pMD18-T载体连接,构建pMD18-T-BMP4克隆质粒。酶切后回收小片段与表达载体pET-22b的多克隆酶切位点连接,构建原核表达载体pET22b-BMP4,酶切及测序鉴定重组子。重组质粒转化至感受态的Rosseta宿主菌,经IPTG诱导表达,SDS-PAGE检测蛋白表达情况。结果显示,从胎盘组织中成功地克隆到人BMP4基因,与NCBI中公布的序列100%相符合,原核表达载体pET22b-BMP4转化至Rosseta构建表达菌体,经IPTG诱导后电泳分析可见重组蛋白表达的条带。

To clone the gene sequences of bone morphogenetic protein 4 and construct the expression vector of this gene,total RNA was extracted from human placenta and reverse transcripted to cDNA.Gene sequence of bone morphogenetic protein 4 was cloned from cDNA with specific primers and linked to pMD18-T vector,and then,gene sequence was cloned to the expression vector pET-22b through NdeⅠ和SalⅠ double digestion.The sequence of BMP4 was 100% matched with the sequence reported in NCBI.The expression vector pET22b-BMP4 was transferred to Rosetta,and the recombined protein was expressed successfully after induced by IPTG.