猪BMP4基因原核表达及优化  

Prokaryotic Expression and Optimization of Bone Morphogenetic Protein 4 from Pigs

作　　者：周建设[1] 李明[1] 陈其新[1] 张驰[1] 石晓卫[2] 赵巧辉 

机构地区：[1]河南农业大学,郑州450002 [2]新乡医学院,河南新乡453003 [3]郑州安图绿科生物工程有限公司,郑州450016

出　　处：《西北农业学报》2011年第1期24-28,64,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：河南省重点科技攻关项目(82102130004);河南农业大学博士基金(30300338)

摘　　要：构建猪BMP4基因pET32a-BMP4原核表达质粒,在E.coli中表达并分离纯化目的蛋白。采用PCR技术以pMD18-T-BMP4重组质粒为模板扩增获得BMP4基因编码成熟肽片段,并插入到原核表达载体pET32a中,构建原核表达重组质粒pET32a-BMP4。将阳性重组质粒转化到表达宿主菌中,在不同的温度、IPTG浓度和时间下进行诱导表达,SDS-PAGE分析鉴定。构建的重组表达质粒经PCR、酶切和DNA测序鉴定与预期的结果一致,表达宿主菌经IPTG诱导表达分子量约为31 ku的融合蛋白,表达产物占菌体总蛋白的30%以上。成功构建了pET32a-BMP4原核表达质粒,并经纯化得到BMP4目的蛋白,为进一步研究该蛋白的结构和功能奠定基础。The pig＇s pET32a-BMP4prokaryotic expression plasmid was constructed to express and purify BMP4 protein in E.coli.The mature peptide fragment of BMP4 was amplified from the pMD18-T-BMP4 recombinant plasmid preserved the laboratory,and cloned into prokaryotic expression vector pET32 a.The positive recombinant plasmid was transformed into expression host strains,and then was induced by different temperatures,concentrations of IPTG（isopropyl-β-thiogalactoside） and times.The purpose protein was identified by SDS-PAGE.The recombinant expression plasmid was identified by PCR method,digested with restricted endoenzymes and subjected to DNA sequencing.It was found to be consistent with the predicted result.The host cells containing the recombinant plasmid expressed fusion protein of 31 ku after being induced by IPTG.The prokaryotic expression plasmid pET32a-BMP4 was successfully constructed and the target recombinant protein BMP4 was expressed,which lays a foundation for the further study of the structure and functions of BMP4 protein.

关 键 词：猪 骨形态发生蛋白4 原核表达 条件 

分 类 号：Q78[生物学—分子生物学]