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目的 克隆人骨保护素 (OPG)成熟肽段编码区基因 ,并在大肠杆菌中表达。方法 采用RT PCR法 ,扩增人OPG成熟肽段编码区cDNA ,并克隆入原核表达载体pMAL c2x中 ,转化BL2 1(DE3)PlysS大肠杆菌感受态细胞。经 0 .1mmol/LIPTG诱导后 ,收集菌体蛋白 ,进行SDS PAGE及Westernblot鉴定。结果 获得人OPG成熟肽段编码区cDNA ,以构建的原核表达载体pMAL OPG转化菌株后 ,可表达人OPG和麦芽糖结合蛋白 (MBP)的融合蛋白 ,相对分子质量 (Mr)为 85 0 0 0。表达产物的蛋白量约为菌体总蛋白的 13%。Westernblot表明 ,融合蛋白能与抗人OPG多克隆抗体特异性结合。结论 获得人OPG成熟肽全长cDNA ,并在大肠杆菌中以OPG MBP融合蛋白的形式表达
Objective To clone the coding region of human osteoprotegerin (OPG) mature peptide and express it in Escherichia coli. Methods cDNA of coding region of human OPG mature peptide was amplified by RT PCR and cloned into prokaryotic expression vector pMAL c2x. The recombinant plasmid was transformed into BL21 (DE3) PlysS competent cells. After induced by 0.1 mmol / L IPTG, the bacterial proteins were collected and identified by SDS PAGE and Western blot. Results The cDNA of coding region of human OPG mature peptide was obtained. After being transformed with the constructed prokaryotic expression vector pMAL OPG, the fusion protein of human OPG and maltose binding protein (MBP) was expressed. The relative molecular mass (Mr) was 85 000. The expression product of the protein is about 13% of the total bacterial protein. Western blot showed that the fusion protein can specifically bind to anti-human OPG polyclonal antibody. Conclusion The full-length cDNA of human OPG mature peptide was obtained and expressed in E. coli as OPG MBP fusion protein