OPG与MBP融合蛋白在大肠杆菌中的表达

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BL21（DE3）PlysS大肠杆菌感受态细胞，经0.1mmol/L IPTG诱导后，收集菌体蛋白，进行SDS－PAGE及Western blot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（M）为85000。表达产物的蛋白量约为菌体总蛋白的13％。Western blot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OPC－MBP融合蛋白的形式表达。

Expression of human OPG-MBP fusion protein in E.coli

Aim To clone the mature peptide cDNA of human osteoprotegenin ( OPG) and express it in E.Coli . Methods Using the isolated total RNA from human osteosacoma cell line MG63 as a template, cDNA encoding the human OPG mature peptide was amplified by RT PCR. The PCR product was cloned into pUC19 and sequenced. Then, gene encoding mature region of OPG was inserted into prokaryotic expression vector pMAL c2x and transformed into competent E.coli BL21(DE3)PlysS to express via induction of IPTG. OPG MBP fusion protein was identified by SDS PAGE and Western blot. Results The cloned gene sequence of OPG mature peptide was identical with that reported. SDS PAGE and Western blot analysis indicated that the relative molecular mass ( M r) of the fusion protein was about 85 000, Fusion protein accounted for about 13% of total bacteria protein and could react specifically with anti OPG antibody. Conclusion cDNA encoding OPG mature peptide has been cloned and expressed in E.coli .