鸡骨保护素（chOPG）的原核表达、纯化及抗体制备.pdfVIP

中国农业科学 2008,41(4):1207-1212 Scientia Agricultura Sinica 鸡骨保护素（chOPG）的原核表达、纯化及抗体制备 姚 静，侯加法，王 艳，张风荣 （南京农业大学动物医学院，南京 210095 ） 摘要：【目的】骨保护素（OPG）是一种新发现能抑制前体破骨细胞分化及成熟破骨细胞活性的蛋白，本文旨 在从体外扩增、表达鸡骨保护素（chOPG）蛋白，并制备其多克隆抗体，为深入研究它的生物学功能奠定基础。【方 法】以体外培养的鸡胚成骨细胞中提取的总RNA为模板，利用RT-PCR 方法，扩增出chOPG基因，测序后克隆至原 核表达载体pET-32a（+），成功构建了原核表达质粒pET-32a（+）-OPG，重组质粒转化Rosetta-gami（DE3）pLysS， 经 IPTG 诱导表达。【结果】表达产物经 SDS电泳，发现目的蛋白大小约为 63 kD，占菌体总蛋白的 11.9%， Western blotting分析显示，表达的chOPG 蛋白具有良好的免疫反应性。用镍离子亲和层析的方法可获得纯化的 蛋白，以纯化的蛋白免疫家兔制备多克隆抗体，ELISA 结果显示效价达到 1 ﹕10240。Western 印迹结果表明，该 抗体可以和chOPG酵母表达产物特异性结合。【结论】成功地实现了鸡骨保护素在大肠杆菌中的表达，并获得其多 克隆抗体。 关键词：鸡；骨保护素；原核表达；纯化；抗体 Prokaryotic Expression and Purification of Recombinant Chicken OPG Protein and Preparation of Rabbit Anti-OPG Antibody YAO Jing, HOU Jia-fa, WANG Yan, ZHANG Feng-rong (College of Veterinary Medicine，Nanjing Agricultural University ，Nanjing 210095) Abstract: 【Objective 】Osteoprotegerin (OPG) is a newly found protein which can inhibit the osteoclast differentiation and activation, the objective of this study is to amplify and express the chicken OPG (chOPG) protein in vitro for the further research about its biological function. 【Method 】 The total RNA were extracted from osteoblasts cultured in vitro for chOPG amplification by RT-PCR method. After the sequence testing was correct, the chOPG gene was inserted to the expression plasmid pET-32a (+) and expressed in Rosetta-gami (DE3) pLysS with IPTG inducement. 【Result 】 The SDSresult showed that the cloned recombinant protein expressed in the form of inclusion bodies in Rossetta with molecular weight of 63 kD and amounted to 11.9% of the whole protein, and western blotting indicated that the expressed protein had satisfied immunobiological activity. Pu