鸡骨保护素（chOPG）的原核表达、纯化及抗体制备

 【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-PAGE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。

Prokaryotic Expression and Purification of Recombinant Chicken OPG Protein and Preparation of Rabbit Anti-OPG Antibody

Osteoprotegerin(OPG) is a new found protein which can inhibit the osteoclast differentiation and activation. In order to do further research about its biological function, the total RNA were extracted from osteoblasts cultured in vitro for OPG amplification by RT-PCR method. After the sequence testing was correct, the OPG gene was inserted to the expression plasmid pET-32a(+) and expressed in Rosetta-gami（DE3）pLysS with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in Rossetta with molecular weight of 63kD and amounted to 11.9% of the whole protein, and western blotting indicated that the expressed protein had satisfied immunobiological activity. Pure protein was obtained by Ni2+-NTA chelating column for preparation of anti-OPG polyclonal antibody by immunizing rabbit, the titer of antiserum generated was 1:10240 by ELISA. Western blotting analysis showed that it could bind with OPG protein specially expressed in Pichia Yeast.