鸡骨保护素chopg的原核表达纯化及抗体制备.doc

鸡骨保护素（）rokaryotic 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 new 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. 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 1﹕10 240 by ELISA. Western blotting analysis showed that it could bind with chOPG protein specially expressed in Pichia Yeast. 【Conclusion】In this recombinant chOPG protein in E.coli and its polyclonal antibody are successfully achieved. Key words: Chicken; chOPG; Prokaryotic expression; Purification; Antibody 0 引言 【研究意义】骨保护素（steoprotegerin，OPG）是一种分泌型糖蛋白，属于TNF受体超家族成员，具有TNF受体超家族特征性的N端富含半胱氨酸区域，但不含跨膜疏水区，鸡骨保护素（chOPG）与人、小鼠与大鼠一致性分别为68.76%、68.60%和68.29%，提示该基因在进化过程有高度的保守性[1]。OPG可抑制破骨细胞分化的最终阶段，抑制成熟破骨细胞的活性并诱导其凋亡[2,3]，提示对OPG的深入研究和应用，将为骨质疏松等骨骼疾病的发病机制、预防及治疗开创一新的途径。【前人研究进展】Hofbauer等[4]研究发现骨保护素在人成骨细胞系中表达量较高，在雌激素作用下上调成骨细胞OPG的表达水平。Vidal等[5]也发现IL-1α可使人成骨细胞中OPG mRNA表达水平增加。国内苏欣等[6]也发现17-β雌二醇可增加成骨细胞OPG的表达，此效应可为雌激素受体拮抗剂阻断。Morinaga[7]