盘状结构域受体2胞外区的可溶性表达、纯化和功能鉴定

盘状结构域受体2（discoidin domain receptor 2,DDR2）是一种与肿瘤细胞转移相关的蛋白酷氨酸激酶，其配体为纤维性胶原，胶原对DDR2的活化上调细胞中基质金属蛋白酶1（MMP－1）的表达。为研究DDR2在类风温性关节炎(rteumatoid arthritis,RA）软骨破坏和肿瘤转移中的作用，尝试了在大肠杆菌中表达一段DDR2胞外区（命名DB），并进行了可溶性部分的纯化和功能鉴定，以备将来用作DDR2的特异性阻断剂。获得了一株表达GST－DB融合蛋白的大肠杆菌克隆；其表达的蛋白质中可溶性部分约占全部融合蛋白的13％；经GST融合蛋白特异性亲和珠纯化后，获得了纯度约86.1%的可溶性GST－DB融合蛋白；竞争结合抑制实验显示，GST－DB具有阻断Ⅱ型胶原和细胞表面天然DDR2受体结合的功能；细胞实验表明，GST－DB有抑制Ⅱ型胶原刺激下的类风湿性关节炎滑膜细胞和NIH3T3细胞分泌MMP－1的作用。以上结果提示，表达的融保蛋白GST－DB具有抑制天然DDR2功能的作用；DDR2在滑膜细胞和NIH3T3细胞中介导Ⅱ型胶原刺激下的MMP－1的分泌。

Expression, Purification and Functional Identification of Extracellular Part of Discoidin Domain Receptor 2

Discoidin domain receptor 2 (DDR2) is a new type of receptor tyrosine kinases, and was thought to be involved in the metastasis of some tumors. Its ligand is fibrillar collagen. The activation of DDR2 induced by collagen mediates the over expression of matrix metalloproteinase 1 (MMP 1) in cells. A specific inhibitor of DDR2 was necessary for the study of DDR2 function. Theoretically, a soluble receptor could possibly be used as specific inhibitor for the native receptor on cell membrane. In this report, a fragment (DB) of extracellular part of DDR2 was cloned and expressed for the use as potential inhibitor. This DB fragment corresponded to the polypeptide from the 23rd amino acid residue to the 293rd amino acid residue of DDR2. The fragment was amplified by RT PCR from human lung cancer tissue, and the product was cloned into pMD18 T vector. After identification by sequence analysis, the fragment was sub cloned into pGEX 4T 1 vector. Fusion protein of GST DB was expressed in JM109 E.coli cells as expected and the soluble part accounted for about 13% of the total fusion protein. The soluble fusion protein was then purified with glutathione affinity resin, and GST DB with purity of 86.1% was obtained. Competitive combination inhibitory test showed that the purified GST DB inhibited the interaction between collagen II and DDR2 on the surface of RA synovial fibroblasts. Zymography analysis showed that the level of MMP 1 of both NIH 3T3 cell and RA synovial fibroblasts with collagen II stimulation decreased after adding GST DB fusion protein. The results indicated that the fusion protein GST DB could inhibit the function of DDR2 on cells, and DDR2 might mediate collagen II induced over expression of MMP 1 in these cells.