鼠盘状结构域受体真核表达载体的构建与表达  

Construction and expression of different functional forms of mouse discoidin domain receptor 2

作　　者：周洁[1] 刘新平[2] 贺小舟[1] 张远强[2] 

机构地区：[1]第四军医大学组织学与胚胎学教研室,西安710032 [2]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《解放军医学杂志》2005年第7期599-602,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家重点基础研究发展规划项目(编号19990756)资助课题

摘　　要：目的分别构建DDR2的野生型(FLDDR2)、嵌和体(FcDDR2)和截短体(ttDDR2)的真核表达载体,并在哺乳动物细胞表达,为进一步研究DDR2与类风湿性关节炎发病机制的关系奠定基础。方法FLDDR2和ttDDR2通过RTPCR的方法获得,测序正确后亚克隆入pCDNA3.1(+)。FcDDR2是利用人IgG1的Fc段替代部分胞浆区,测序正确后亚克隆入pMKITNeo。重组产物经脂质体2000介导瞬时转入COS7细胞,48h后收取蛋白,分别利用蛋白印迹法和免疫沉淀法检测重组产物的表达。转入重组质粒的细胞经胶原刺激3h后收取蛋白,利用免疫印迹和免疫沉淀法检测磷酸化水平的变化。结果所构建的三种真核表达载体均可在COS7细胞中正确表达。将ttDDR2和FLDDR2共转染COS7细胞并给予胶原刺激后FLDDR2的磷酸化水平有所下降,而FcDDR2在无胶原刺激时也可自身活化,其程度与FLDDR2经胶原刺激后的活化程度相仿。结论成功构建了FLDDR2、ttDDR2和FcDDR2三种真核表达载体,其中FcDDR2可以作为一种激活剂,而ttDDR2在一定程度上具有负性竞争的作用。Objective Discoidin domain receptor 2 is a kind of receptor tyrosine kinases, which was found to be over-expressed in synovial fibroblasts in rheumatoid arthritis (RA). The present study was to construct and express wild type (FLDDR2), Fc chimera (FcDDR2) and truncated form of mouse discoidin domain receptor 2 (ttDDR2) for further study. Methods Full-length, truncated form of mouse DDR2 were amplified by RT-PCR. A chimera form of mouse DDR2 was constructed by replacing part of the extracellular domain with Fc fragment of human immunoglobulin G1 (IgG1). Eukaryotic expression vectors of the different forms of mouse DDR2 were constructed by subcloning the PCR products into pcDNA3.1(+) or pMKIT-Neo. Then COS-7 cells were transient transfected with the eukaryotic expression vectors of full-length (FLDDR2), truncated (ttDDR2) and chimeric form of mouse DDR2 (FcDDR2). Successful transfection and expression was confirmed by Western blot(WB) and Immunoprecipitation (IP)/WB. With or without stimulation with soluble type I collagen for 3h, phosphorylation level of transfected cells were detected by IP/WB. Eukaryotic expression vectors of full-length, truncated form and chimeric forms of mouse DDR2 were successfully constructed and confirmed by sequencing. After transient transfection, the expression of these three forms in the respectively transfected cells was observed by Western blot. Results The result of IP/WB suggested that the chimeric form of mouse DDR2(FcDDR2) could be properly expressed in the COS-7 cells. Under the condition of collagen, decreased tyrosine phosphorylation of FLDDR2 was detected in the COS-7 cells that were cotransfected with ttDDR2 and FLDDR2, comparing with that in COS-7 cells transected with only FLDDR2. There was no obvious difference in phosphorylation level between FcDDR2 without collagen stimulation and FLDDR2 with the collagen stimulation. Conclusion Three different forms of mouse DDR2 were successfully constructed. FcDDR2 could be an activator for its auto-phosphorylation. And ttDDR2 could

关 键 词：盘状结构域受体2 类风湿性关节炎 

分 类 号：R593.22[医药卫生—内科学]