可溶性VEGF受体-3原核表达载体的构建与蛋白表达  

Gene cloning of soluble VEGF receptor-3 and expression in procaryotic system

作　　者：杨婉华[1] 汪蕊[1] 陈睿[1] 马湘一[1] 王世宣[1] 卢运萍[1] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,湖北武汉430030

出　　处：《中国现代医学杂志》2005年第23期3547-3550,共4页China Journal of Modern Medicine

基　　金：国家重点基础研究发展规划项目(2002CB513100)

摘　　要：目的克隆并原核表达可溶性血管内皮细胞生长因子受体-3(sVEGFR-3),用于受体阻断剂的筛选及制备。方法利用RT-PCR技术从人胎盘组织中扩增VEGFR-3胞外Ⅰ～Ⅲ区cDNA片段,通过基因重组技术将该片段克隆至原核表达载体PTrcHisA中,连接产物转化大肠杆菌TOP10,IPTG诱导融合蛋白表达,纯化获得可溶性受体。结果成功构建表达载体,经IPTG诱导获得大量稳定表达的sVEGFR-3融合蛋白,纯化后获得纯度达到80%以上的可溶性受体。结论大量可溶性VEGFR-3的获得有助于进一步探索肿瘤淋巴道转移的靶向性治疗方案。[Objective] Due to its high affinity to VEGF-C/D, soluble VEGFR-3 is expected to be a prospective candidate used in dominant-negative way. This study was aimed at expressing soluble VEGFR-3 in prokaryotic system for panning its inhibitors. [Methods] In this study, cDNA fragments of VEGFR-3 Ⅰ -Ⅲ extracellular domains were amplified through RT-PCR from human placenta tissue with a pair of specific primers and cloned to PTrcHis A. The fusion protein was expressed stably in E.coh TOP10. [Results] SDS-PAGE showed that the soluble fusion protein which molecular weight was identical to expeetation was remarkably expressed after induced by IPTG and purified through the ProbondTM. [Conclusion] Gaining a large quantity of soluble VEGFR-3 makes it possible to find out the way of tumor lymphatic metastasis targeting therapy.

关 键 词：可溶性受体 VEGFR-3 原核表达 

分 类 号：R730.3[医药卫生—肿瘤]