摘要
目的探讨可溶性血管内皮生长因子受体2（sKDR）抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞（HUVEC）总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜（CAM）试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子（VEGF）刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。
Objective To explore the inhibiting effect of soluble vascular endothelial growth factor （VEGF）receptor-2 （solubility kinase insert domain receptor, sKDR） on the proliferation of vascular endothelial cells and its role in angiogenesis. Methods Extract the total RNA of human umbilical vein endothelial cells （HUVECs） for amplification of extracellular domians 1 - 4 of KDR gene. Insert the amplified genc fragment into plasmid pQE4O, and transform the constructed recombinant plasmid pQFE40-KDR to E. coil M15 for expression under induction of IPTG. The expressed sKDR protein was purified by nickel ion affinity chromatography,then re-naturalized and identified by Western blot. Determine the effect of sKDR protein on proliferation of HUVEC and angiogenesis by MTT method and chick chorioallantoic membrane （CAM） test respectively.Results The sKDR gene fragment at a length of about 1150 bp was amplified by RT-PCR and expressed in a form of inclusion body in pQE40 prokaryotic expression system. The expressed product, with a relative molecular mass of about 50 000 and a protein content of 80 μg/ml,contained 98% of total somatic protein and showed a single band on electrophoretic profile. It was proved as recombinant sKDR protein by Western blot. The protein inhibited the proliferation of HUVECs stimulated by VEGF and blocked the CAM angiogenesis induced by VEGF. Conclusion The prokaryotic expression vector for sKDR was successfully constructed and expressed in E. coli M15. The purified sKDR showed binding capacity to VEGF and might be an ideal target for gene therapy of angiogenesis of tumors.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第2期85-89,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金资助项目(30571857)
吉林省科学技术厅资助项目(200505118)
关键词
血管内皮生长因子受体2
血管生成
人脐静脉内皮细胞
鸡胚尿囊膜
Vascular endothelial growth factor receptor 2
Angiogenesis
Human umbilicalo vein endothelial cells
Chick chorioallantoic membrane
