摘要
目的 观察血管内皮生长因子受体 (VEGFR)结合肽 -颗粒酶 B(Gra B)融合蛋白抗血管生成的作用 .方法 抽提人外周血淋巴细胞总 RNA,进行 RT- PCR克隆 Gra B c DNA;抽提 L o Vo细胞总 RNA,进行 RT- PCR克隆 VEGFR结合肽c DNA,用限制性酶切和 DNA测序进行鉴定 ;通过聚丙烯酰胺凝胶电泳 (SDS- PAGE)分析表达产物 .表达产物经亲和层析纯化后 ,以人血管内皮细胞 (ECV30 4 )和鸡胚尿囊膜血管测定其生物学活性 .结果 表达产物以可溶性分子形式存在于菌体中 ,具有良好的抗原性和特异性 ,且具有抑制血管内皮细胞增殖及破坏鸡胚尿囊膜血管形成的活性 .结论 VEG-FR结合肽 - Gra B融合蛋白具有抑制血管生成的功能 。
AIM To develop an inducible system for expression of VEGF receptor binding domain GraB in E. coli on the basis of expressive specifity of VEGF receptor on the vascular endothelial of tumor and of the effect that GraB induces cell apoptosis. The biological function of VEGF receptor binding domain GraB was studied for the purpose of antiangiogenesis research. METHODS After GraB cDNA and hVEGF receptor binding domain cDNA were amplified by RT PCR via extracting lymphocyte total RNA and LoVo cell total RNA respectively, the fusion gene was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid PtrcHis2A/VEGFD GraB was constructed and transformed into TOP10F. RESULTS After 8 h of IPTG induction, the VEGF receptor binding domain GraB was expressed to 15% of total proteins. Western blot assay proved the expressed protein to be of good antigenicity and high specificity. The recombinant protein purified by affinity chromatography was proved to inhibit ECV303 proliferation and destroy neovascularization of the chick chorioallantoic membrance. CONCLUSION VEGF receptor binding domain GraB fusion protein may be a potent inhibitor of tumor angiogenesis and metastasis.
出处
《第四军医大学学报》
北大核心
2002年第21期1919-1932,共14页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 (3 9870 72 3 )
