Cloning of cDNA Encoding Extra-Cellular Domain of Human VEGF Receptor KDR and Effect of Its Recombinant Protein on Endothelial Cell Proliferation and Angiogenesis / 中国肿瘤生物治疗杂志

Objective: To clone cDNA encoding Ⅴ-Ⅶ extra-cellular domains of VEGF receptor KDR and investigate biological activity of the recombinant protein. Methods: Total RNA from the umbelical venous endothelial cells was obteined. From it, KDR cDNA was cloned into the BamH1 and Kpn 1 sites of pQE31 and made expressed with induction of IPTG. The recombinant protein was purified, refolded and determined for its biological activities. Results: The expression system could express the human recombinant extra-cellular protein of KDR with expression level accounting for 5% of the total bacterial protein. After renaturation, the recombinant protein could bind to 125 I-VEGF and suppress endothelial cell proliferation and angiogenesis of chick chorioallantoic membrance induced by VEGF. Conclusion: Human KDR extra-cellular domain can be expressed in E coli and after being refolded, has an action of binding to VEGF.

Gene Cloning of Soluble VEGF Receptor FLT-1 and the Effect of Its Recombinant Protein on Angiogenesis / 中国肿瘤生物治疗杂志

Objective: To clone and express cDNA fragment of domains 1 and 3 of soluble VEGF receptor FLT-1 in E. coli and investigate effect of its recombinant protein on endothelial proliferattion and angiogenesis. Methods: Total RNA from the umbelical venous endothelial cells was obtained. From it, soluble FLT-1(sFLT-1) cDNA fragment of domians 1 and 3 was cloned and expressed in QIA expressionist. The recombinant protein was purified by chromatography and refolded. The methods of MTT and angiogenesis test of chick chorioallantoic membrance were used to determine bioactivities of sFLT-1 recombinant protein. Results: The expression system could express the sFLT-1 cDNA fragment with a low expression level. After purification and renaturation, the recombinant protein could specifically bind to 125 I-VEGF. 1 ?g recombinant protein sFLT-1 could suppress endothelial cell proliferation induced by 10 ng VEGF and angiogenesis of chick chorioallantoic membrance induced by 20 ng VEGF.Conclusions: sFLT-1 cDNA fragment can be expressed in QIAexpressionist. After being refolded, it can bind to VEGF and serve as an antagonist of VEGF.