Résumé
Objective: To observe the inhibitory effect of gefitinib combined with DNA vaccine targeting EGFR against implanted Lewis tumors in mice.Methods: Chicken EGFR L2 domain and rabbit IgG Fc domain fusion pVAX1/cEGFR-rFc DNA vaccine was injected into mice and the titer of anti-EGFR in serum was determined by ELISA.The growth of Lewis cells was measured by MTT.Lewis lung cancer mouse models were established and were randomly divided into vaccine,gefitinib,gefitinib+vaccine,and control groups.The tumor volume and weight and survival of mice were examined in different groups.Results: The titer of anti-EGFR in mice vaccinated with pVAX1/cEGFR-rFc plasmid was 1∶1 000.The proliferation of Lewis cells in anti-EGFR combined with gefitinib was significantly inhibited compared with those in anti-EGFR and gefitinib groups(P
Résumé
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.
Résumé
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.