| Antiangiogenic therapy represents a new strategy for tumor therapy and vascular endothelial growth factor (VEGF) appears to be the most important regulator of angiogenesis. To explore the anti-antitumor of the antibody against VEGF, the quail VEGF (qVEGF) cDNA was amplified from embryo tissue using RT-PCR and cloned into pMD18-T vector for sequence analysis. The cDNA was then subcloned into pGEX-4T-2vector in frame with GST tag. The recombinant pGEX4T2-qVEGF was transformed into E.coil BL21(DE3) and expression of GST fusion protein was induced by IPTG, which was purified by Ni-NTA column. After dialyzation and renaturation, soluble qVEGF-GST was obtained and tested by Western blotting. C57BL/6J mice are randomly divided into3groups of ten:qVEGF-GST fusion protein group, qVEGF protein group and normal saline (NS) control group. Each mouse was hypodermically injected with100μg of recombinant antigen mixed with Freund’s adjuvant and boosted with on weeks2and3, followed by inoculation with Lewis lung carcinoma cells. Tumor growth and mouse’s physical status were observed during immunization. Tumor weight and serum anti-VEGF antibody level were measured by enzyme-linked immuno-sorbent assay (ELISA).The results showed that an expected555bp qVEGF cDNA was amplified and the recombinant vector pGEX4T2-qVEGF was constructed successfully. After IPTG induction, an expected48kDa fusion protein was detected in the recombinant E.coli lysate by SDS-PAGE. The purity of the affinity-purified fusion protein was large than81%and the recovery of the soluble protein was about59%, which was recognized by the antiserum in Western-blot assay. Animal experimental results showed that the tumor weight of qVEGF-GST group, qVEGF group and NS group was2.29±0.429,2.95±0.510and3.37±0.021g, respectively. Serum anti-VEGF antibody level of qVEGF-GST group was1:2000.These results indicate that qVEGF-GST and qVEGF are immunogenic and that their antibodies can inhibit the growth of Lewis lung cancer cells in mice. |
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