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目的 构建非洲爪蟾和小鼠血管内皮细胞生长因子重组表达载体,进行蛋白质原核表达,并对表达产物进行分离、纯化和鉴定。方法 首先通过限制性核酸内切酶酶切及连接反应,构建非洲爪蟾和小鼠血管内皮细胞生长因子的原核表达载体p ET- x VEGF和p ET- m VEGF。筛选的重组质粒经酶切和DNA序列测定等证实正确性后,再转染感受态大肠杆菌BL 2 1(DE3) ,经IPTG诱导蛋白质表达。表达产物经Ni- NTA亲合层析及肠激酶特异性酶切进行分离纯化,最后用SDS- PAGE和蛋白免疫印迹法进行鉴定。结果 限制性核酸内切酶酶切和DNA序列测定等显示目的基因片段和阅读框架正确无误,表明非洲爪蟾和小鼠血管内皮细胞生长因子的原核表达载体p ET-x VEGF和p ET- m VEGF构建成功。重组蛋白质在大肠杆菌中获得稳定表达,分离纯化的表达产物x VEGF和m VEGF 的纯度达到95 %以上,其相对分子质量与预期值一致。抗小鼠VEGF抗体可特异性识别x VEGF和m VEGF。结论 重组非洲爪蟾和小鼠血管内皮细胞生长因子的原核表达、分离纯化及鉴定成功,为进一步研究异种VEGF蛋白质疫苗抗小鼠肿瘤模型奠定了基础。
Objective To construct recombinant expression vector of vascular endothelial growth factor (VEGF) in Xenopus laevis and mice and prokaryotic expression of the protein, and to isolate, purify and identify the expressed product. Methods The prokaryotic expression vectors p ET-x VEGF and p ET-m VEGF of Xenopus laevis and mouse vascular endothelial growth factor were constructed by restriction endonuclease digestion and ligation. The recombinant plasmids were confirmed by enzyme digestion and DNA sequencing, then transfected into competent E. coli BL21 (DE3) and induced by IPTG. The expressed product was isolated and purified by Ni-NTA affinity chromatography and enterokinase-specific digestion, and finally identified by SDS-PAGE and Western blotting. Results Restriction endonuclease digestion and DNA sequencing showed that the target gene fragment and reading frame were correct, indicating that the prokaryotic expression vectors of VEGF and p ET-m of Xenopus laevis and mouse vascular endothelial growth factor VEGF was successfully constructed. The recombinant protein was stably expressed in E.coli. The purity of the expressed products of x VEGF and mVEGF reached more than 95%, and the relative molecular mass was consistent with the expected value. Anti-mouse VEGF antibodies specifically recognize x VEGF and m VEGF. Conclusion The prokaryotic expression, isolation, purification and identification of vascular endothelial growth factor in Xenopus laevis and mice are successful, which lays the foundation for further study on anti-mouse tumor model induced by VEGF protein vaccine.