重组非洲爪蟾和小鼠血管内皮细胞生长因子的原核表达、纯化及鉴定

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pET—xVEGF和pET—mVEGF。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BL21(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pET—xVEGF和pET—mVEGF、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xVEGF和mVEGF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xVEGF和mVEGF。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。

Prokaryotic Expression, Purification and Identification of Recombinant Xenopus Laevis and Mouse Vascular Endothelial Growth Factors

OBJECTIVE: To perform prokaryotic expression, purification and identification of Xenopus laevis and mouse vascular endothelial growth factors (VEGF). METHODS: The prokaryotic expression vectors were constructed by restriction endonuclease digestion and ligation, then the recombinant vector pET-xVEGF and the control vector pET-mVEGF were identified by restriction enzymatic digestion and DNA sequencing. The confirmed vectors were transformed into Escherichia coli. BL21 (DE3) and recombinant protein expression was induced by Isopropy-beta-D-thiogalactoside. The recombinant xVEGF and mVEGF were obtained and purified by Ni-NTA affinity chromatography under denature conditions and enterokinase specific digestion, respectively. Finally, the purified proteins' molecular weights and specificity were detected by SDS-PAGE and western blot analysis. RESULTS: Two new recombinant expression vectors, pET-xVEGF and pET-mVEGF, were constructed successfully. The xVEGF and mVEGF fusion proteins were expressed in E. coli. BL21 (DE3) stably, and the molecular weights of the purified xVEGF and mVEGF were identical to the expected values. The purity of final products reached a level higher than 95%. In addition, these two purified proteins could react with a specific antibody against mouse VEGF as expected. CONCLUSION: Recombinant Xenopus laevis VEGF and its control mouse VEGF protein may provide tools for further study of anti-tumor active immunity with this xenogeneic protein vaccine in mouse tumor models.