【摘要】 目的研究血管内皮生长因子(VEGF)与小分子泛素样修饰蛋白(SUMO)融合并转入大肠杆菌BL21中进行VEGF的可溶性高效表达。方法采用PCR扩增方法,获得编码VEGF与SUMO融合蛋白的DNA片段并插入表达载体pET3c中,构建重组表达载体pET3c-SUMO-VEGF,转化到大肠杆菌BL21中,获得重组菌株BL21/pET3c-SUMO-VEGF。以IPTG为诱导剂,对阳性重组菌株的诱导表达参数如诱导剂浓度、诱导温度、诱导时间等进行优化。结果融合蛋白的最佳表达条件为当OD600约0.6时,加入0.5mMIPTG,20℃诱导24h,获得了纯度达70%以上的SUMO-VEGF。结论通过融合表达,解决VEGF独立表达时表达量低和可溶性差的问题,使其商业化的步伐进一步加快。更多还原

【Abstract】 Objective To obtain a great quantity of soluble vascula epidermal growth factor (VEGF) by fusion of VEGF gene with small ubiquitin-related modifier (SUMO) gene in Escherichia coli BL21 strain for high expression. Methods The SUMO gene and VEGF gene were fused by PCR. The fused fragment was cloned into the expression vector pET3c. The expression of SUMO-hEGF in Ogarami (DE3)/pET3c-SUMO- VEGF was induced by IPTG. Results The optimal soluble expression condition was OD600 get to 0.6 and then induced by 0.5mM IPTG at 20℃ for 24h. The result of SDS-PAGE electrophoresis showed that the purity of SUMO-hEGF was 70%. Conclusion The problem of low expression and insoluble of VEGF have been solved and this will offer experience for high production of VEGF.更多还原