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利用组氨酸标签系统提高抗糖尿病疫苗HSP65-6P277融合蛋白的产量,简化纯化工艺。通过PCR方法,在融合蛋白HSP65-6P277的N端添加6个组氨酸标签后,定向克隆到pET28a原核表达载体中成功构建了组氨酸标签融合表达载体pET28a-HIS-HSP65-6P277。通过乳糖诱导,融合蛋白HIS-HSP65-6P277在大肠杆菌BL21(DE3)宿主中以可溶形式表达,镍柱亲和层析纯化后,经SDS-PAGE和Western blotting鉴定表明表达产物是约70 k具有6个组氨酸标签肽的融合蛋白;分离纯化融合蛋白的时间从原来的15～20 d缩短为2～3 d,产量提高到56 mg/L。组氨酸标签系统可以较好的简化抗糖尿病疫苗HSP65-6P277融合蛋白的纯化工艺,提高产量。
Using histidine tagging system to increase the yield of anti-diabetic vaccine HSP65-6P277 fusion protein, simplifying the purification process. Six histidine tag was added to the N terminus of fusion protein HSP65-6P277 by PCR and cloned into pET28a prokaryotic expression vector to successfully construct histidine tag fusion expression vector pET28a-HIS-HSP65-6P277. Through lactose induction, the fusion protein HIS-HSP65-6P277 was expressed in soluble form in E. coli BL21 (DE3) host. The purified product was purified by nickel affinity chromatography and identified by SDS-PAGE and Western blotting. The expressed product was about 70 k The fusion protein with 6 histidine tag peptide was obtained. The time for separating and purifying the fusion protein was shortened from 15 to 20 days to 2 to 3 days and the yield increased to 56 mg / L. Histidine tagging system can be better to simplify the anti-diabetic vaccine HSP65-6P277 fusion protein purification process, increase production.