【摘要】 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。更多还原

【Abstract】 The HIS tag purification system is used to improve the production of recombinant protein and to simplify production technique.HSP65-6P277 was a kind of recombinant protein which prevented the type 1 diabetes.Here,a study with a purpose of high level expression and convenient procedure of this vaccine in Escherichia coli was presented.The 6 × His-tag was artificially added at HSP65-6P277 N-terminal using PCR(polymerase chain reaction) amplification technique.Then this gene cassette was cloned into pET28a and the HIS affinity tagged HSP65-6P277 fusion protein expression plasmid pET28a-HIS-HSP65-6P277 was successfully constructed.By lactose induction,the fusion protein HIS-HSP65-6P277 was expressed as soluble form in the E.coli BL21(DE3).The recombinant HIS-HSP65-6P277 was purified only by HIS Bind Resin affinity chromatography.The electrophoretic behavior in SDS-PAGE and the result shown in the Weston blotting illustrated that the purified HIS-HSP65-6P277 was a HIS tag fusion protein with molecular weight of 70 k.As a result,the time period of the whole production technique was reduced from 15 ～ 20 days to 2 ～ 3 days.And the production was increased from 12 mg to 56 mg per liter culture.In conclusion,the HIS tag purification system provides a novel approach to simplifying the production technique and amplifying production of a vaccine against type 1 diabetes.更多还原