中文摘要：

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B121（DE3）宿主中以可溶形式表达，镍柱亲和层析纯化后，经SDS-PAGE和Westernblotting鉴定表明表达产物是约70k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15～20d缩短为2—3d，产量提高到56mg／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。

英文摘要：

The HIS tag purification system is used to improve the production of recombinant protein and to simplify production tech- nique. 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 BI21 （DE3）. The recombinant HIS-HSP65-6P277 was purified only by HIS Bind Resin affinity chromatography. The eleetrophoretic 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 euhure. 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.