一种带有组氨酸标签的抗糖尿病疫苗的构建、表达、纯化及鉴定  

Construction,Expression,Purification and Identification of a Protein Vaccine with HIS Tag

作　　者：杨雪[1] 刘晓锐[1] 邢芸[1] 徐茂磊[1] 金亮[1] 刘景晶[1] 吴洁[1] 

机构地区：[1]中国药科大学 生命科学与技术学院 微基因实验室,江苏南京210009

出　　处：《药物生物技术》2013年第3期189-193,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.81172973;31270985);中央高校基本科研业务费研究生专项(No.JKY2011076)

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。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.

关 键 词：疫苗 Ⅰ型糖尿病 大肠杆菌 重组表达 His-Hsp65-6p277融合蛋白 分离纯化 组氨酸标签 

分 类 号：I1587.1[文学—世界文学]