柱上切除GST标签制备幽门螺杆菌Lpp20蛋白  

Preparation of Lpp20-GST fusion protein of Helicobacter pylori with thrombin-cleavage of GST tag on column

作　　者：姜茵[1] 王小平[1] 何殿殿[1] 李妍[1] 

机构地区：[1]南方医科大学生物技术学院生物治疗研究所,广东广州510515

出　　处：《中国公共卫生》2012年第7期987-989,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(31070119);广东省自然科学基金(S2011010006070)

摘　　要：目的表达幽门螺杆菌Lpp20-GST融合蛋白,获取切除GST标签的重组蛋白。方法采用异丙基硫代半乳糖苷(IPTG)诱导重组表达质粒Lpp20/pGEX-4T-1在大肠埃希菌BL21(DE3)中表达,收集菌体并采用反复冻融、溶菌酶裂解及超声破菌3种细胞破碎方法,表达产物在谷胱甘肽琼脂糖树脂4B柱上纯化,利用凝血酶切除GST标签,用鼠抗Lpp20单克隆抗体进行纯化产物western blot鉴定。结果高效表达出Lpp20-GST融合蛋白,相对分子质量约为4.5 kDa,产物以部分可溶性形式表达,凝血酶成功切除GST标签,纯化产物能被鼠抗Lpp20单克隆抗体识别。结论凝血酶柱上切除GST标签获得目的蛋白。Objective To express Lpp20-GST fusion protein with glutathione-S-transferase（GST）fusion gene expression system and the cleavage of GST-tag on glutathione sepharose 4B column using thrombin.Methods The recombinant expression plasmid Lpp20/pGEX4T-1 was induced in E.coli BL21（DE3）by isoproythio β D-galacoside（IPTG）and the bacterial sediment was lysed by repeating freezing and thawing,lysozyme lysis,and ultrasonic wave.The soluble supernatant was loaded on glutathione sepharose 4B column and GST-tag was cleavaged on column using thrombin.Purified Lpp20 was proved by mouse anti-Lpp20 monoclonal antibody（mAb）with western blot.Results The fusion protein Lpp20-GST was partly expressed in soluble form with relative molecular mass of 45 kDa.Thrombin cleavaged GST-tag on column and purified Lpp20 was recognized by mouse anti-Lpp20 mAb.Conclusion Target protein can be obtained by thrombin-cleavage of GST-tag on column.

关 键 词：幽门螺杆菌 LPP20 凝血酶 纯化 GST标签 

分 类 号：Q786[生物学—分子生物学]