霍乱毒素B亚单位与谷氨酸脱羧酶抗原表位肽融合蛋白的表达及鉴定  

Expression and Identification of the Fusion Protein of the CTB-GAD Epitope

作　　者：刘利成[1] 方宏清[1] 彭远义[2] 李彦英[1] 张艳红[1] 刘传喧[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]西南大学动物科技学院,重庆400716

出　　处：《生物技术通讯》2006年第3期318-321,共4页Letters in Biotechnology

摘　　要：目的:在大肠杆菌中表达霍乱毒素B单位(CTB)与谷氨酸脱羧酶(GAD)抗原表位肽段(531～545)的融合蛋白。方法:通过PCR技术将CTB基因与GAD基因融合在一起,插入pET22b载体,转化大肠杆菌BL21(DE3)后经诱导获得融合基因的表达;对表达产物进行包涵体复性后,纯化得到融合蛋白分子;对该融合蛋白分子进行了Western印迹和GM1-ELISA分析。结果:表达的融合蛋白的相对分子质量约为14000,Western印迹表明该融合蛋白具有霍乱毒素抗原性;GM1-ELISA实验表明该融合蛋白能够特异性地结合神经节苷酯GM1,表明该蛋白具有与CTB相似的五聚体结构。结论:融合蛋白CTB-GAD的成功表达,为后续动物实验提供了充足的抗原。Objective： To express the fusion protein of cholera toxin B subunit（CTB） and the epitope peptide（531-545） of glutamate acid decarboxylase（GAD） in E.coli. Method： The gene of GAD epitope was fused to the gene of CTB by PCR, then the fused gene was subcloned into pET22b vector to construct recombinant expression plasmid and transformed into E.coli BL21（DE3）. After induced by IPTG, the fusion protein was expressed and accumulated as inclusion bodies, The insoluble fusion protein was denatured and refolded, then the protein was purified. Finally, the protein was examined by Western-blotting and GM1-ELISA. Results： The relatively molecular weight of fusion protein was about 14 kD. The results of Western-blotting indicated that the fusion protein could bind specially to the anti-CTB serum. The fusion protein retain GM1-ganglioside binding affinity showed that it exits as a pentamer as the pentameric CTB configuration can bind to the receptor. Conculsion： This study had successfully expressed the fusion protein CTB-GAD and provided abundant antigen for the animal experiments.

关 键 词：霍乱毒素B亚单位 谷氨酸脱羧酶 糖尿病 口服耐受 

分 类 号：Q786[生物学—分子生物学]