霍乱毒素B亚单位与胰岛素B链结构类似多肽融合蛋白的制备  

Expression and Identification of the Fusion Protein of CTB-Ins B

作　　者：杨洁[1,2] 王华倩[1,3] 熊祺琰[1,4] 黄文睿[1] 李泰明[1] 吴洁[1] 曹荣月[1] 刘景晶[1] 

机构地区：[1]中国药科大学生命科学与技术学院微基因药物实验室,江苏南京210009 [2]徐州医学院肿瘤生物治疗实验室,江苏徐州221002 [3]广东工业大学轻工化工学院,广东广州510006 [4]江苏省农业科学院兽医研究所,江苏南京210014

出　　处：《药物生物技术》2012年第2期113-116,共4页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.30872393;No.30672464;No.30701023)

摘　　要：为了在大肠杆菌中表达霍乱毒素B亚单位(Cholera toxin B subunit,CTB)与胰岛素B链(9-23)肽段结构类似多肽(Ins B(9-23))的融合蛋白,使用基因工程方法构建了CTB与Ins B(9-23)的融合基因CTB-Ins B-X若干,并将融合基因克隆到大肠杆菌表达载体pET28a(+)中,获得的重组质粒转化大肠杆菌BL21(DE3)。重组菌株经乳糖诱导后,其表达产物经过12%SDS-PAGE分析表明该菌株可以包含体形式表达融合蛋白,并制备得到纯度较高的重组蛋白。该重组蛋白的包含体经过变性、复性后,可以在体外自组装成五聚体结构。GM1-ELISA分析结果表明,重组蛋白在体外可以与神经节苷脂GM1(monosialoganglioside)特异性结合,表明该融合蛋白保持了CTB形成五聚体的生物活性。To express the fusion protein of cholera toxin B subunit（CTB） and insulin B peptide（9-23） in E.coli,the recombinant expression plasmid pET28a-CTB-Ins B was constructed in which Ins B（9-23） genes were fused to the 3′end of CTB gene and cloned into pET28a（＋） to obtain a prokaryotic expression vector pET28a-CTB-Ins B.Subsequently the recombinant plasmid was transformed into E.coli strain BL21（DE3）.After being induced with 5 mmol/L lactose for 5 h,the expression products were analyzed by sodium dodecyl sulphate-polyacrylamide gel（12%） electrophoresis（SDA-PAGE）,and the results indicated that the recombinant protein CTB-Ins B-X was expressed and accumulated as inclusion bodies.Purificated recombinant proteins were obtained after washing,renaturing and purifying via DEAE-52 cellulose and sephadex G-50 chromatography.The proteins were further analyzed for GM1 binding activity in a GM1-ELISA.The results of the GM1 binding assay showed（1）、（2）,that the recombinant proteins could bind to monosialoganglioside specifically,although not as strong as cholera toxin B subunit,that they possessed biological activity in vitro.

关 键 词：霍乱毒素B亚单位 胰岛素B链(9-23)肽段 包含体 五聚体 神经节苷脂GM1 

分 类 号：R392-33[医药卫生—免疫学]