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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
引用本文:杨洁,王华倩,熊祺琰,黄文睿,李泰明,吴洁,曹荣月,刘景晶.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[J].药物生物技术,2012(2):113-116.
作者姓名:杨洁  王华倩  熊祺琰  黄文睿  李泰明  吴洁  曹荣月  刘景晶
作者单位:中国药科大学生命科学与技术学院微基因药物实验室;徐州医学院肿瘤生物治疗实验室;广东工业大学轻工化工学院;江苏省农业科学院兽医研究所
基金项目: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。

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

Expression and Identification of the Fusion Protein of CTB-Ins B
YANG Jie,WANG Hua-qian,XIONG Qi-yan,HUANG Wen-rui,LI Tai-ming,WU Jie,CAO Rong-yue,LIU Jing-jing.Expression and Identification of the Fusion Protein of CTB-Ins B[J].Pharmaceutical Biotechnology,2012(2):113-116.
Authors:YANG Jie  WANG Hua-qian  XIONG Qi-yan  HUANG Wen-rui  LI Tai-ming  WU Jie  CAO Rong-yue  LIU Jing-jing
Institution:1(1.Laboratory of Minigene Pharmacy,School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,China;2.Laboratory of Biological Cancer Therapy,Xuzhou Medical College,Xuzhou 221002,China;3.Department of Pharmacy Engineering,Faculty of Chemical Engineering and Light Industry,Guangdong University of Technology,Guangzhou 510006,China;4.Institute of Veterinary Medicine Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)
Abstract: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.
Keywords:Cholera toxin B subunit  Ins B(9-23)  Inclusion body  Pentamer  Monosialoganglioside
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