白喉毒素DAB_(389)-αMSH重组毒素的构建及表达  

Construction and Expression of Recombinant Toxin DAB_(389)-αMSH

作　　者：李泽鸿[1,2] 刘文涛[1] 李昌昊[1] 张国利[2] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《中国兽药杂志》2010年第9期7-9,37,共4页Chinese Journal of Veterinary Drug

基　　金：长春市科技局资助项目(编号:09YJ47)

摘　　要：利用含有白喉毒素N端序列的基因作上游引物、含有αMSH全序列作下游引物,以pET28a/DAB389-EGF为模板,PCR扩增DAB389-αMSH基因片段,用限制性内切酶EcoR I和NcoI酶切,并插入原核表达载体pET28a的相应位点,构建了重组表达载体pET28a/DAB389-αMSH,在大肠杆菌中表达重组融合蛋白DAB389-αMSH,转化菌经1 mmol/L IPTG、30℃诱导5 h,用SDS-PAGE和Western blot鉴定表达的重组毒素。结果表明扩增的片段与理论值一致,重组质粒的DNA序列分析正确;SDS-PAGE表明重组毒素相对分子量为43.76 KD,且表达量达菌体总蛋白量的31.6%。Western blot分析显示,重组毒素能特异性地与抗白喉抗体结合,表明已成功构建表达了重组DAB389-αMSH的工程菌株,并获得表达蛋白。The DAB389-αMSH gene fragment was amplified from plasmid pET28a/DAB389-EGF by PCR with the upper primer containing diphtheria toxin complementary sequences,and the down primer containing αMSH complementary sequences.The PCR product was digested by the restriction enzymes Nco I and EcoR I,and cloned into Nco I/EcoR I site of expression vector pET28a（＋）,resulting in plasmid pET28a/DAB389-αMSH.The recombinant plasmid was transformed into E.coli BL21 and induced by 1mmol/L IPTG at 30℃ for 5 hours,It was demonstrated by SDS-PAGE and Gel scanned analysis that the recombinant toxin was expressed soluble,and the level of expression reached 31.6% of the total protein,the relative molecular weight was 43.76 KD.So the engineering bacteria of recombinant plasmid DAB389-αMSH with biologic activity was successfully constructed.

关 键 词：DAB389-αMSH 重组毒素 构建 表达 

分 类 号：S852.44[农业科学—基础兽医学]