DAB_(389)(Gly_4Ser)_2αMSH重组融合蛋白的构建及表达  

Construction and expression of recombinant Toxin DAB_(389)(Gly_4Ser)_2αMSH

作　　者：李泽鸿[1] 张国利[1] 岳玉环[1] 朱平[1] 

机构地区：[1]军事医学科学院军事兽医研究所

出　　处：《中国兽医学报》2007年第4期477-480,共4页Chinese Journal of Veterinary Science

基　　金：国家科技部科技型中小企业创新基金(03C26212200869)

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全序列和(Gly4Ser)2互补序列的基因作下游引物,以pET28a/DAB389(Gly4Ser)2EFG为模板,PCR扩增DAB389(Gly4Ser)2αMSH基因片段,用限制性内切酶EcoR和Nco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(Gly4Ser)2αMSH,在大肠杆菌中表达重组融合蛋白DAB389(Gly4Ser)2αMSH,转化菌经IPTG、30℃诱导5h,用SDS-PAGE和Western blot鉴定表达的重组蛋白。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,重组蛋白相对分子质量为45870,且表达量达菌体总蛋白量的29.2%。Western blot分析显示,重组蛋白能特异地与抗白喉抗体结合。成功构建表达了重组DAB389(Gly4Ser)2αMSH的工程菌株,表达产物具有生物学活性。The DAB389 （Gly4Ser）2αMSH gene fragment was amplified from plasmid pET28a/DAB389 （Gly4Ser）2EGF by PCR with the upper primer containing diphtheria toxin complementary sequences,and the down primer containing αMSH and （Gly4Ser）2 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 （Gly4Ser）2αMSH. The recombinant plasmid was then transformed into E. coli BL21 （λDE3） and induced by IPTG 30 ℃ 5 h. It was demonstrated by SDS-PAGE and Gel scanned analysis that the recombinant toxin was expressed to be soluble and reached 29.2% of the total protein. So the engineering bacteria of recombinant plasmid DAB389 （Gly4Ser）2αMSH with biologic activity was successfully constructed.

关 键 词：DAB389(Gly4Ser)2αMSH 重组蛋白 构建 表达 

分 类 号：S852.61[农业科学—基础兽医学]