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当前位置: 首页 > 文章 > Stx1A-LHRH融合毒素基因的构建及高效表达 中国兽医学报 2010,30 (5) 620-623
Position: Home > Articles > Construction and expression of Stx1A-LHRH fusion toxin Chinese Journal of Veterinary Science 2010,30 (5) 620-623

Stx1A-LHRH融合毒素基因的构建及高效表达

作  者：
王文东;刘军;孙洋;祝令伟;郭学军;周博;冯书章
单  位：
吉林大学畜牧兽医学院;军事医学科学院军事兽医研究所
关键词：
志贺毒素;LHRH;融合毒素;高效表达
摘  要：
通过PCR方法,从肠出血性大肠杆菌O157:H7的基因组DNA中扩增出Stx1A基因序列,并将之与编码LHRH的基因序列连接起来,构建编码Stx1A-LHRH的重组融合基因片段,并定向克隆到表达质粒pET28a的NcoⅠ和EcoRⅠ位点之间,构建重组表达质粒pET28a::stx1A-LHRH,将重组质粒转化到宿主菌BL21(DE3)中。对重组菌株用IPTG进行诱导表达。SDS-PAGE电泳检测结果表明重组菌株表达出了23700的目的融合蛋白Stx1A-LHRH,目的蛋白为包涵体表达,经薄层扫描分析表明表达量约占菌体总蛋白的37.6%。为了将来更好的进行目的蛋白的功能研究,本研究再将融合基因片段克隆到表达质粒pMAL-p2x中实现了重组蛋白的可溶性表达,表达的重组蛋白经amylose亲和层析柱一步纯化后纯度可达93.4%,为下一步进行重组蛋白的活性分析及其生物学作用研究奠定了基础。
译  名：
Construction and expression of Stx1A-LHRH fusion toxin
作  者：
WANG Wen-dong1,2,LIU Jun2,SUN Yang2,ZHU Ling-wei2,GUO Xue-jun2,ZHOU Bo2,FENG Shu-zhang1,2 (1.College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China;2.Institute of Veterinary Science,The Academy of Military Medical Science,Changchun 130062,China)
关键词：
shiga toxin;LHRH;fusion toxin;expression
摘  要：
To construct the recombinant toxin Stx1A-LHRH,the fragment of stx1A was amplified from genome of EHEC O157:H7 by PCR.The PCR product was fused to structural sequence of LHRH gene.The fusion gene of stx1A-LHRH was digested with restriction endonuclease NcoⅠ/EcoRⅠ,and then inserted into expression vector pET28a.The resultant recombinant plasmid pET28a::stx1A-LHRH was transformed into host E.coli BL21(DE3).IPTG was used to induce the expression of the target protein and SDS-PAGE was used to analyze the expression of fusion protein.The fusion protein Stx1A-LHRH was high level expressed in inclusion bodies of E.coli and covered about 37.6% of total bacterial proteins.To further study the function of recombinant toxin Stx1A-LHRH,the fusion gene was subcloned into vector pMAL-p2x,and the target protein was expressed in soluble form.The purity of target protein is up to 93.4% after amylose affinity chromatography purification.This study established a foundation for the further study of recombinant toxin Stx1A-LHRH.

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