Stx1A-LHRH融合毒素基因的构建及高效表达  

Construction and expression of Stx1A-LHRH fusion toxin

作　　者：王文东[1,2] 刘军[2] 孙洋[2] 祝令伟[2] 郭学军[2] 周博[2] 冯书章[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2010年第5期620-623,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30270985)

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。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.

关 键 词：志贺毒素 LHRH 融合毒素 高效表达 

分 类 号：S852.61[农业科学—基础兽医学]