B型肉毒毒素轻链蛋白的原核表达及纯化  

Prokaryotic expression and purification of botulinus neurotoxin serotype B light chain protein

作　　者：刘雪[1,2,3] 张国利[2,3] 陈萍[1] 田园[2,3] 岳玉环[2,3] 吴广谋[2,3] 张亮[1,2] 冯越[1,2] 朱平[2,3] 

机构地区：[1]吉林农业大学食品科学与工程学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林省人兽共患病预防与控制重点实验室省部共建重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2013年第5期630-633,共4页Chinese Journal of Biologicals

摘　　要：目的克隆B型肉毒毒素轻链Bont-B基因,使其在大肠杆菌内表达,并对表达的重组蛋白进行纯化。方法设计并人工合成Bont-B基因,克隆入表达载体pMD19-T中,构建质粒pMD19-T-Bont-B,经酶切后与pET-28a载体连接,构建重组表达质粒pET-28a-Bont-B,将重组表达质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot分析后,经金属螯合层析Cu2+柱进行纯化,纯化产物经SDS-PAGE分析纯度。结果重组表达质粒pET-28a-Bont-B经PCR、双酶切及测序鉴定,证明构建正确;表达的重组蛋白相对分子质量约50 000,主要以可溶性形式表达,表达量约占菌体总蛋白的6%;纯化后的重组蛋白纯度可达90%以上。结论已成功克隆并在大肠杆菌BL21(DE3)中原核表达了Bont-B轻链基因,为制备抗Bont-B轻链单克隆抗体及研究肉毒中毒机制和治疗奠定了基础。Objective To clone botulinus neurotoxins serotype B（Bont-B）light chain gene,express in E.coli and purify the expressed recombinant protein.Methods Bont-B gene was designed and synthesized,and cloned into vector pMD19T.The constructed recombinant plasmid pMD19-T-Bont-B was digested with restriction enzyme and inserted into vector pET-28a.The constructed recombinant plasmid pET-28a-Bont-B was transformed to competent E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by copper ion metal chelating chromatography,and analyzed for purity by SDS-PAGE.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-28a-Bont-B was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 50 000,mainly existed in a soluble form,contained 6% of total somatic protein and reached a purity of more than 90% after purification.Conclusion Bont-B light chain gene was successfully cloned and expressed in E.coli BL21（DE3）,which laid a foundation of preparation of monoclonal antibody against Bont-B light chain as well as study on mechanism and treatment of botulism.

关 键 词：B型肉毒毒素轻链 原核细胞 基因表达 纯化 

分 类 号：R378.83[医药卫生—病原生物学] Q786[医药卫生—基础医学]