铜绿假单胞菌外毒素A基因突变体的构建及其在大肠埃希菌中的表达  

Construction of Pseudomonas aeruginosa exotoxin A gene mutant and its expression in E. coli

作　　者：姚立红[1] 徐一[1,2] 陈爱珺[1] 郭建强[1] 薄洪[1] 尉玉杰 张智清[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京102206 [2]中国动物疫病预防控制中心,北京100125 [3]莱阳市畜牧兽医局,山东莱阳265200

出　　处：《中国生物制品学杂志》2014年第1期23-27,共5页Chinese Journal of Biologicals

基　　金：艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2009ZX10004-710)

摘　　要：目的将铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A,PEA)基因突变为无毒性的ntPE基因,并在大肠埃希菌中进行表达。方法利用Primer Premier 5.0软件设计PEA基因引物及PEA基因第553位氨基酸缺失的突变引物,以铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)基因组DNA为模板,PCR扩增PEA基因,插入pGEM-T载体中,构建重组克隆质粒pGEM-PEA,以其为模板,利用突变引物,扩增ntPE基因(无毒性PEA),插入pET-30a载体中,构建原核表达质粒pET-ntPE,转化大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Western blot分析。结果测序结果证明已成功获得突变基因;原核表达质粒经双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约69 000,主要以包涵体形式存在,表达量占菌体总蛋白的30%,可被小鼠抗PEA单克隆抗体特异性识别,具有较好的抗原性。结论已成功获得无毒性的PEA突变基因,为构建以PEA为蛋白佐剂的融合蛋白疫苗奠定了基础。Objective To mutate Pseudomonas aeruginasa exotoxin A （PEA） gene into non-toxic mutant ntPE gene and express in E. coll. Methods The primers for PEA gene and the mutant in which the amino acid at site 553 was deleted were designed by using Primer Premier 5. 0 software, with which PEA gene was amplified by PCR using the genomic DNA of P. aeruginosa as a template and inserted into pGEM-T vector. The ntPE gene was amplified with mutant primer using the constructed recombinant cloning plasmid pGEM-PEA as a template and inserted into pET-30a. The constructed recombinant plasmid pET-ntPE was transformed to E. coli BL21 （DE3） for expression under induction of IFFG, and the expressed product was analyzed by SDS-PAGE and Western blot. Results Sequencing result proved that the mutant gene was successfully obtained. Restriction analysis proved that prokaryotic expression vector was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 69 000, mainly existed in a form of inclusion body, contained about 30% of total somatic protein, was recognized specifically by mouse anti-PEA monoclonal antibody, and showed good antigenicity. Conclusion Non-toxic PEA gene mutant was successfully obtained, which laid a foundation of construction of fusion protein vaccine using PEA as a protein adjuvant.

关 键 词：铜绿假单胞菌 外毒素 基因 突变体 原核细胞 基因表达 

分 类 号：R378.991[医药卫生—病原生物学] Q786[医药卫生—基础医学]