牛源大肠杆菌F41菌毛蛋白的原核表达和多克隆抗体的制备  

Prokaryotic expression of F41 pilus protein of enterotoxigenic Escherichia coli from bovine origin and preparation of its polyclonal antibody

作　　者：倪宏波[1] 郎秀艳[1] 邵光喜[1] 周玉龙[1] 焉秋龙[1] 刘银冰[1] 宫大庆[1] 王春仁[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2013年第1期48-50,55,共4页Chinese Journal of Biologicals

基　　金：黑龙江省高校科技创新团队建设计划(2010td05);黑龙江八一农垦大学博士后基金(农业部农产品加工质量监督检验测试中心)

摘　　要：目的原核表达及纯化牛源产肠毒素性大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)F41菌毛蛋白,并制备F41菌毛蛋白的多克隆抗体。方法以ETEC基因组为模板,采用PCR法扩增F41菌毛基因,克隆入表达载体pQE-30,转化大肠杆菌XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析,表达产物经切胶纯化,免疫新西兰大白兔,制备多克隆抗体,采用间接ELISA法测定抗血清效价,Western blot分析F41菌毛蛋白与多抗的反应原性。结果重组表达质粒pQE-30-F41经双酶切及测序证实构建正确;F41菌毛重组蛋白以包涵体形式表达,相对分子质量约为32 000;重组蛋白的表达量约占菌体总蛋白的35%,纯化的重组蛋白纯度可达92%;制备的抗血清效价达1∶2.56×106以上,Western blot结果表明F41菌毛蛋白与制备的多抗具有良好的反应原性。结论已成功原核表达并纯化了F41菌毛重组蛋白,且制备了高效价的多克隆抗体,为单克隆抗体制备及其免疫检测方法的建立奠定了基础。Objective To express F41 pilus protein of enterotoxigenic Escherichia coli（ETEC） from bovine origin in prokaryotic cells and prepare its polyclonal antibody.Methods F41 pilus gene was amplified by PCR using the genome of ETEC as a template,and inserted into prokaryotic expression vector pQE30.The constructed recombinant plasmid was transformed to E.coli XL1-Blue for expression under induction of IPTG.The expressed protein was identified by SDS-PAGE,purified by cutting the gel and inoculated to New Zealand rabbits.The prepared polyclonal antibody was determined for titer by indirect ELISA and for reactogenicity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pQE-30-F41 was constructed correctly.The expressed F41 pilus protein existed in a form of inclusion body,with a relative molecular mass of about 32 000,which contained about 35% of total somatic protein and reached a purity of 92% after purification.The prepared antiserum reached a titer of more than 1 ∶ 2.56 × 106,and showed good reactogenicity as proved by Western blot.Conclusion Recombinant F41 pilus protein was successfully expressed in prokaryotic cells and purified,and high titer polyclonal antibody was prepared,which laid a foundation of preparation of monoclonal antibody and development of immunological method for determination of E.coli F41 pilus protein.

关 键 词：产毒素性大肠杆菌 F41菌毛 原核细胞 基因表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]