巴氏杆菌toxA基因的原核表达及生物学特性研究  

Expression and biological characteristic studies of toxA gene of Pasteurella multocida

作　　者：张健淞 夏雨婷[1] 沈懿娟 梁中洋 李玉峰[1] ZHANG Jian-song;XIA Yu-ting;SHEN Yi-juan;LIANG Zhong-yang;LI Yu-feng(College of Veterinary Medicine,Nanjing Agricultural University/Key Laboratory of Bacteriology,Ministry of Agriculture,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/农业部动物细菌学重点开放实验室

出　　处：《中国兽医科学》2018年第8期991-997,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31672565);国家重点研发项目(2016YFD0500701-3);江苏省优势学科项目(PAPD)

摘　　要：为了探讨多杀性巴氏杆菌皮肤坏死毒素（PMT）对真核细胞的致病机制,将编码PMT的tox A基因以及其N端tox N（1~1461 bp）和C端tox C（2 958~3 858 bp）分别克隆到原核表达载体p ET-32a中进行蛋白表达。3个重组蛋白在大肠杆菌中均以可溶性形式表达,其分子质量大小分别为176 ku、88 ku和67 ku,均能与His单克隆抗体发生特异性免疫反应。体外细胞毒性试验和豚鼠皮肤坏死试验均表明,r PMT与天然PMT具有相同的生物学功能。此外,以原核表达蛋白r PMT-N和r PMT-C分别制备的鼠多抗为一抗,对转染表达N端和C端真核质粒的Vero细胞进行IFA和Western-blot检测。结果表明,2种蛋白特异性良好,制备的多克隆抗体免疫反应条带单一。该研究的成功开展将为后续探究PMT细胞内作用机制提供可靠的生物材料。To investigate PMT of Pasteurella mutocida in pathogenesis of mammal cells,the prokaryotic expression vectors of entire tox A gene,N-terminal（1—1 461 bp） and C-terminal genes（2 958—3 858 bp） were constructed by molecular cloning with p ET-32 a.Three recombinant proteins are expressed and their molecular weighs are 176 ku,88 ku and 67 ku,respectively.In result,the expressed product bands could specifically bind to the anti-HIS-labeled monoclonal antibody.Both the cellular toxicity in vitro test and the dermonecrotic test in guinea pigs showed that the r PMT had equal bioactivities with natural PMT.The anti-r PMT-N and anti-r PMT-C serums were obtained by immunizing ICR mice.PMT-N and PMT-C expressed in Vero cells can be bind to these polyclonal antibodies,indicating that these polyclonal antibodies had high specificity.The aboved-mentioned results provide reliable biomaterials for further studies on PMT mechanisms in eukaryotic cells.

关 键 词：产毒素巴氏杆菌 多杀性巴氏杆菌皮肤坏死毒素 生物学活性 截短表达 多克隆抗体 

分 类 号：S852.612[农业科学—基础兽医学]