副猪嗜血杆菌CDT亚基原核表达及其抗原性鉴定  

Prokaryotic expression and antigenicity identification of recombinant CDT subunits of Haemophilus parasuis

作　　者：李军星[1] 王一成[1] 袁秀芳[1] 徐丽华[1] 曹会敏[1] 申世川[1] 张鲁滨[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《中国预防兽医学报》2012年第9期746-748,共3页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省自然科学基金(Y3100491);浙江省农业科学院创新能力提升项目(2010RY01D06)

摘　　要：为原核表达副猪嗜血杆菌(H.parasuis)CDT毒素并检测其在体外培养时的分泌表达情况,本实验将切除信号肽的CDT毒素的3个亚基基因分别克隆于pET-28a载体中在大肠杆菌进行表达,并将重组蛋白经亲和层析纯化后,免疫兔制备抗血清,用于鉴定H.parasuis体外培养时CDT分泌表达情况。结果表明CDT毒素3个亚基均在Rosetta菌株中得到表达,其中cdtB约为28.2 ku,符合预期大小,而cdtA和cdtC亚基比预期的23.5 ku和17.4 ku略大。表达的3个重组蛋白可以被自然感染猪血清识别,表明其具有良好的反应原性,同时也表明H.parasuis在猪体内定殖或感染过程中分泌CDT。制备的抗血清可以与体外培养H.parasuis的分泌蛋白中的CDT亚基反应,表明重组蛋白具有良好的免疫原性,同时也表明H.parasuis体外培养时也分泌CDT。Cytolethal distending toxin （CDT） is a virulence factor of gram negative bacteria. To investigate the antigenicity of recombinant CDT subunits, the encoding sequences of 3 CDT subunits （without signal peptide） were cloned into pET-28a and expressed in E. coli and the antiserum were prepared against the CDT subunits by immunization of rabbits with purified proteins, respectively. SDS-PAGE analysis showed that the cdtA, cdtB and cdtC were about 3.5 ku, 28.22 ku and 17.4 ku which all reacted with serum from naturally infected pigs, indicating that the CDT subunits were secreted in infected pigs. Furthermore, the prepared antisera agaist CDT subunits were able to react positively with H. parasuis culture supernatant, indicationg that the CDT subunits were secreted proteins. These results provided a basis for further study of the biological activity of H. parasuis CDT.

关 键 词：副猪嗜血杆菌 重组CDT亚基 原核表达 抗原性 

分 类 号：S852.61[农业科学—基础兽医学]