牛荚膜A型多杀性巴氏杆菌强菌株rpoE蛋白的原核表达及其免疫保护效力研究  

Prokaryotic expression and efficacy of immune protection of rpoE of bovine capsular type A Pasteurella multocida virulent strains

作　　者：嵇少泽 高云航[1] 勾长龙 张喜庆[1] 王羽[1] 田佳琪[1] 李晨 JI Shao-ze;GAO Yun-hang;GOU Chang-long;ZHANG Xi-qing;WANG Yu;TIAN Jia-qi;LI Chen(Department of Preventive Veterinary Medicine,Jilin Agricultural University,Jilin 130118,China)

机构地区：[1]吉林农业大学动物科学技术学院

出　　处：《中国预防兽医学报》2019年第7期740-743,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：肉牛产业技术体系废弃物处理与环境控制岗位(CARS-38);畜禽重大疫病防控与高效安全养殖综合技术研发(2016YFD0501400);畜禽养殖固体废弃物无害化处理及资源化利用技术研究(20170101073JC);养殖场固体废弃物处理功能性菌剂研发及资源化利用技术研究(JJKH20180643KJ)

摘　　要：为研究牛荚膜A型多杀性巴氏杆菌(PM)强菌株rpoE蛋白的免疫保护性,本研究利用PCR方法扩增牛荚膜A型PM的rpoE基因,构建pET-28a-rpoE重组表达质粒,经诱导表达后SDS-PAGE检测结果显示,表达的rpoE蛋白相对分子量约为20 ku,与理论值一致;western blot结果显示该蛋白具有很好的特异性和反应原性;纯化的rpoE蛋白经皮下免疫BALB/c小鼠,间接ELISA检测融合蛋白的免疫原性,结果显示rpoE蛋白可以诱导小鼠产生较高水平的特异性抗体;PM攻毒后,免疫组保护率为70%。本研究获得的牛荚膜A型PM重组rpoE蛋白具有良好的免疫原性,免疫小鼠后能够提供一定的保护,可以作为研发PM亚单位疫苗的候选抗原。In order to analyze the immunogenicity of the rpoE protein of bovine Pasteurella multocida(PM),the rpoE gene of PM was amplified by PCR and cloned into the prokaryotic expression vector pET-28a.The recombinant plasmid(pET-28a-rpoE)was transformed into Rosetta(DE3)for protein expression.SDS-PAGE results showed that the recombinant protein was about 20ku,which was consistent with its theoretical value.The recombinant protein was further identified by western blot,and the results revealed that it has highly specificity and immunoreactivity.The BALB/c mices were immunized subcutaneously with the purified PM RpoE protein and the immunogenicity was detected using indirect ELISA.The results showed that rpoE protein can induce mice to produce high levels of specific antibodie.The protective rate of immune group was 70%after challenging with PM.The recombinant protein purified in this study with good cimmunogenicity and providing certain immune protection for BALB/c mices,which suggests that the rpoE may be used as a candidate antigen for developing subunit vaccine of PM.

关 键 词：荚膜A型PM rpoE基因 原核表达 免疫原性 免疫保护力 

分 类 号：S852.61[农业科学—基础兽医学]