山东省副猪嗜血杆菌分子流行病学调查及其外膜蛋白P2基因的表达纯化  

MOLECULAR EPIDEMIOLOGICAL INVESTIGATION OF HAEMOPHILUS PARASUIS IN SHANDONG PROVINCE AND EXPRESSION, PURIFICATION OF OMPP2 GENE

作　　者：杨杰[1,2] 李建达[1,2] 曾昊 任素芳 郭立辉[1,2] 孙文博[1,2] 陈智[1,2] 丛晓燕[1,2] 于江[1,2] 吴家强[1,2,3] YANG Jie;LI Jian-da;ZENG Hao;REN Su-fang;SUN Wen-bo;CHEN Zhi;CONG Xiao-yan;YU Jiang;WU Jia-Qiang(Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Shandong Provincial Key Laboratory of Animal Disease Control and Breeding,Jinan 250100,China;College of Life Sciences,Shandong Normal University,Jinan 250014,China)

机构地区：[1]山东省农业科学院畜牧兽医研究所,济南250100 [2]山东省畜禽疫病防治与繁育重点实验室,济南250100 [3]山东师范大学生命科学学院,济南250014

出　　处：《中国动物传染病学报》2018年第5期40-47,共8页Chinese Journal of Animal Infectious Diseases

基　　金：山东省农业科学院青年科研基金项目(2015YQN51);山东省农业科学院重大科技成果培育计划(2014CGPY04);山东省农业科学院农业科技创新工程(CXGC2016B14);国家自然科学基金(31600106);国家重点研发计划项目(2017YFD0500602);山东省现代农业产业技术体系(SDAIT);山东省农业重大应用技术创新项目;泰山学者工程经费资助

摘　　要：根据副猪嗜血杆菌(Haemophilus parasuis,Hps)外膜蛋白(outer membrane proteins,Omp)P2基因序列(GenBank登录号:FJ685754.1)设计引物,对2015～2016年在山东省不同地区采集的225份病料进行PCR扩增和遗传演化分析,结果发现2015年和2016年Hps检出阳性率分别为7.63%、7.48%,细菌分离确定225份样品中17份为阳性;Hps菌株之间的氨基酸同源性在86.7%～99.7%,同源性较高的蛋白具有交叉反应性。构建Hps omp P2基因原核表达载体,确定OmpP2蛋白的最佳诱导温度,通过亲和层析Ni-IDA树脂纯化,获得高纯度的OmpP2蛋白,Western blot确定纯化的OmpP2蛋白与不同血清型的Hps阳性血清均存在良好的反应原性。本研究为进一步探索Hps OmpP2的生物学特性及对副猪嗜血杆菌病的血清学诊断、亚单位疫苗的开发奠定了基础。A total of 225 samples were collected in 2015 and 2016 from different regions of Shandong province for molecular epidemiology of Haemophilus parasuis(Hps).The primers were designed for PCR amplification and sequence analysis according to the ompP 2 gene sequence(FJ685754.1).The results showed that the positive rate of Hps in 2015 and 2016 were 7.63%and 7.48%,respectively.The amino acid homology among 17 Hps isolates was 86.7%-99.7%.The prokaryotic expression vector of ompP 2 gene constructed and the optimal expression temperature of OmpP2 protein was determined.The highly purified OmpP2 protein was obtained with affinity chromatography Ni-IDA resin.The purified OmpP2 protein had good reactivity in Western blot with Hps positive sera of different serotypes.This study laid the foundation for further research on the biological characteristics of OmpP2,serological diagnosis and development of subunit vaccine against Hps.

关 键 词：副猪嗜血杆菌 流行病学 ompP2基因 表达 纯化 

分 类 号：S852.612[农业科学—基础兽医学]