猪源多杀性巴氏杆菌的分离鉴定及其主要抗原蛋白的原核表达  

Isolation and Identifcation of Porcine Pasteurella multocida Strain and Prokaryotic Expression of Its Major Antigen Proteins

作　　者：姜伟 郭明佳 吴树康 金彩虹 Jiang Wei;Guo Mingjia;Wu Shukang;Jin Caihong(Longkou Center for Animal Disease Prevention and Control,Longkou,Shandong 265701,China)

机构地区：[1]龙口市动物疫病预防控制中心,山东龙口265701

出　　处：《中国动物检疫》2022年第4期66-71,共6页China Animal Health Inspection

摘　　要：2021年3月,山东省龙口市某养猪场发生了一起以败血症为主要临床症状的烈性传染病。为对引起此次疫病的病原进行确诊,无菌采集发病死亡仔猪的肝脏、脾脏等脏器进行染色镜检、病原分离培养、生化鉴定及PCR检测。结果显示,本次分离株的形态结构、培养特性及生化结果与多杀性巴氏杆菌较为一致,且可特异性扩增出多杀性巴氏杆菌特异性基因Kmt,因此本次分离菌株为一株猪源多杀性巴氏杆菌,命名为LK01。随后,对该分离株的5个主要抗原蛋白基因(OmpH、OmpA、AspA、pIpE和TAA)进行克隆与原核表达。先将PCR成功扩增出的目的片段与双酶切回收后的pCold-I载体进行同源重组连接,测序结果正确的重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中,随后经IPTG诱导后进行Western blot检测。结果显示,在30.0、21.5、52.0、37.5和45.5 kDa处出现特异性条带,表明各重组蛋白均成功表达。本研究为开展猪源多杀性巴氏杆菌病的流行病学研究,开发新型重组疫苗与诊断试剂奠定了基础。In March 2021,a severe infectious disease with major symptom of septicemia occurred in a swine farm in Longkou City,Shandong Province.In order to identify the pathogen leading to the disease,livers and spleens of dead piglets were aseptically collected for staining microscopic examination,bacterial isolation and culture,biochemical identifcation and PCR detection.The results showed that the morphological structure,culture characteristics and the results of biochemical test of the isolate were relatively consistent with those of Pasteurella multocida(PM),then Kmt,the specifc gene of PM was successfully amplifed,it was identifed that the isolate was a porcine PM named as LK01.Subsequently,its fve major antigen genes(including OmpH,OmpA,AspA,pIpE and TAA)were cloned and expressed in prokaryotic expression system.By homologous recombination,the target fragments amplifed by PCR were linked with the pCold-I that was after double digestion.The recombinant plasmid with correct sequencing results was transformed into E.coli BL21(DE3)cells and induced by IPTG for Western blot detection.It was observed that specifc bands appeared at 30.0,21.5,52.0,37.5 and 45.5 kDa,indicating that all recombinant proteins were successfully expressed.A foundation was thus provided for further epidemiological study on porcine PM and for development of new recombinant vaccines and diagnostic reagents.

关 键 词：多杀性巴氏杆菌 分离鉴定 抗原蛋白 克隆 原核表达 

分 类 号：S855.1[农业科学—临床兽医学]