摘要
为研究副猪嗜血杆菌(H.parasuis)外膜蛋白P5(OmpP5)的抗原特性,通过原核表达技术对H.parasuis血清5型的OmpP5基因进行表达,用蛋白质免疫印迹技术和酶联免疫吸附试验检测重组OmpP5与种内和部分革兰氏阴性菌的交叉反应性,同时对这些菌株之间OmpP5(A)氨基酸序列进行同源性及系统进化分析,通过免疫小鼠验证OmpP5与免疫保护力的相关性。结果显示,重组OmpP5可刺激机体产生高水平抗体,它既可与15个血清型H.parasuis全菌超免疫兔抗血清发生交叉反应,也可与流感嗜血杆菌、胸膜肺炎放线杆菌、志贺氏菌、沙门氏菌、巴氏杆菌和李斯特杆菌等发生交叉反应;通过对各交叉反应菌株的OmpP5(A)氨基酸序列比较发现,H.parasuis的15个血清型菌株之间OmpP5(A)序列的同源性为88.4%～100%;和与其发生交叉反应的其他种属细菌的OmpP5(OmpA)同源性在45.9%～72.7%之间,系统进化分析表明具有较近的亲缘关系;免疫保护试验结果显示,重组OmpP5免疫组小鼠经同型菌株攻击后第7天有2只存活,而未免疫组则在攻击后16h内全部死亡。表明,H.parasuis OmpP5抗原高度保守,不具有种属特异性,且具有较强的抗原性和部分免疫保护作用。
The aim of this study was to learn antigenic characterization of the outer membrane protein P5（OmpP5） of Haemophilus parasuis.OmpP5 gene was cloned from H.parasuis servoar 5 and expressed in Escherichia coli.The cross-reaction of OmpP5 in different serotype of H.parasuis and with related gram-positive bacteria was analyzed by Western-blot and ELISA.The amino acid sequence identity and the phylogenetic analysis between these strains were analyzed.Lastly,the immunoprotection of OmpP5 was evaluated by a lethal challenge dose of H.parasuis serovar 5 in mice.As a result,recombinant OmpP5 could react with the antibodies of all the 15 serovars reference strains of H.parasuis,and it also react with antibodies of some other gram-negative and gram-positive bacteria,including Haemophilus influenzae,Acdnobacillus pleuropneumoniae,Salmonella,Pasteurella multocida,Shigella and Listeria monocytogenes.The bioinformatics analysis showed that the amino acid sequences identities of the 15 serovars of H.parasuis were from 88.4% to 100% and they shared from 45.9% to 72.7% with the cross-reaction bacteria.This demonstrated that the H.parasuis OmpP5 is highly conserved in the different serotype of H.parasuis and also conserved in the different kind of gram-positive bacteria.In addition,this study demonstrated that whilst all the unimmunized mice as control died by 16 hour post-challenge,two mice（out of 7） from the immunized groups survived beyond 7 days post-challenge.These result showed that the recombinant OmpP5 could excite strong antibody response and provide partially immunoprotection.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2012年第12期1230-1236,共7页
Chinese Veterinary Science
基金
“十二五”国家高技术研究发展(863)计划项目(2012AA101304)
