猪多杀性巴氏杆菌OmpW、TbpA蛋白的原核表达及免疫原性研究  

Study on Prokaryotic Expression and Immunogenicity of OmpW and TbpA Proteins in Swine Pasteurella multocida

作　　者：张哲玮 代小童 韦祖丹 陈云鹏 甘晶 贝为成 ZHANG Zhewei;DAI Xiaotong;WEI Zudan;CHEN Yunpeng;GAN Jing;BEI Weicheng(State Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China;Guangxi Yangxiang Co.,Ltd.,Guigang 537100,China;The Cooperative Innovation Center for Sustainable Pig Production,Wuhan 430070,China)

机构地区：[1]华中农业大学动物医学院,农业微生物学国家重点实验室,武汉430070 [2]湖北洪山实验室,武汉430070 [3]广西扬翔农牧有限责任公司,贵港537100 [4]生猪健康养殖协同创新中心,武汉430070

出　　处：《中国畜牧兽医》2023年第4期1489-1498,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家生猪产业技术体系(CARS-35);动物基因工程疫苗国家重点实验室开放课题(AGVSK-ZD-201805);畜禽病原微生物学湖北省重点实验室开放课题(KLPCAAB-2020-03)。

摘　　要：【目的】研究猪多杀性巴氏杆菌OmpW和TbpA蛋白的免疫原性,探索其作为疫苗候选抗原的可能。【方法】对NCBI中不同血清群猪多杀性巴氏杆菌中OmpW和TbpA蛋白氨基酸序列进行相似性比对,并对OmpW和TbpA蛋白进行生物信息学分析。基于此,构建重组表达菌株pET28a-OmpW-BL21和pET28a-TbpA-BL21,重组OmpW和TbpA蛋白经镍柱亲和层析纯化后与氢氧化铝佐剂混匀制备亚单位疫苗,免疫小鼠后使用猪多杀性巴氏杆菌GX-PmD4菌株攻毒,评估OmpW和TbpA蛋白的免疫原性。【结果】相似性比对发现,OmpW和TbpA蛋白在不同血清群猪多杀性巴氏杆菌中高度保守;SignalP 6.0 Server和TMHMM Server v.2.0软件预测发现OmpW蛋白有信号肽和跨膜区,而TbpA蛋白有信号肽无跨膜区;ABCpred软件分析显示,OmpW和TbpA蛋白分别有14和22个B细胞抗原表位;SOPMA和SWISS-MODEL软件进一步预测蛋白结构表明:OmpW和TbpA蛋白的α-螺旋、β-转角、延伸链和无规则卷曲分别占16.18%、5.39%、36.76%、41.67%和40.42%、5.69%、16.77%、37.13%。经原核表达获得的重组OmpW和TbpA蛋白均具有较强的抗原性。在免疫小鼠后的14和28 d,与对照组相比,血清中OmpW、TbpA特异性抗体水平极显著上升(P<0.01)。小鼠免疫攻毒试验结果显示,重组OmpW组和TbpA组的小鼠存活率分别为50.0%和37.5%。【结论】本研究成功表达了猪多杀性巴氏杆菌OmpW和TbpA蛋白,且重组OmpW蛋白免疫原性较强,可作为猪多杀性巴氏杆菌亚单位疫苗的有效候选抗原。【Objective】This study was aimed to investigate the immunogenicity of OmpW and TbpA proteins of swine Pasteurella multocida and explore their potential as vaccine candidate antigens.【Method】The similarity of amino acid sequences of OmpW and TbpA proteins in different serotypes of swine Pasteurella multocida were compared by NCBI.Further bioinformatics analysis of OmpW and TbpA proteins were carried out.Based on this,the recombinant expression strains pET28a-OmpW-BL21 and pET28a-TbpA-BL21 were successfully constructed,which were purified and mixed with aluminum hydroxide adjuvant to prepare subunit vaccine.After immunizing mice,the immunogenicity of OmpW and TbpA proteins was evaluated using swine Pasteurella multocida GX-PmD4 strain to challenge.【Result】The results of similarity analysis indicated that OmpW and TbpA proteins were highly conserved in different serotypes of swine Pasteurella multocida.SignalP 6.0 Server and TMHMM Server v.2.0 softwares predicted that OmpW protein had signal peptide and transmembrane region,while TbpA protein only had signal peptide without transmembrane region.ABCpred software analysis showed that OmpW and TbpA proteins had 14 and 22 B cell antigen epitopes,respectively.SOPMA and SWISS-MODEL softwares further predicted the protein structure of OmpW and TbpA,which showed that the alpha helix,beta turn,extended strand and random coil accounted for 16.18%,5.39%,36.76%,41.67%,and 40.42%,5.69%,16.77%,37.13%,respectively.The recombinant OmpW and TbpA proteins obtained by prokaryotic expression technique had strong antigenicity.Compared with control group,the serum specific antibody levels of OmpW and TbpA in serum were extremely significantly increased on 14 and 28 d after immunizing mice(P<0.01).The survival rate of mice in OmpW and TbpA groups was 50.0%and 37.5%,respectively.【Conclusion】In this study,the OmpW and TbpA proteins of swine Pasteurella multocida were successfully expressed,the recombinant OmpW protein had strong immunogenicity and could be used as an effectiv

关 键 词：猪多杀性巴氏杆菌 OmpW TbpA 表达 免疫原性 

分 类 号：S852.61[农业科学—基础兽医学]