摘要
旨在通过分析溶血性曼氏杆菌重要抗原的免疫原性,为疫苗和诊断试剂研制提供依据。在生物信息学分析的基础上,构建溶血性曼氏杆菌lktA、ompA、plpE、plpF、ompP2和gs60基因的原核重组表达载体,并将其转化至大肠杆菌BL21(DE3)中进行表达,获得重组蛋白质。随后用被抗溶血性曼氏杆菌感染的绵羊血清进行Western-blot和酶联免疫吸附测定(ELISA),评价各蛋白质的免疫原性并筛选优势免疫原;用各重组蛋白质免疫小鼠,评价免疫小鼠后诱导其产生的抗体水平,以评价各重组蛋白质作为重组疫苗的潜在价值。Western-blot和ELISA检测结果表明,各重组蛋白质均具有良好的免疫原性,其中rLktA、rOmpA、rGs60与感染的绵羊血清结合能力最强,为优势免疫原;各重组蛋白质免疫小鼠后均能诱导其产生体液免疫应答,其中rOmpA、rPlpE和rPlpF免疫小鼠后诱导其产生的抗体在免疫后35 d仍能与全菌蛋白质结合。研究结果为溶血性曼氏杆菌感染血清学诊断试剂的开发和新型疫苗的研制提供了重要的试验数据。
The purpose of this study is to compare and analyze the immunogenicity of major antigens of Mannheimia haemolytica,and to provide a basis for the development of vaccines and diagnostic reagents.Based on bioinformatics analysis,prokaryotic expression vectors of lktA,ompA,plpE,plpF,ompP2 and gs60 genes were constructed and transformed into Escherichia coli BL21(DE3),and recombinant proteins were expressed and purified.Subsequently,Western-blot and enzyme-linked immunosorbent assay(ELISA)were performed by using sheep anti-Mannheimia haemolytica serum to evaluate the immunogenicity of each protein,and to screen the dominant immunogens.Meanwhile,mice were immunized with each recombinant protein,and the antibody levels were tested to evaluate potential value of each protein as a recombinant vaccine.The results demonstrated that all tested proteins were good immunogens,while rLktA,rOmpA and rGs60 showed the strongest binding ability to infected sheep serum,suggesting that they were the dominant immunogens of Mannheimia haemolytica.All recombinant proteins could induce humoral immune response in mice,while rOmpA,rPlpE and rPlpF could induce high titer antibody after 35 days post immunization.These results provide important data for developing serological diagnostic tools and new vaccines.
作者
陆奕彤
李垚
杨发龙
LU Yi-tong;LI Yao;YANG Fa-long(Key Laboratory of the Ministry of Education for the Protection and Utilization of Animal Genetic Resources on the Qinghai-Tibet Plateau,Southwest Minzu University,Chengdu 610041,China)
出处
《江苏农业学报》
CSCD
北大核心
2022年第2期446-452,共7页
Jiangsu Journal of Agricultural Sciences
基金
四川省重点研发项目(2021YFN0008)
西南民族大学研究生创新项目(CX2020SZ57)。
关键词
溶血性曼氏杆菌
重要抗原
免疫原性
Mannheimia haemolytica
important antigen
immunogenicity
