牛A型多杀性巴氏杆菌新型保护性抗原的筛选及鉴定

Screening and identification of new protective antigens of bovine Pasteurella multocida serotype A

为筛选牛A型多杀性巴氏杆菌新型保护性抗原,采用生物信息学方法,对牛A型多杀性巴氏杆菌Pm CQ 2基因组中功能未知基因进行分析,从中筛选出22个具有信号肽的蛋白编码基因进行克隆表达。以Pm CQ2基因组D N A为模板,22个基因经PCR扩增分别克隆到载体p ET-30a(+)或p ET-32a(+)上,转入E.coli BL21(DE3)中进行IPTG诱导表达,经SDS-PAGE检测,22个基因均获得表达。W estern-blot检测发现有8个重组蛋白与感染Pm CQ2的犊牛血清具有良好的免疫反应,以其制备抗原免疫昆明小鼠,经ELISA检测免疫小鼠血清抗体水平后,采用2 LD50的Pm CQ2腹腔攻毒测定其免疫保护效果。结果表明,8个重组蛋白均能诱导小鼠产生较高水平的抗体,其中3号和9号蛋白免疫小鼠后抗体效价达到1∶12 800,且3号和9号蛋白免疫保护效果最好,免疫保护率均为30%,其余重组蛋白的免疫保护率均较低。

The aim of this study is to screen a new protective antigens of bovine Pasteurella multo- cidaeapsular serotype A. Using bioinformatics tools,here 22 genes coding for proteins with signal pep- tide were screened out from the genes of unknown function in P. multocidacapsular serotype A isolate PmCQ2. The DNA fragments of 22 genes were amplified from PmCQ2 genome DNAby PCR,and were cloned into pET-30a（-5） or pET-32a（-5） plasmid,the recombinant plasmids were transformed into E. coli BL21 （DE3） and induced by IPTG for fusion gene expression. All of 22 genes can express the protein in E coli BL21（DE3） detected by SDS-PAGE. Using the serum of calf infected with PmCQ2 as the primary antibody, Western-blot showed that 8 of 22 recombinant proteins had a good reaction,and then chosed these 8 pro- teins to immunize Kunming mice, respectively. After sampling blood by tail vein for serum to detect the production of antibodies against proteins by ELISA,the mice were challenged intraperitoneally with 2 LD50 PmCQ2. The results showed that all of 8 protein antigens induced high titers of antibody production in mice,and the antibody titers of protein No. 3 and No. 9 induced were up to 1：12 800, protein No. 3 and No. 9 also showed the best immune protection against PmCQ2 than other proteins,its rate of immune pro- tection were both to 30%.