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为评价鸭源鸡杆菌(Gallibacterium anatis,G.anatis)外膜蛋白A(Outer membrane proteins A,OmpA)的免疫原性及免疫保护力,本研究通过PCR技术扩增去除信号肽的ompA基因序列,酶切后与载体pET-32a(+)连接,构建重组表达质粒pET-32a(+)-OmpA,在IPTG诱导下成功表达出约40 ku的包涵体蛋白。Western-blot分析显示重组蛋白rOmpA具有抗原性;以纯化的rOmpA免疫小鼠,分别在一免后第2周和第4周进行加强免疫。三免后进行抗体检测并以G.anatis PDS-RZ-1-SLG株的5×LD_(50)攻毒,同时设立全菌灭活组和PBS组作为对照。结果显示,rOmpA可诱导小鼠产生较高水平的抗体,可提供50%的免疫保护力。结果表明,鸭源鸡杆菌外膜蛋白A是一种保守性共同抗原,可作为鸭源鸡杆菌疫苗的候选抗原。
In order to evaluate the immunogenicity and immunopotency of OmpA (OmpA) of Gallibacterium anatis (G.anatis), the ompA gene sequence of the signal peptide was amplified by PCR. After digested with pET-32a (+) vector, the recombinant plasmid pET-32a (+) - OmpA was constructed and expressed in the presence of IPTG at about 40 ku. Western-blot analysis showed that recombinant protein rOmpA was antigenic. Immunized mice with purified rOmpA were boosted at the second week and the fourth week respectively. Antibody test was carried out after three immunizations and challenged with 5 × LD_ (50) of G.anatis PDS-RZ-1-SLG strain. At the same time, whole-body inactivated group and PBS group were set as control. The results show that rOmpA can induce mice to produce higher levels of antibodies, providing 50% of the immune protection. The results showed that the outer duck protein A of duck origin was a conservative common antigen and could be used as a candidate antigen for the Agrobacterium ducklings vaccine.