鸭疫里默氏菌DnaK、OmpA和OmpA-DnaK蛋白的免疫原性

Immunogenicity of Riemerella anatipestifer DnaK, OmpA and OmpA-DnaK proteins

摘要:

【背景】鸭疫里默氏菌(Riemerella anatipestifer，RA)可引起鸭等多种禽类败血症和浆膜炎，给禽养殖业造成严重经济损失。蛋白疫苗是预防RA感染的重要策略之一。目前，有关RA重组蛋白免疫原性报道较少，且其应用也受到单一蛋白抗原诱导的特异性免疫反应不足的限制。【目的】探究分子伴侣DnaK、外膜蛋白A (outer membrane protein A，OmpA)和OmpA-DnaK蛋白疫苗在鸭体内诱导的免疫应答，评估其免疫原性，为RA疫苗抗原研发提供依据。【方法】克隆DnaK和OmpA基因并分别与pET-32a (+)载体相连，利用限制性酶切位点Nco I和BamH I将OmpA连接至DnaK基因上游，经原核表达和纯化制得重组蛋白DnaK、OmpA和OmpA-DnaK。3种重组蛋白分别皮下免疫雏鸭2次，检测其血清抗体滴度、淋巴细胞增殖和细胞因子(IL-2和IL-4)水平；以RA-GH5肌肉注射攻毒，检查其组织病理学变化及免疫保护率。【结果】成功表达了DnaK、OmpA和OmpA-DnaK重组蛋白，分子量分别约为90、60和130 kDa。3种蛋白疫苗均能诱导宿主产生体液和细胞免疫应答，并且诱导的免疫保护反应减轻了RA引起的组织病理学损伤。与单独DnaK或OmpA免疫相比，OmpA-DnaK显著提高了血清抗体滴度、淋巴细胞增殖指数，以及IL-2和IL-4细胞因子水平。OmpA-DnaK对鸭的免疫保护率为50%，DnaK和OmpA均为30%。【结论】RA重组OmpA、DnaK和OmpA-DnaK蛋白疫苗均能诱导鸭免疫反应，并且DnaK与OmpA融合表达可引起更强的体液免疫和细胞免疫应答。

Abstract:

[Background] Riemerella anatipestifer (RA) causes septicemia and infectious serositis in a variety of birds such as duck, leading to serious economic losses in the poultry industry. Protein vaccination is considered to be one of the most important strategies for preventing RA infection in ducklings. At present, there are few studies on the immunogenicity of RA recombinant proteins, and their application is also limited by the insufficient specific immune response induced by the single protein antigen. [Objective] The study aims to explore the immune response induced by the molecular chaperone DnaK, outer membrane protein A (OmpA) and OmpA-DnaK vaccines in ducklings, and to evaluate whether the recombinant fusion protein OmpA-DnaK has enhanced immunogenicity, which is conducive to the further development of RA vaccines. [Methods] We amplified the DnaK and OmpA genes and linked them to the pET-32a(+) vector, respectively. OmpA gene was ligated to the upstream region of DnaK gene through the restriction sites of Nco I and BamH I. DnaK, OmpA and OmpA-DnaK proteins were prepared by prokaryotic expression and purification. The DnaK, OmpA and OmpA-DnaK vaccines were respectively used to immunize ducklings twice via subcutaneous injection, and then the serum antibody titers, lymphocyte proliferation and cytokine (IL-2 and IL-4) levels were determined. The ducklings were then challenged by RA-GH5 strain through intramuscular injection, and the histopathological changes and immune protection rate were examined. [Results] The recombinant DnaK, OmpA and OmpA-DnaK proteins were expressed, with molecular weights of approximately 90, 60 and 130 kDa, respectively. The three recombinant protein vaccines induced the humoral and cellular immune responses of ducklings, and mitigated the histopathological damage caused by RA. Compared with DnaK or OmpA alone, OmpA-DnaK significantly increased serum antibody titer, lymphocyte proliferation index, and IL-2 and IL-4 levels after vaccination. The immune protection rate of OmpA-DnaK to ducklings was 50%, and those of both DnaK and OmpA were 30%. [Conclusion] The recombinant OmpA, DnaK and OmpA-DnaK protein vaccines all induced immune responses in ducklings. Moreover, the fusion of DnaK and OmpA caused stronger humoral and cellular immune responses.