pUC18-CpG重组质粒对鸭疫里默氏菌灭活疫苗的免疫增强效应

Immune enhancement of Riemerella anatipestifer inactivated vaccine by the recombinant plasmid pUC18-CpG

摘要:

【背景】鸭疫里默氏菌(Riemerella anatipestifer, RA)是造成禽养殖业损失的重要病原体，可引起鸭等多种禽类浆膜炎和败血症。疫苗存在反应弱、免疫原性低、使机体产生抗体水平低等问题。【目的】探究pUC18-CpG佐剂的免疫反应及保护作用，为CpG佐剂疫苗预防鸭浆膜炎提供依据。【方法】通过鸭外周血淋巴细胞增殖试验和实时荧光定量PCR试验确定最优序列。构建重组质粒pUC18-CpG，并与CpG ODN进行免疫原性比较。以灭活RA-GH5为抗原，pUC18-CpG重组质粒为佐剂，分别皮下免疫雏鸭2次，检测其血清抗体滴度和细胞因子水平；以RA-GH5肌肉注射攻毒，观察组织病理学变化及免疫保护率。【结果】筛选出免疫刺激活性最佳序列CpG-3，含CpG-3的pUC18-CpG重组质粒与CpG-3的免疫原性无明显差异。与RA疫苗相比，添加pUC18-CpG重组质粒组显著提高了血清抗体滴度以及IL-2和IL-4细胞因子水平。80 μg pUC18-CpG佐剂疫苗对鸭的免疫保护率为70%，20、40 μg pUC18-CpG佐剂疫苗均为60%。【结论】pUC18-CpG佐剂与RA-GH5灭活疫苗联用能够引起更强的体液免疫和细胞免疫应答。

关键词:鸭疫里默氏菌;CpG ODN;pUC18-CpG;佐剂

Abstract:

[Background] Riemerella anatipestifer (RA), a major pathogen that causes losses in the poultry industry, can cause serositis and septicemia in ducks and other birds. The available vaccines generally have weak reaction and low immunogenicity and induce low antibody levels. [Objective] To explore the immune response and protective effect of CpG adjuvant and provide evidence for the prevention of duck serositis with CpG adjuvant. [Methods] The proliferation test of lymphocytes in duck peripheral blood and real-time fluorescence quantitative PCR were employed to determine the optimal sequence. The recombinant plasmid pUC18-CpG was constructed and compared with CpG ODN for immunogenicity. Ducklings were immunized via subcutaneous injection twice with inactivated RA-GH5 as the antigen and pUC18-CpG as the adjuvant, and then the serum antibody titer and cytokine level 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 optimal sequence CpG-3 was screened out. The immunostimulatory activity of pUC18-CpG containing CpG-3 was not significantly different from that of CpG-3. Compared with RA vaccine alone, the addition of pUC18-CpG significantly increased the serum antibody titer and the levels of interleukin-2 (IL-2) and IL-4. The immune protection rates of 80 μg, 20 μg, and 40 μg pUC18-CpG to the ducklings were 70%, 60%, and 60%, respectively. [Conclusion] The combination of pUC18-CpG and RA-GH5 inactivated vaccine can induce stronger humoral and cellular immune responses.

Key words:Riemerella anatipestifer;CpG ODN;pUC18-CpG;adjuvant