禽致病性大肠杆菌菌影负载鸭源鸡杆菌flfA基因核酸疫苗的研制及免疫效果评价
作者:
作者单位:

1.河南农业大学 动物医学院,河南 郑州;2.动物病原与生物安全教育部重点实验室,河南 郑州;3.商丘美兰生物工程有限公司,河南 商丘

作者简介:

马立静:实验设计和操作、数据收集和处理、论文撰写和修改;彭泽宇:协助实验操作、数据收集和处理;翟金丽:协助实验操作;于静玥:协助实验操作;王增:技术支持;隗梦蝶:协助实验操作;王新卫:论文讨论、技术支持;陈陆:论文讨论、技术支持;杨霞:实验方案的设计、论文讨论、技术支持、论文撰写和修改。

基金项目:

河南省高等学校重点科研项目(22A230004)


Development and immune efficacy evaluation of a Gallibacterium anatis flfA DNA vaccine with avian pathogenic Escherichia coli ghost as the vector
Author:
Affiliation:

1.College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan, China;2.Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou, Henan, China;3.Shangqiu Meilan Biotechnology Co., Ltd., Shangqiu, Henan, China

Fund Project:

This work was supported by the Key Scientific Research Project of Colleges and Universities in Henan Province (22A230004). *Corresponding author. E-mail: yangxia66@163.com

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    摘要:

    目的 研制禽致病性大肠杆菌菌影负载鸭源鸡杆菌flfA基因的核酸疫苗,并评估其在鸡体内的免疫保护效果。方法 以鸭源鸡杆菌的菌毛基因flfA为目的基因,采用携带鸡β肌动蛋白启动子的pCAGGS-HA质粒作为表达载体,构建pCAGGS-flfA真核表达质粒;构建温控质粒PBV-E-SN,并将其转化至对氨苄青霉素敏感的禽致病性大肠杆菌分离株制备菌影;将pCAGGS-flfA质粒装载入菌影制备菌影疫苗,设计菌影负载pCAGGS-flfA质粒组、pCAGGS-flfA质粒组、空菌影组、PBS组及正常对照组。对7日龄雏鸡进行首次免疫,首免2周后二次加强免疫,首免4周后对鸡进行鸭源鸡杆菌攻毒试验,检测疫苗的免疫保护效果。结果 所构建的真核表达质粒pCAGGS-flfA能在体外细胞中成功表达;构建的禽致病性大肠杆菌菌影菌株在42 ℃热诱导210 min后,裂解率达到99.94%;免疫保护试验结果显示,无论是通过ELISA检测的特异性IgG抗体水平,还是对鸡泄殖腔拭子和咽拭子的排菌情况以及组织脏器中细菌载量的测定,菌影负载pCAGGS-flfA质粒组的免疫保护效果均显著高于其他免疫组,包括单独免疫pCAGGS-flfA质粒组。与单独免疫pCAGGS-flfA质粒组相比,菌影负载pCAGGS-flfA质粒组的免疫保护效果更为优越。结论 本研究表明菌影作为pCAGGS-flfA的负载载体显著增强了该核酸疫苗的免疫保护效果。

    Abstract:

    Objective To develop a DNA vaccine by loading avian pathogenic Escherichia coli ghosts with the fimbrium gene flfA of Gallibacterium anatis and evaluate the immune effect of this vaccine in chickens.Methods Taking flfA of G. anatis as the target gene, we constructed the eukaryotic expression plasmid pCAGGS-flfA from the plasmid pCAGGS-HA carrying the chicken β-actin promoter, which enhanced the efficacy of vaccine. The temperature-sensitive plasmid PBV-E-SN was then constructed and transformed into the ampicillin-sensitive avian pathogenic E. coli isolate to prepare a bacterial ghost. Finally, pCAGGS-flfA was loaded into the bacterial ghost to prepare a bacterial ghost vaccine. Seven-day-old chickens were assigned into five groups: bacterial ghost loading pCAGGS-flfA, pCAGGS-flfA, empty bacterial ghost, PBS, and normal control. The primary vaccination was carried out for 7-day-old chickens, and the booster immunization was performed two weeks after the primary immunization. The chickens were challenged with G. anatis four weeks after the primary immunization, and the immune effect of the vaccine was evaluated.Results The eukaryotic expression plasmid pCAGGS-flfA was successfully expressed in cells in vitro. The lysis rate of the constructed avian pathogenic E. coli ghosts heated at 42 °C for 210 min reached 99.94%. The specific IgG antibody titer measured by ELISA, the number of shedding bacteria in chicken cloacal swabs and throat swabs, and the bacterial loads in tissue and organs of challenged chickens showed that the immune effects in the bacterial ghost loading pCAGGS-flfA group and the pCAGGS-flfA group were much higher than those in other groups. Moreover, the bacterial ghost loading pCAGGS-flfA showed stronger immune effect than pCAGGS-flfA.Conclusion Bacterial ghosts as the carriers of pCAGGS-flfA significantly enhanced the immune effect of the DNA vaccine.

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马立静,彭泽宇,翟金丽,于静玥,王增,隗梦蝶,王新卫,陈陆,杨霞. 禽致病性大肠杆菌菌影负载鸭源鸡杆菌flfA基因核酸疫苗的研制及免疫效果评价[J]. 微生物学报, 2025, 65(3): 1181-1196

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  • 收稿日期:2024-09-27
  • 在线发布日期: 2025-03-10
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