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  

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

作　　者：马立静 彭泽宇 翟金丽 于静玥 王增 隗梦蝶 王新卫[1] 陈陆[1] 杨霞[1,2] MA Lijing;PENG Zeyu;ZHAI Jinli;YU Jingyue;WANG Zeng;WEI Mengdie;WANG Xinwei;CHEN Lu;YANG Xia(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan,China;Ministry of Education Key Laboratory for Animal Pathogens and Biosafety,Zhengzhou,Henan,China;Shangqiu Meilan Biotechnology Co.,Ltd.,Shangqiu,Henan,China)

机构地区：[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

出　　处：《微生物学报》2025年第3期1181-1196,共16页Acta Microbiologica Sinica

基　　金：河南省高等学校重点科研项目(22A230004)。

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pCAGGSflfA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210min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。[Objective]To develop a DNA vaccine by loading avian pathogenic Escherichia coli ghosts with the fimbrium gene fifA of Gallibacterium anatis and evaluate the immune effect of this vaccine in chickens.[Methods]Taking fifA of G.anatis as the target gene,we constructed the eukaryotic expression plasmid pCAGGS-fifA from the plasmid pCAGGS-HA carrying the chickenβ-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-fifA 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-fifA was successfully expressed in cells in vitro.The lysis rate of the constructed avian pathogenic E.coli ghosts heated at 42℃ 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-fA group and the pCAGGS-fifA group were much higher than those in other groups.Moreover,the bacterial ghost loading pCAGGS-fifA showed stronger immune effect than pCAGGS-fifA.[Conclusion]Bacterial ghosts as the carriers of pCAGGS-fifA significantly enhanced the immune effect of the DNA vaccine.

关 键 词：禽致病性大肠杆菌菌影 flfA基因 核酸疫苗 免疫效果 

分 类 号：S859.797[农业科学—临床兽医学]