鸡白痢沙门菌pagC基因缺失株菌蜕疫苗的制备和免疫保护效果评价

`Assessment of Immune Protective Effect of Bacterial Ghost Vaccine Developed from Salmonella pullorumΔPagC Mutant Strain

为了在鸡白痢沙门菌pagC基因缺失突变株的基础上制备可用于鉴别诊断的菌蜕疫苗并评价其免疫保护效果,本研究利用细胞穿膜肽MAP制备ΔPagC菌蜕,透射电镜和荧光显微镜观察菌蜕形态和核酸残留情况;将ΔPagC菌蜕与佐剂ISA206混合乳化制备菌蜕疫苗并进行无菌检测;基于ICR小鼠评价菌蜕疫苗对鸡白痢沙门菌CVCC1800、肠炎沙门菌ATCC19585和鼠伤寒沙门菌CVCC542感染的交叉保护效果;结合基于PagC蛋白的沙门菌血清学检测方法评价pagC基因缺失株菌蜕疫苗的鉴别诊断效果。结果显示,菌蜕制备最佳条件为用终浓度为50μmol/L MAP溶液处理OD_(600 nm)=0.3的菌液1 h以上,灭活效果可达99.00%;菌蜕保持基本细胞形态的同时内容物大量释出,核酸残留水平较低;菌蜕疫苗针对上述3种血清型沙门菌的免疫保护率分别为25%、100%和100%;菌蜕疫苗与基于PagC蛋白的沙门菌血清学检测方法联用可在一定程度上起到鉴别诊断作用。结果表明,制备所得菌蜕疫苗在保护小鼠的同时,能够在一定程度上区分人工免疫和自然感染,为沙门菌的净化工作提供帮助。

In order to produce a bacterial ghost vaccine based on Salmonella pullorumΔPagC mutant strain and evaluate its immune protective effect and potential diagnostic utility,this study employed cell penetrating peptides MAP(model amphipathic peptide)to generateΔPagC bacterial ghost,and transmission electron microscope and fluorescence microscope to observe the ghost morphology and detect nucleic acid residue.Specifically,theΔPagC bacterial ghost was emulsified with adjuvant ISA206 for bacterial ghost vaccine preparation and sterility detection;its cross-protective effect against Salmonella pullorum CVCC1800,Salmonella enteritidis ATCC19585 and Salmonella typhimurium CVCC542 was examined in ICR mice model;and its potential for differential diagnosis was analyzed with the PagC protein-based Salmonella serological detection method.The results showed that treatment of Salmonella pullorumΔPagC(OD 600 nm=0.3)with 50μmol/L MAP solution for 1 h led to the killing of 99.00%of bacteria;the bacterial ghost maintained basic cell morphology with large amount of cell contents left,and the level of nucleic acid residue was low.The immune protection rates of this ghost vaccine against three serotypes of Salmonella mentioned above were 25%,100%and 100%,respectively.Thus,combined with the PagC protein-based Salmonella serological detection method,this bacterial ghost vaccine has potential to be used for differential diagnosis.Thus,the bacterial ghost vaccine prepared in this study provides protection in mice against Salmonella infection,and facilitates discrimination between artificial immunity and natural infection to a certain extent,could help for the purification of Salmonella.