荚膜B型多杀性巴氏杆菌外膜囊泡生物学特性分析与免疫效果评价  

Biological Characteristics and Immune Effect Evaluation of Outer Membrane Vesicles ofCapsular Type B Pasteurella multocida

作　　者：高洁 李晓成[2] 穆杨 张慧[2] 魏荣[2] 李劼 GAO Jie;LI Xiaocheng;MU Yang;ZHANG Hui;WEI Rong;LI Jie(Department of Preventive Veterinary Medicine,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;The Spirit Jinyu Biological Pharmaceutical Co.,Ltd,Huhhot 010111,China)

机构地区：[1]西北农林科技大学动物医学院预防兽医系,杨凌712100 [2]中国动物卫生与流行病学中心,青岛266032 [3]金宇保灵生物药品有限公司,呼和浩特010111

出　　处：《畜牧兽医学报》2024年第5期2168-2175,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划(2021YFD1800300)。

摘　　要：旨在探究荚膜B型多杀性巴氏杆菌外膜囊泡(outer membrane vesicles, OMVs)的生物学特性及免疫保护效果。本研究利用超高速离心和密度梯度离心方法提取荚膜B型多杀性巴氏杆菌的OMVs,通过透射电子显微镜(transmission electron microscope, TEM)观察OMVs的形态及大小,采用SDS-PAGE、LC-MS/MS分析OMVs主要蛋白,以蛋白定量试剂测定其蛋白浓度以及鲎试剂法测定其内毒素含量,然后以新西兰白兔为动物模型,开展免疫保护效果评价。结果显示,荚膜B型多杀性巴氏杆菌的OMVs呈完整规则的球形结构,平均粒径108.3 nm,其中的蛋白质主要集中在33、40、53 ku处,内毒素含量为3.31×10^(6) EU·mL^(-1),用制备的OMVs以50μg·只^(-1)免疫新西兰白兔2次后,免疫兔血清抗体效价可达1∶405 600,外周血淋巴细胞IFN-γ和IL-4转录水平明显升高,对荚膜B型多杀性巴氏杆菌攻击的保护效率为75%。综上表明,荚膜B型多杀性巴氏杆菌的外膜囊泡具有良好的免疫保护效果,有作为候选疫苗的潜质,本研究为外膜囊泡疫苗的研究奠定了基础。In order to explore the biological characteristics and immune effect of outer membrane vesicles(OMVs)of capsular type B Pasteurella multocida.Outer membrane vesicles were extracted by ultra-high speed and density gradient centrifuge methods.The shape and size of the outer membrane vesicles were observed by transmission electron microscope(TEM),the proteins of OMVs were analyzed by SDS-PAGE and LC-MS/MS,the protein concentration was determined by the BCA method,and the endotoxin content was determined by limulus lysate test.Antibody titers and immune protection test were proceeded in New Zealand white rabbits.The results showed that OMVs had a regular spherical structure,the average particle size was 108.3 nm.OMVs contained a variety of protein bands,the protein bands were mainly 33,40,53 ku.The endotoxin content was 3.31×10^(6) EU·mL^(-1).The serum antibody titer of immunized New Zealand white rabbits was 1∶405600 when the injection dose was 50μg per rabbit.The transcription levels of IFN-γand IL-4 in peripheral blood lymphocytes increased.The immune protection efficiency against capsular tybe B Pasteurella maltocida was 75%.In conclusion,the OMVs of capsular type B Pasteurella multocida have good immunoprotective effcet and has the potential to be an candidate vaccine.This study lay the foundation for the research of OMVs vaccines.

关 键 词：多杀性巴氏杆菌 外膜囊泡 生物学特性 疫苗 

分 类 号：S852.612[农业科学—基础兽医学]