牛溶血性曼氏杆菌白细胞毒素、脂蛋白E和外膜蛋白A的免疫原性研究  

The immunogenicity of leukotoxin, PlpE and OmpA of bovine Mannheimia haemolytica

作　　者：李奕欣 姜志刚[1] 于力[1] LI Yi-xin;JIANG Zhi-gang;YU Li(Division of Livestock Infections Disease,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/牛羊传染病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第7期753-758,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：内蒙古自治区科技重大专项(2020ZD0006)。

摘　　要：在应激条件下,溶血性曼氏杆菌(Mh)能够引起牛的纤维素性肺炎或胸膜肺炎,对全球养牛业危害严重,免疫接种是防控Mh病的主要手段。为了评价牛源A1型Mh脂蛋白E(PlpE)、外膜蛋白A(OmpA)以及白细胞毒素(LKT)的免疫原性,本研究分别将PlpE、OmpA以及LKT的毒力效应蛋白LKTA的编码基因克隆至原核表达载体pET-28a,并在大肠杆菌中表达,采用亲和层析或电洗脱的方法对重组蛋白进行提纯。将纯化的重组蛋白rPlpE、r OmpA和rLKTA分别或组合接种BALB/c小鼠,首次免疫14 d后采用相同方法加强免疫,分别于首次免疫后14 d、21 d、28 d和35 d采集小鼠血清。采用间接ELISA方法检测小鼠血清中针对Mh的特异性IgG抗体水平,结果显示各重组蛋白均能够诱导小鼠产生针对Mh的特异性IgG抗体,而rPlpE和rLKTA诱导的抗体水平明显高于r OmpA。采用牛淋巴细胞中和试验检测rLKTA免疫小鼠血清中针对LKT的中和抗体水平,结果显示rLKTA能够诱导小鼠产生高水平的中和抗体,二免后21 d小鼠血清中的平均中和抗体滴度可达363。以致死剂量的A1型Mh菌液对免疫小鼠进行攻击,结果显示rPlpE对小鼠的保护率为50%(2/4),而rOmpA对小鼠无保护作用。综上所述,r LKTA和r PlpE能够诱导小鼠产生针对Mh的保护性免疫应答,是A1型Mh的优势保护性免疫原组分,本研究为全面了解牛源A1型Mh主要免疫保护性抗原以及Mh新型疫苗的研究奠定了实验基础。Mannheimia haemolytica(Mh) can cause bovine fibrinous pneumonia or pleuropneumonia under stress conditions,which is a serious harm to the global cattle industry. Immunization is the main method to prevent and control Mh disease. In this study, in order to evaluate the immunogenicity of leukotoxin(LKT), lipoprotein E(Plp E) and outer membrane protein A(Omp A)of A1 bovine Mh, the encoding genes of Plp E, Omp A and the virulence effector protein of LKT(LKTA) were cloned into the prokaryotic expression vector p ET-28 a, respectively. Each recombinant protein was successfully expressed in E. coli and purified by affinity chromatography or electroelution. Purified r LKTA, r Plp E and r Omp A recombinant proteins were immunized to BALB/c mice separately or in combination twice on days 0 and 14. Sera from immunized mice were collected at 14, 21, 28 and 35 days after the first immunization. Indirect ELISA was used to measure the level of Mh-specific Ig G antibodies in the sera from immunized mice, and the results showed that all recombinant proteins could induce mice to produce Mh-specific Ig G antibodies,while the level of Ig G antibodies induced by r Plp E and r LKTA were significantly higher than those induced by r Omp A. The bovine lymphocyte neutralization test was used to evaluate the level of neutralizing antibodies against LKT in the sera from r LKTAimmunized mice, and the results showed that r LKTA could induce mice to produce high levels of LKT neutralizing antibodies with the average neutralizing titer of 363 on 21 days after the boost immunization. The immunized mice were challenged with a lethal dose of Mh A1, and the results showed that immunization with r Plp E produced a protection rate of 50%(2/4) for mice, while r Omp A had no protective effect on mice. In summary, r LKTA and r Plp E can induce protective immune responses against Mh in mice, and are the dominant protective antigen components of Mh A1. This study lays an experimental basis for comprehensive understanding of the main immunoprotective antig

关 键 词：牛溶血性曼氏杆菌 白细胞毒素 脂蛋白E 外膜蛋白A 免疫原性 

分 类 号：S852.61[农业科学—基础兽医学]