鱼类致病荧光假单胞菌外膜蛋白LolB多克隆抗体制备、鉴定与免疫活性分析  

Preparation,Identification and Immunoactivity Analysis of Polyclonal Antibody of Outer Membrane Protein LolB of Fish Pathogenic Pseudomonas fluorescens

作　　者：晁嘉 简思杰 孙薇 陈锐 丁锐 陈琛[1,3,5] 刘祥 CHAO Jia;JIAN Sijie;SUN Wei;CHEN Rui;DING Rui;CHEN Chen;and LIU Xiang(Chinese-German Joint Institute for Natural Product Research,College of Biological Sciences and Engineering,Shaanxi University of Technology,Hanzhong Shaanxi 723001,China;Fuyang Normal University,School of Biology and Food Engineering,Fuyang Anhui236037,China;Collaborative Innovation Center for Comprehensive Development of Biological Resources in Qinba Mountain Area,South Shaanxi,Hanzhong Shaanxi 723001,China;The State Key Laboratory of Biological Resources and Ecological Environment of Qinba,Shaanxi University of Technology(cultivation)Hanzhong Shaanxi 723001,China;Shaanxi Key Laboratory of Resources and Biology,Hanzhong Shaanxi 723001,China)

机构地区：[1]陕西理工大学生物科学与工程学院中德天然产物研究所,陕西汉中723001 [2]阜阳师范大学生物与食品工程学院,安徽阜阳236037 [3]陕南秦巴山区生物资源综合开发协同创新中心,陕西汉中723001 [4]陕西理工大学,秦巴生物资源与生态环境省部共建国家重点实验室(培育),陕西汉中723001 [5]陕西省资源生物重点实验室,陕西汉中723001

出　　处：《西北农业学报》2023年第10期1521-1533,共13页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省科技厅面上项目(2022JM-120);安徽省教育厅重点项目(2022AH051330);国家外国专家局项目(GDT20186100426)。

摘　　要：以荧光假单胞菌的外膜蛋白LolB为研究对象,评价其免疫学功能。生物信息学分析各菌株间LolB的亲缘关系;分子克隆构建LolB蛋白的表达菌株并确定最佳诱导表达条件;SDS-PAGE切胶纯化LolB蛋白并免疫红鲫;Western blotting检测红鲫LolB血清的特异性与效价,ELISA体外模拟红鲫LolB血清与荧光假单胞菌的相互识别作用,免疫因子(ACP、AKP、LZM、IgM)与细胞吞噬作用测定,抗氧化因子(CAT、SOD、MDA、GSH-Px)、炎症因子(IL-1β、TNF-α)表达分析及组织病理学探究LolB蛋白的免疫保护作用。结果显示,LolB在假单胞菌属间同源性和亲缘性更近,LolB抗血清可能提供交叉免疫保护作用。原核表达纯化LolB蛋白,获得最佳诱导表达条件。红鲫LolB抗血清具有较好特异性,效价达1∶200;且抗血清与荧光假单胞菌在体外存在相互识别作用;免疫因子以及细胞吞噬作用均呈上升趋势,表明LolB激活红鲫的非特异性免疫。红鲫免疫LolB蛋白攻毒荧光假单胞菌后,抗氧化因子以及炎症因子呈下降趋势,表明LolB蛋白具有抗氧化和消炎作用;组织病理学切片显示LolB蛋白保护红鲫的肾脏、脾脏、肠道的完整性。荧光假单胞菌LolB具有良好的免疫原性,有望成为渔用疫苗的候选抗原。Pseudomonas fluorescens is a pathogenic bacterium that affects fishery economy.To investigate the development of a potential fish vaccine against P.fluorescens,outer membrane protein LolB was selected as the research object to evaluate its immunologic function.The genetic relationship of LolB among different strains was analyzed by bioinformatics.Molecular cloning techniques were used to construct the LolB protein-expressing strain,and optimal conditions for its expression were determined.After purified by the inclusion body washing and SDS-PAGE gel-cutting,Carassius auratus was immunized with LolB protein to prepare polyclonal antibody.The specificity and titer of LolB serum of C.auratus were detected by Western blotting,while the recognition effect between LolB serum and P.fluorescens was simulated using enzyme-linked immunosorbent assay(ELISA)in vitro.Non-specific immunity of C.auratus was assessed by immunologic factors(ACP,AKP,LZM,IgM)and cell phagocytosis.Antioxidant factors(CAT,SOD,MDA,GSH-Px),inflammatory factors(IL-1β,TNF-α)and histopathological sections were analyzed to explore the immunoprotective effects of LolB protein.The results showed that LolB exhibited a closer homology and conservative evolution among Pseudomonas spp,suggesting that LolB antiserum may possess a cross-protective capabilities.The successful cloning,expression,and purification of the LolB protein were achieved.The optimal conditions for induction expression were determined as follows:OD 600=0.8,IPTG=0.1 mmol/L,inducing temperature at 37℃,and inducing time for 12 h.C.auratus displayed enhanced specificity with the titer of 1∶200.In vitro experiments demonstrated immune recognition between the LolB antiserum and P.fluorescens,exhibiting a titer of 1∶3200.After the C.auratus was immunized with LolB and challenged with P.fluorescens,the immunologic factors(ACP,AKP,LZM,IgM)and cell phagocytosis exhibited increasing trend,suggesting that LolB activated the non-specific immunity of C.auratus.Antioxidant factors(GSH-Px,MDA,SOD,CAT

关 键 词：荧光假单胞菌 外膜蛋白LolB 主动免疫 组织病理学 红鲫 

分 类 号：S943[农业科学—水产养殖]