嗜水气单胞菌外膜蛋白AHA2991的原核表达、纯化与免疫原性研究  

Prokaryotic expression,purification and immunogenicity studies of outer membrane protein AHA2991 Aeromonas hydrophila

作　　者：简思杰 晁嘉 孙薇 陈春琳 陆娟 刘勇 刘祥 JIAN Si-jie;CHAO Jia;SUN Wei;CHEN Chun-lin;LU Juan;LIU Yong;LIU Xiang(School of Biology and Food Engineering,Fuyang Normal University,Fuyang,Anhui 236037,China;Chinese-German Joint Institute for Natural Product Research/College of Biological Sciences and Engineering,Shaanxi University of Technology,Hanzhong,Shaanxi 723001,China)

机构地区：[1]阜阳师范大学生物与食品工程学院,安徽阜阳236037 [2]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

出　　处：《西南农业学报》2023年第4期876-884,共9页Southwest China Journal of Agricultural Sciences

基　　金：国家外国专家局项目(GDT20186100426);安徽省教育厅科学研究重点项目(2022AH051330);陕西省科技厅面上项目(2022JM-120);阜阳师范大学人才项目(2022KYQD0003)。

摘　　要：【目的】评价嗜水气单胞菌外膜蛋白AHA2991的免疫学功能,为探索嗜水气单胞菌渔用疫苗候选抗原提供理论依据。【方法】通过生物信息学分析AHA2991氨基酸序列,揭示不同菌株间的亲缘关系;分子克隆构建AHA2991表达菌株并确定最佳诱导条件;包涵体洗涤及SDS-PAGE切胶纯化获得AHA2991并免疫红鲫;Western blotting检测AHA2991红鲫血清的特异性与效价;ELISA模拟AHA2991红鲫血浆与嗜水气单胞菌的体外相互识别作用;酸性、碱性磷酸酶(ACP、AKP)测定与细胞吞噬作用评价AHA2991非特异性免疫功能;组织病理学切片探究AHA2991免疫对红鲫内脏的影响情况。【结果】AHA2991蛋白家族在不同菌株间具有同源性,不同菌株间亲缘关系较近,尤其在气单胞菌间亲缘关系更近;AHA2991的最佳诱导条件为:菌液浓度OD600=1.0,IPTG终浓度0.1 mmol/L,在28℃下诱导8 h。红鲫AHA2991抗血清具有良好的特异性,其效价为1∶800;体外AHA2991血浆对嗜水气单胞菌具有识别作用,滴度可达1∶3200;ACP、AKP指标及细胞吞噬作用均显示AHA2991激活红鲫非特异性免疫;组织病理学切片表明AHA2991免疫对红鲫内脏结构无影响。【结论】嗜水气单胞菌AHA2991具有良好的免疫原性,有望成为嗜水气单胞菌渔用疫苗的候选抗原成分。[Objective]The immunogenicity of outer membrane protein AHA2991 of Aeromonas hydrophila was evaluated to provide a theoretical basis for the application of candidate vaccine antigen to prevent A.hydrophila infection in fish.[Method]The amino acid sequence of AHA2991 was performed to analyze the relationship among bacteria by bioinibnnatics.The AHA2991 expression strain was constructed by molecular cloning and the optimal induction conditions were identified.AHA2991 was purified by inclusion body washing and SDS-PAGE,and immunized to Carassius auratus.Western blotting was used to assess the specificity and titer of the C.auratus serum,and ELISA was used to simulate the recognition effect between AHA2991 serum and A.hydrophila.Nonspecific immunity of AHA2991 was assessed by acid phosphatase(ACP),alkaline phosphatase(AKP)and leukocyte phagocytosis.Furthermore,the toxicity of AHA2991 immunization to C.auratus was investigated by histopathological sections of visceral organ.[Result]AHA2991 had homology related among different bacteria,relatively close relationship between different strains,especially in Aeromonas.AHA2991 was cloned,expressed and purified successfully,and the optimum expressing conditions of AHA2991 were as follows:Strain ODgo value of 1.0,IPTG concentration of 0.1 mmol/L,inducing temperature of 28℃,and inducing time of 8 hours.The AHA2991 serum of C.auratus had a high specificity and the titer was 1:800,and the AHA2991 plasma could recognize to A.hydrophila in vitro with the titer to 1:3200.The results of AKP,ACP and leukocyte phagocytosis indicated that AHA2991 stimulated the non-specific immune response to C.auraius.AHA2991 immunization had no toxicity to the visceral structure of C.auratus by histopathological sections.[Conclusion]A.hydrophila AHA2991 has immunogenicity and is expected to be a candidate vaccine against A.hydrophila infection in fish.

关 键 词：嗜水气单胞菌 外膜蛋白AHA2991 红鲫血浆 组织病理学 主动免疫 

分 类 号：Q71[生物学—分子生物学]