摘要
为探究嗜水气单胞菌外膜蛋白AHA1182的免疫原性,依据AHA1182氨基酸序列,采用生物信息学分析嗜水气单胞菌、杀鲑气单胞菌、简氏气单胞菌、多样气单胞菌、舒氏气单胞菌、产碱普罗维登斯菌以及爱德华氏菌之间的亲缘关系;构建AHA1182原核表达菌株并确定最佳诱导表达条件;采用包涵体洗涤及SDS-PAGE切胶纯化方法获得AHA1182蛋白并免疫红鲫;利用Western blot检测红鲫抗AHA1182蛋白血清的特异性与效价;采用酶联免疫吸附(ELISA)法模拟红鲫抗AHA1182蛋白抗体血清与嗜水气单胞菌的体外相互识别作用;通过测定酸性、碱性磷酸酶(ACP、AKP)活性与细胞吞噬作用,评价非特异性免疫;利用组织病理学切片探究AHA1182蛋白免疫对红鲫内脏的影响。结果显示,AHA1182蛋白家族在不同菌株间均具有亲缘性,尤其在气单胞菌间亲缘关系较近,由此推测,AHA1182蛋白抗血清具有交叉免疫保护作用;成功克隆、表达、纯化AHA1182,最佳诱导条件:菌液浓度OD_(600)=1.0,IPTG终浓度为0.5 mmol/L,在28℃条件下诱导8 h;红鲫AHA1182蛋白抗血清具有良好的特异性,AHA1182蛋白抗体与嗜水气单胞菌特异性结合(滴度可达1∶3200);AKP、ACP、细胞吞噬作用均显示,AHA1182蛋白可激活红鲫非特异性免疫。从组织病理学切片可以看出,AHA1182蛋白对红鲫内脏结构无影响。综上,嗜水气单胞菌AHA1182蛋白具有良好的免疫原性。
To evaluate the immunogenicity of outer membrane protein AHA1182 of Aeromonas hydrophila,bioinformatics method was firstly performed to analyze the relationship among A.hydrophila,A.salmonicida,A.schubertii,A.diversa,A.jandaei,Providencia alcalifaciens and Edwardsiella ictaluri.Recombinant expression strain of AHA1182 was constructed by molecular cloning and the optimal expression conditions were identified.AHA1182 was purified by inclusion body washing and SDS-PAGE,and was used to immunize Carassius auratus.Western blot was used to assess the specificity and titer of the C.auratus serum,and enzyme-linked immunosorbent assay(ELISA)was used to simulate the recognition effect between anti-AHA1182 serum and A.hydrophila.Nonspecific immunity was assessed by acid phosphatase(ACP),alkaline phosphatase(AKP)and leukocyte phagocytosis.Furthermore,the toxicity of AHA1182 immunization to C.auratus was investigated by histopathological sections of visceral organ.The results showed that AHA1182 was genetically related among different bacteria,especially in Aeromonas,which meant that AHA1182 serum might have cross-immune protective ability.AHA1182was cloned,expressed and purified successfully,and the optimal expression conditions for AHA1182included strain OD_(600)value of 1.0,isopropyl-β-D-thiogalactoside(IPTG)concentration of 0.5 mmol/L,inducing temperature of 28℃and inducing time of 8 hours.Western blot showed that AHA1182 serum had a high specificity and could recognize A.hydrophila in vitro with the titer of 1∶3200.The results of AKP,ACP,and leukocyte phagocytosis indicated that AHA1182 stimulated the non-specific immune of C.auratus.AHA1182 immunization had no toxicity to the visceral structure of C.auratus.The results suggest that A.hydrophila AHA1182 has good immunogenicity.
作者
简思杰
晁嘉
孙薇
陈锐
丁锐
陈琛
刘祥
JIAN Sijie;CHAO Jia;SUN Wei;CHEN Rui;DING Rui;CHEN Chen;LIU Xiang(Chinese-German Joint Institute for Naturel Product Research/College of Biological Sciences and Engineering,Shaanxi University of Technology,Hanzhong 723001,China;School of Biology and Food Engineering,Fuyang Normal University,Fuyang 236037,China;Qinling-Bashan Mountains Bioresources Comprehensive Development C.I.C.,Hanzhong 723001,China;Qinba State Key Laboratory of Biological,Hanzhong 723001,China;Shaanxi Province Key Laboratory of Bio-resources,Hanzhong 723001,China)
出处
《河南农业科学》
北大核心
2022年第11期135-144,共10页
Journal of Henan Agricultural Sciences
基金
陕西省科技厅面上项目(2022JM-120)
国家外国专家局项目(GDT20186100426)。
