迟缓爱德华菌FliC-TolC融合蛋白表达及免疫特性研究  

Fusion expression and immune characteristics of FliC and TolC proteins in Edwardsiella tarda

作　　者：张志强 杜万年[2] 王苗 于秀剑 田占云 刘晨 李昕甜 吴同垒 康元环[3] 史秋梅[2] 刘莉[1] ZHANG Zhi-qiang;DUWan-nian;WANG Miao;YU Xiu-jian;TIAN Zhan-yun;LIU Chen;LI Xin-tian;WU Tong-lei;KANG Yuan-huan;SHI Qiu-mei;Liu Li(Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,China;Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;College of Animal Science and technology,Jilin agriculture University,Changchun 130118,China)

机构地区：[1]浙江省淡水水产研究所农业部淡水渔业健康养殖重点实验室/浙江省鱼类健康与营养重点实验室,浙江湖州313001 [2]河北省预防兽医学重点实验室河北科技师范学院,河北秦皇岛066004 [3]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国预防兽医学报》2020年第1期18-22,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：“农业部淡水渔业健康养殖重点实验室、浙江省鱼类健康与营养重点实验室或浙江省淡水水产遗传育种重点实验室”开放基金课题(ZJK201812);中国博士后科学基金(2017M611935);河北科技师范学院海洋专项(2018HY007)。

摘　　要：鞭毛蛋白FliC与外膜蛋白TolC均具有免疫保护效果,其中FliC蛋白具有疫苗佐剂的特性,但目前尚未有关迟缓爱德华菌(E.tarda)FliC-TolC融合产物免疫动物的免疫效果的相关报道。为研究E.tarda FliC-TolC融合蛋白的免疫特性,本研究利用融合PCR方法扩增获得fliC-tolC片段,构建重组载体pET-28a-fliC-tolC,并利用E.coli BL21表达系统表达了融合蛋白FliC-TolC。将纯化的FliC-TolC、TolC、FliC蛋白免疫小鼠后,以E.tarda强毒株攻毒评估免疫效果;并利用斑马鱼模型进行了平行试验。结果显示,本研究克隆了E.tarda fliC-tolC基因并表达和纯化了相应重组蛋白FliC-TolC;FliC-TolC蛋白激发小鼠产生的抗体水平优于FliC、TolC蛋白单独免疫组,表明FliC-TolC蛋白具有优良的免疫原性。攻毒试验表明FliC-TolC蛋白组小鼠对强毒株可产生较好的抵抗力,相对保护率为95%;斑马鱼攻毒试验相对保护率为60%。本研究证实了E.tarda重组蛋白FliC-TolC的免疫效力,为进一步研制E.tarda亚单位疫苗提供了参考和借鉴。Flagellin protein FliC and outer membrane protein TolC have immune protective effects.FliC protein has the characteristics of vaccine adjuvants,but there are no reports about the immune effect of Edwardsiella tarda FliC-TolC fusion products on animals.To study the immune characteristics of FliC-TolC fusion protein of Edwardsiella tarda,the fliC-tolC fragment was amplified by fusion PCR,and the recombinant plasmid pET-28 a-fliC-tolC was constructed.The fusion protein FliC-TolC was expressed by E.coli BL21 expression system.After immunizing mice with purified FliC-TolC,or individual TolC and FliC proteins,the immune efficacy was evaluated with the virulent strain of Edwardsiella tarda.Parallel experiments were carried out with the zebrafish model.The results showed that the fliC-tolC gene of Edwardsiella tarda was successfully cloned and the corresponding recombinant protein FliC-TolC was expressed and purified in this study.FliC-TolC protein stimulated mice to produce more antibodies than individual FliC and TolC protein groups,indicating that FliC-TolC protein exhibited had excellent immunogenicity.The results showed that the mice immunized with FliC-TolC protein had better resistance to virulent strains,the relative protection rate was 95%,and the relative protection rate of zebrafish was 60%.This study confirmed the immune efficacy of the recombinant protein FliC-TolC of Edwardsiella tarda,and provided a reference for further development of subunit vaccines against Edwardsiella tarda.

关 键 词：迟缓爱德华菌 FliC-TolC融合蛋白 蛋白表达 免疫保护力 

分 类 号：S852.61[农业科学—基础兽医学]