迟缓爱德华菌鞭毛蛋白FlgJ原核表达及免疫原性
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摘要
为研究迟缓爱德华菌鞭毛蛋白FlgJ的免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌flgJ基因,构建重组载体pET-32a-flgJ,将其转化大肠杆菌BL21后进行诱导表达,表达产物经SDS-PAGE和Western blot分析显示,重组蛋白大小约54 000;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌分离株ET-13进行攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为70%。本研究克隆了迟缓爱德华菌外膜蛋白flgJ基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组FlgJ蛋白亚单位疫苗的研制奠定基础。
To study the immunoprotection of FlgJ against Edwardsiella tardain mice,the encoding gene,flgJ,was amplified by PCR fromE.tardaisolate ET-13,and furthered cloned into the pET-32 avector.The recombinant plasmid was transformed into E.coli BL21(DE3)cells,in which a recombinant FlgJ(rFlgJ)protein was expressed by inducing with IPTG.SDS-PAGE and Western blot analysis showed that the expressed rFlgJ was about 54 000,which was recognized by the positive serum against E.tarda.The mice were immunized intramuscularly with purified rFlgJ,and challenged with the E.tardaisolate ET-13.Result showed that the rFlgJ provided a 70% protection for immunized mice.The data demonstrated that the FlgJ could be used as the antigen for developing the subunit vaccine
To study the immunoprotection of FlgJ against Edwardsiella tardain mice,the encoding gene,flgJ,was amplified by PCR fromE.tardaisolate ET-13,and furthered cloned into the pET-32 avector.The recombinant plasmid was transformed into E.coli BL21(DE3)cells,in which a recombinant FlgJ(rFlgJ)protein was expressed by inducing with IPTG.SDS-PAGE and Western blot analysis showed that the expressed rFlgJ was about 54 000,which was recognized by the positive serum against E.tarda.The mice were immunized intramuscularly with purified rFlgJ,and challenged with the E.tardaisolate ET-13.Result showed that the rFlgJ provided a 70% protection for immunized mice.The data demonstrated that the FlgJ could be used as the antigen for developing the subunit vaccine
引文
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[5]李宁求,余露军,付小哲,等.3种免疫途径下迟缓爱德华菌菌蜕疫苗对欧洲鳗的免疫保护效果[J].水产学报,2014(11):1910-1916.
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[8]郭志燕,周明旭,段强德,等.细菌鞭毛的致病性及其免疫学应用的研究进展[J].微生物学报,2014(3):251-260.
[9]LI X,XU J,XIE Y,et al.Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544infection in BALB/c mice[J].Vet Microbiol,2012,161(1/2):137-144.
[10]李洪涛,张新涛,刘明,等.A型禽流感病毒不同拷贝数基质蛋白胞外区基因的原核表达及免疫保护力研究[J].中国预防兽医学报,2010(10):795-799.
[11]徐全圆,石明,杨夷平,等.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,2015(2):132-135.
[12]王宝松,李明义,单虎,等.猪链球菌2型MRP-FBP串联表达蛋白对小鼠的免疫保护力[J].中国兽医科学,2009(6):522-526.
[13]邵峰.病原细菌和宿主天然免疫系统相互拮抗的机制研究[J].中国基础科学,2012(1):7-13.
[14]潘志明,文科,游猛,等.沙门菌鞭毛蛋白增强新城疫病毒融合蛋白在小鼠中的免疫原性[J].生物技术通讯,2009(6):827-829.
[15]焦新安,刘秀梵,王志亮,等.沙门氏菌鞭毛蛋白属共同抗原表位及其免疫生物学特性[J].动物医学进展,1997(1):17-20.
[16]罗佳,李薇,段云峰,等.Toll样受体5和鞭毛蛋白的相互作用影响宿主区分病原菌和益生菌(英文)[J].微生物学通报,2014(7):1368-1375.
[2]房文红,周凯.诺氟沙星对溶藻弧菌和恩诺沙星对迟缓爱德华菌的抗生素后效应[J].海洋渔业,2005(1):44-48.
[3]SUEZAWA C,YASUDA M,NEGAYAMA K,et al.Identification of genes associated with the penetration activity of the human type of Edwardsiella tarda Edw GⅡthrough human colon epithelial cell monolayers[J].Microb Pathog,2016,95:148-156.
[4]HIRAI Y,ASAHATA-TAGO S,AINODA Y,et al.Edwardsiella tarda bacteremia.A rare but fatal water-and foodborne infection:Review of the literature and clinical cases from a single centre[J].Can J Infect Dis Med Microbiol,2015,26(6):313-318.
[5]李宁求,余露军,付小哲,等.3种免疫途径下迟缓爱德华菌菌蜕疫苗对欧洲鳗的免疫保护效果[J].水产学报,2014(11):1910-1916.
[6]KATHARIOS P.Virulence regulation during late infection by a fish pathogen;sense and sensibility of bacteria may lead to novel vaccine development strategy[J].Virulence,2017(1):1-3.
[7]黄新新,陆承平.迟缓爱德华菌外膜蛋白抗原性分析[J].中国免疫学杂志,2002(6):385-387.
[8]郭志燕,周明旭,段强德,等.细菌鞭毛的致病性及其免疫学应用的研究进展[J].微生物学报,2014(3):251-260.
[9]LI X,XU J,XIE Y,et al.Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544infection in BALB/c mice[J].Vet Microbiol,2012,161(1/2):137-144.
[10]李洪涛,张新涛,刘明,等.A型禽流感病毒不同拷贝数基质蛋白胞外区基因的原核表达及免疫保护力研究[J].中国预防兽医学报,2010(10):795-799.
[11]徐全圆,石明,杨夷平,等.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,2015(2):132-135.
[12]王宝松,李明义,单虎,等.猪链球菌2型MRP-FBP串联表达蛋白对小鼠的免疫保护力[J].中国兽医科学,2009(6):522-526.
[13]邵峰.病原细菌和宿主天然免疫系统相互拮抗的机制研究[J].中国基础科学,2012(1):7-13.
[14]潘志明,文科,游猛,等.沙门菌鞭毛蛋白增强新城疫病毒融合蛋白在小鼠中的免疫原性[J].生物技术通讯,2009(6):827-829.
[15]焦新安,刘秀梵,王志亮,等.沙门氏菌鞭毛蛋白属共同抗原表位及其免疫生物学特性[J].动物医学进展,1997(1):17-20.
[16]罗佳,李薇,段云峰,等.Toll样受体5和鞭毛蛋白的相互作用影响宿主区分病原菌和益生菌(英文)[J].微生物学通报,2014(7):1368-1375.