鳗弧菌四种鞭毛蛋白的重组表达、分离纯化及免疫应用研究-生药学专业论文.docxVIP

具有最好的抑菌效应。为了检测不同鞭毛蛋白作为佐剂的应用可能，我们选用之前报道的具有一定 免疫保护效应的亚单位疫苗 rEsal，进行免疫佐剂效应研究。为此目的，我们分 别用rEsal以及rEsal加四种鞭毛蛋白免疫牙鲆。结果表明，添加rFlaE 作为佐剂 时，实验组的相对免疫保护率提高20%以上，对应的抗血清中特异抗体效价上升 2倍以上，免疫相关基因IL-β、TNF、IgM、MHCⅠα、CD8α、CD40、TLR5等 的表达水平也明显上调3-7倍。总之，以上结果表明鳗弧菌四种鞭毛蛋白FlaA,FlaB,FlaD和FlaE均具有一 定的免疫保护效应，其中rFlaB的免疫保护效率最高；同时，rFlaE作为亚单位 疫苗的佐剂能显著提高疫苗的免疫保护效率。这些研究结果证明鳗弧菌的不同鞭 毛蛋白具有不同的免疫学功能，为鳗弧菌的病害防治提供了新的途径。关键词：鳗弧菌；鞭毛蛋白；重组表达；免疫保护；佐剂Thestudyoffour flagellinsofVibrioanguillarum:expression, purificationoftheflagenllinsinEscherichiacoliand their immuneapplication researchAbstractWiththerapiddevelopmentofscienceandtechnology,moreandmoreadvanced breedingtechniquesandmeansareusedinaquaculture.Butontheotherhand,the frequentfishdiseasesseriouslyrestrictthedevelopmentofaquaculturebecauseit’s startrapidlyandhardtocontrol.Thepathogenicbacteriaespeciallythevibriohave causedsevereeconomicloss,andincreasinglybecomethefocus ofattention.VibrioanguillarumisamarinepathogenofVibrioswhichcausevibriosisin closeto50species offish,includingculturedandwildfish,mollusks,andcrustaceans, inmarine,brackish,andfreshwater.Moreandmorereportsindicatethatthe flagenllum-mediatedmotilityisrequiredforoptimalbacterialvirulenceinmany species ofVibrio,includingVibrioanguillarum.ThegenomeofV.anguillarumcontainsfiveflagellingenes,i.e.flaA,flaB, flaC, flaD,andflaE.Inthisstudy,weanalyzedthevaccinepotentialandadjuvanticityof FlaA,FlaB,FlaD,andFlaEinamodelofJapaneseflounder(Paralichthysolivaceus). Forthispurpose,recombinantFlaA,FlaB,FlaD,andFlaEwereexpressedinand purifiedfromEscherichiacoli(BL21).InvivoimmunogenicityanalysisshowedthatantibodiesagainstrFlaA,rFlaB, rFlaD,andrFlaEweredetectedinratantiserumraisedagainstliveV.anguillarum, withthehighestantibodylevelbeingthatagainstrFlaB,about2-4timeshigherthan rFlaA,rFlaD,orrFlaE.Whenadministeredintoflounderviaintraperitonealinjection, rFlaA,rFlaD,andrFlaEinducedcomparablerelativepercentsurvival(RPS)rates,whichweresignificantlylowerthanthatinducedbyrFlaB;rFlaB75%，rFlaA53.9%,rFlaD55.7%，rFlaE49.9%.Specif