重組細(xì)粒棘球蚴親肌肉抗原抗小鼠感染細(xì)粒棘球蚴免疫保護(hù)性及其機(jī)制的研究

1、（（（寧夏醫(yī)科大碩士學(xué)位論文中文摘要重組細(xì)粒棘球蚴親肌肉抗原抗小鼠感染細(xì)粒棘球蚴的免疫保護(hù)性及其機(jī)制研究摘要目的:對(duì)構(gòu)建的純化重組親肌肉抗原（rEg.myophilin）進(jìn)行免疫保護(hù)和免疫機(jī)制的研究，為包蟲病分子疫苗的研制提供新的候選分子。方法：(1)疫苗制備：重組Eg.myophlilinpET28a工程菌株的大量表達(dá)、純化及疫苗制備。（2）動(dòng)物分組方案：選取111只小鼠隨即分為A、B對(duì)照組和實(shí)驗(yàn)組C組，分別對(duì)其進(jìn)行皮下PBS、PBS

2、佐劑和PBS加佐劑加候選疫苗的注射，之后分別用同樣的試劑對(duì)其對(duì)應(yīng)組進(jìn)行兩次加強(qiáng)免疫；最后用1500只細(xì)粒棘球蚴的原頭蚴腹腔攻擊感染。（3）免疫保護(hù)力計(jì)算：（免疫保護(hù)力計(jì)算公式：減囊率=1實(shí)驗(yàn)組平均包囊數(shù)對(duì)照組平均包囊數(shù)100%）。4）對(duì)小鼠進(jìn)行免疫保護(hù)機(jī)制的研究，①體液免疫抗體指標(biāo)的測定：從領(lǐng)取小鼠開始按不同的時(shí)間點(diǎn)，分別分批次對(duì)小鼠采血，進(jìn)行血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平的測定；②細(xì)胞免疫檢測：從領(lǐng)

3、取小鼠即開始按不同的時(shí)間點(diǎn)，分別分批次對(duì)小鼠取脾臟，并脾細(xì)胞刺激培養(yǎng)，監(jiān)測其細(xì)胞因子IFNγ、IL4和IL10的水平。結(jié)果：(1)rEg。myophilin能誘導(dǎo)小鼠產(chǎn)生86.11%以上的免疫保護(hù)力。2）采集血清進(jìn)行ELISA血清抗體IgG、IgG1、IgG2a、IgG2b、IgG3和IgE以及細(xì)胞因子IFNγ、IL4和IL10的檢測，結(jié)果表明：抗體IgG、IgG1、IgG2a以及IgE，和細(xì)胞因子IFNγ、IL4分別在免疫之后出現(xiàn)了增

4、高，對(duì)照組IL10在攻擊感染后出現(xiàn)了明顯的增高，而在實(shí)驗(yàn)組則沒有相應(yīng)的增高。結(jié)論：(1)rEg.myophilin為極具應(yīng)用潛質(zhì)的疫苗候選分子。2）對(duì)rEg.myophilin產(chǎn)生的免疫保護(hù)性可能機(jī)制：①抗體介導(dǎo)的體液免疫，IgG及其亞類IgG1和IgE介導(dǎo)的體液免疫，在抗細(xì)粒棘球蚴感染過程中發(fā)揮重要作用；②細(xì)胞免疫中，細(xì)胞因子水平變化顯示Th1類免疫應(yīng)答在試驗(yàn)組免疫保護(hù)中占優(yōu)勢作用，有利于機(jī)體抵御細(xì)粒棘球蚴的感染；③該疫苗候選分子的注

5、入使得免疫抑制機(jī)制得到了有效地阻遏，有利于機(jī)體的抗寄生蟲。④體液免疫和細(xì)胞免疫相輔相成，相互促進(jìn)，表現(xiàn)在：IL4在IgE亞型類別轉(zhuǎn)換中起到了II寧夏醫(yī)科大碩士學(xué)位論文英文摘要Studiesofimmunoptectionitsmechanismofrecombinantmyophilin（rEg.myophilin）againstEchinococcusGranulosusinmiceAbstractObject:Toidentifyv

6、accinecidateacterofrecombinantmyophilinagainsthydatidosis，throughdetectingtheimmunoprotectionitsmechanism.Methods:(1)WeexpressedpurifytheproteinfromrecombinantengineeringbacteriarEg.myophlilinpET28athenidentifieditsimmun

7、oacter.(2)WepreparatedthevaccinebyemulsifyingrEg.myophilinwithcompleteimcompleteFreundsadjuvantfimmunizationbooster.(3)ThreegroupsoffemalemicewereusedgroupedthemasABCwhichwereimmunizedsubcutaneouslywithphosphatebuffereds

8、aline(PBS)Freund’scompleteadjuvant(FCA)inPBSrEg.myophilinwithFCArespectively.Twoweeksafteradditionaltwoboostersmicewerechallengedwith1500Egprotoscoleces(PSCs)intraperitoneally.(4)immunoprotectionwascomputedbyfmula:reduct

9、ionincystload=1(averageofcystsintestgroupaverageofcystsincontrolgroup)100%.Serumspleenswerecollectedatdifferenttimepointbysacrificing5miceineachgroup.Antibodiesinserumcytokinesinsupernatantoflymphocytesculturesweremeasur

10、ed.Result:miceimmunizedwithrEg.myophilinproducedeffectiveimmunityprotection(reductionsincystloadare89.86%86.11%comparedwithgroupAB).ComparedtocontrolgroupsmiceimmunizedwithrEg.myophilinshowedthatspecificIgGIgG1IgG2aIgEac

11、companiedbyIFNγIL4significantlyincreasedafteronemetimesofimmunizationbutneitherIgG2bnIgG3increased.NeverthelessIL10significantlyincreasedinthecontrolgroupscomparedwithrEg.myophilingroupinwhichitappearedadeclinetrend.Conc

12、lusion：rEg.myophilinwasidentifiedasavaccinecidatefitseffectiveprotection(reductionincystload86.11%)rEg.myophilinshowedaeffectivelyprotectiveimmunitywhichmayattributeto:(1)IgGsIgEmediatedhumalimmuneresponse(2)cellmediated