雞IL-2受體α亞基mRNA在柔嫩艾美耳球蟲感染中的變化.pdf

1、中文摘要雞IL2受體0t亞基mRNA在柔嫩艾美耳球蟲感染中的變化摘要雞球蟲病(Coccidiosis)是由艾美耳球蟲(Eimeria)l起的一種嚴(yán)重危害養(yǎng)禽業(yè)發(fā)展的胞內(nèi)寄生原蟲病，主要引起宿主細(xì)胞免疫反應(yīng)白細(xì)胞介素2(IL2)是主要的調(diào)節(jié)機(jī)體細(xì)胞免疫的淋巴因子IL2通過與其受體(IL2R)結(jié)合發(fā)揮調(diào)節(jié)免疫細(xì)胞的作用，所以IL2R基因的誘導(dǎo)表達(dá)是淋巴細(xì)胞克隆擴(kuò)增的基礎(chǔ)和關(guān)鍵。研究發(fā)現(xiàn)IL2的產(chǎn)生與T細(xì)胞的增值和抗球蟲效應(yīng)密切相關(guān)球蟲感染對(duì)

2、IL2R表達(dá)的影響尚無報(bào)道本論文研究了柔嫩艾美耳球蟲感染過程中雞中樞與外周免疫器官中白細(xì)胞介素2受體a亞基(chIL2Rc【)分子表達(dá)的變化，旨在揭示球蟲感染對(duì)雛雞IL2R表達(dá)的影響以脾淋巴細(xì)胞總RNA為模板，通過RTPCR方法克隆了雞白細(xì)胞確rCr2受體c【亞基(chlL2Ra)編碼區(qū)基因序列分析發(fā)現(xiàn)，雞IL2RⅡ編碼區(qū)基因與GenBank上已發(fā)表序列的核苷酸同源性為992％，氨基酸同源性為995％Chll2Ro【與其它哺乳動(dòng)物112

3、Rc【在核苷酸和氨基酸水平上的同源性分別為296％545％和188％28O％雞IL2RGt成熟多肽鏈由胞外區(qū)(164ilia)，穿膜區(qū)(22aa)和胞漿區(qū)(4aa)三部分組成，其N端4～21區(qū)域氨基酸和C端194212區(qū)域氨基酸疏水性強(qiáng)，中間22～193區(qū)域氨基酸高度親水；將chII廣2Ro【胞外段基因克隆到pET32a()中獲得重組質(zhì)粒pET32aexchlL2Ra，轉(zhuǎn)化宿主茵EcoliRossed刑(DE3)后用IPTG誘導(dǎo)表達(dá)的融

4、合蛋白約34ku為了檢測(cè)雞IL2Ro【亞基(chIL2Ro【)在球蟲感染中的變化。采取柔嫩艾美耳球蟲感染后第一、三、五、七天的三周齡雞外周血、脾臟，盲腸扁桃體和法氏囊無菌分離淋巴細(xì)胞細(xì)胞分成兩份，一份加培養(yǎng)液和ConA刺激劑體外培養(yǎng)，另一份不加ConA培養(yǎng)收集培養(yǎng)的細(xì)胞提RNA用RTPCR方法，以Bactin為內(nèi)參照，定量分析淋巴細(xì)胞中IL2Ra的mRNA豐度。結(jié)果表明，未經(jīng)ConA刺激的實(shí)驗(yàn)組，和對(duì)照組相比，外周血，脾臟和盲腸扁桃體淋

5、巴細(xì)胞IL2RamRNA表達(dá)量感染后第五天顯著升高(P005)，第七天達(dá)到高峰法氏囊中IL2Ra第五天顯著升高(P005)經(jīng)過ConA刺激的實(shí)驗(yàn)組，和對(duì)照組相比，感染柔嫩艾美耳球蟲后第一天，脾臟和盲腸扁桃體淋巴細(xì)胞中IL2RamRNA的相對(duì)濃度顯著降低伊005)隨后外周血和脾臟淋巴細(xì)胞中IL2Rc【mRNA相對(duì)豐度在第三天和第七天均顯著升高fP005)，第七天達(dá)到高峰mCHANGEOFIL2RECEPTOR0【CHAINmRNAOFCH

6、ICKENINFECTEDWITHE懈R陰兀巴吼L4ABSTRACTAviancoecidiosisisacytoplasticentozoicparasitediseaseandmaycausesevereeconomiclosstotheindustryofpoultrycellmediatedimmunityplaysachiefroleinprotectingchickenfromcoecidiosisInterleukine一

7、2(IL2)secretedbyactivatedTcellsisallessentialcytokineformediationofcell—mediatedimmunityItplaysaimportantroleinmediatingimmu∞ceilsbycombiningwithitsreceptor(BER、secretedintargetedcellmembraneSothatIL2Rplaysakeyroleinacti

8、vationofthelymphocyteItwasreportedthatIL2isinfastassociationwithexpansionofactivatedTcellsandanticoccidiosisStillnecognizedpaperonCoccidiosisinducedchangesinchickenⅡ，2RmRNAexpressionForthatChangesininterleukin2receptoral

9、pha(IL2RⅡ)chainmRNAexpressedinimmuneorgansofchjckeninfcctedwithEimeriatenellawasstudiedThenucleotidesequenceencodingmatureproteinofchickeninterleukin2receptoralpha(chlL2RoL、chainwasobtainedfromchickenspleenlymphocytebyRT

10、PCRneidentityofchlL2R0ttothatinGenBankatnucleotideandaminoacidlevelwas992％and995％respectivelyChlL2R?！緃adalowidentitytothoseofothermammalian，rangingfrom296％to545％atnucleotidelevelandfrom188％to280％ataminoacidlevelMaturedpr

11、oteinofchlL2Rawasconstitutedbyexchceliulardomain(164aa)，transmembranedomain(22aa)andintrocellulardomain(4aa)The4～21aminoacidresiduesofN—endand194212ofCendofchIL2Rotproteinwerehydrophilicityplotmiddleofitwerehydrophobicpr

12、opertyeDNAencodingchIL2RⅨ(exchlL2R0t)extracellulardomainwasamplifiedandsubclonedintotheexpressionvectorpET32a()ExchIL2RpET32awasexpressedinEcoliRossetaVM(DE3)whosemagnitudewas34kuToobservethechangeofchickeninerleukine2re

13、ceptorachain(chlL2Ro【、mRNAlevelsofchickeninfectedwithEimeriatenella，blood，tonsilofcaecalspleenandbursaofFabriciussampleswerecollectedasepticallyfromchickenonday1，3，5，7postinfectionwithEimeriatenellaLymphocyteofthemwassep