斯氏艾美耳球蟲MIC-5基因的篩選、表達及重組蛋白免疫原性研究.pdf

1、Y1057399分類號UDC$852723密級編號中國農(nóng)業(yè)科學院博士后研究工作報告斯氏艾美耳球蟲MIC5基因的篩選、表達及重組蛋白免疫原性研究喬軍工作完成日期2005年8月2007年7月報告提交日期2007年7月中國農(nóng)業(yè)科學院研究生院家畜疫病病原生物學國家重點實驗室2007年7月AbstractOccidiosisiSakindofsporozoaldiseasesinrabbitscausedbycoccidianofEimeriid

2、ae，whichhasposedagreatthreattabbitbreedingindustryinofircountryInthispaper，thefollowingstudiesoninvestigationofcoccidiaspeciesofrabbitsinlanzhouisolationofpurespecieofEstiedaibycoccidiafreerabbitandsingicoocysttechniques

3、。constructionofcDNAlibraryofsporozoitesofE。stied口iscreeningandexpressionofmicronemeprotein5(EsMIC51toassayimmunogenicityofrecombinantproteinMIG5wereconductedTheresultscarlbesummarizedasfollows：Firstly，14coceidiaspeciesof

4、Eimeriawereobservedinthissurvey，whichwereEstiedaiEmediaEmagnaEirrestiduaEpiriformisEexigua，EintestinalisEcoecicolaEneoleporis最nagpurensisEper[oransE1eporisEflsvescensandEmatsubayashitThedominantcoceidianspeciesinrabbitsa

5、reEperforans(212％、Eirrestidua(10O％)andEstiedai(90％)，respectivelyn圮infectionrateofEimeriacoceidianWas100percentforrabbitsof14monthsofageSecondlytheeDNAlibraryofsporozoitesofEstiedaiwassuccessfullyconstructed，whichconsiste

6、dof62x10sindependentcloneswitharecombinationrateof949％Theinsertedfragmentswere5001500bpinlengthThirdlytwopositiveplaqueswhichharboring1095bpeDNAfragmentwerescreenedbytheBentonDavishybridizationtechniquefromtheeDNAlibrary

7、Sequenceanalysisrevealedthatthegenefragmentshared891％identitiesinnucleotidesequencewiththesameregionofEtMIC一5gene，harboring903bpencodingre#on，174bp3’UTRand17bppoly(A)tail4appledomains(Adomains)werefoundinitsdeducedaminoa

8、cidsequenceFounhlyEsMIC一5wassuccessfullyexpressedinEcolitransformatedbyprokaryoticexpressionvectorpETMIC5TheexpressedrecombinantproteinMIC一5WaspurifiedwithNi“ioncolumnFifthlyrabbitIFN—ygeneWasamplifiedbyRTPCRTherecombina

9、ntIFNYWasexpressedinBHKtransfectedbyeukaryoticexpressionvectorpVAXIFNYwithliposomeSixtMyMIC5proteinwassuccessfullyexpressedinPichiapastorisintheformofsecretionproteinandprovedtobeagoodimmunogenicityinimmunizedmiceLast，ba

10、sedontheimmunizationoftwentycoccidia—freerabbits，theresultsshowedthattherecombinantproteinEsMIC5couldefficientlyinducespecifichumoralandcellularimmuneresponsesagainstEstiedaiKeywords：Estiedai，Sporozoites，eDNAlibraryMicro