犬gamma型干擾素cDNA的克隆及其在鼠骨髓瘤細(xì)胞(SP2-0)中表達.pdf

1、中國農(nóng)業(yè)大學(xué)碩士學(xué)位論文犬gamma型干擾素cDNA的克隆及其在鼠骨髓瘤細(xì)胞(SP20)中表達姓名：楊琪申請學(xué)位級別：碩士專業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：夏春2002.5.1‘業(yè)!坐些叁望塑1111111：!一ABSTRACTInlerferons(IFNs)arenaturedproteindefensingbodiesagainstviralinfectionsandalsoplayimportantrolesincombatingtum

2、orsandregulatingimmunityForcontrollingcaninedisease，wedevelopedcaninerecombinantIFNTheprimersweredesignedfromconservedregionsdescribedinGeneBankwhichincludingtheHindlllandNotIrestrictionsitesrespectivelyThecanineIFNy(Cal

3、FNy)cDNAwasclonedbyusingreversetranslationpolymerizechainreaction(RTPCR)methodsTheclonedCalFN7genewasinsertedintopGEM—TEasyVectorforselectionThenCalFN—vgeneweredigestedwithHindIIlandNot1，andinsertedintopRc／CMV2expression

4、vectorAfteridentifiedbyenzymaticdigestionandPCR，therecombinantexpressionvector(pRc／CMV2／CalFNy)wassequencedToestablishanlongtimeexpressionsystem，pRc／CMV2／CalFN—Yvectorwastransfectedintomousemyelomacells(SP2／0)andMadin—Da

5、rbyCanineKidney(MDCK)andthenselectedbyG418Thecellsculturesupernatantswasharvestedandtheantiviralactivitywasmeasuredfollowingcytopathic—effectinhibitionassayusingMadinDarbyCanineKidney(MDCK)一vesicularstomatitisvirus(VSV)a

6、ndMadin—DarbyBovineKidney(MDBK)vesicularstomatitisvirus(VSV)systemsThepRc／CMV2／CalFNyvectorwassequenced，andpredictedtoproduceasignalpeptideof23aminoacidsandamatureproteinof143aminoacidswithamolecularweightof19KDTwopotent

7、ialN—glycosylationsitesarelocatedatpositionsI6and83ofthematureproteinComparingtheCalFNyproteinsequencewiththatofCalFN—YreposedfromDDBJ／GenBankrevealedahomologyof99％TheantiviralactivityofrecombinantCalFN_丫wastested，theins

8、tantexpressingproduce’Santiviralactivityis7，000U／mlofculturemedium；thesteadyexpressingproduce’Santiviralactivityis5，000U／mlofeultnremediumThisresearchisusefulforstudyingnotonlyonproducingrecombinantCalFN丫butalsoonthechar