AIV地方分離株（H9N2）HA和NA基因克隆、序列分析及HA的原核表達(dá).pdf

1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文AIV地方分離株（H9N2）HA和NA基因克隆、序列分析及HA的原核表達(dá)姓名：唐志芬申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：賈永清王君偉20040601，，，，』耋些奎蘭至莖鶩薹堡堅(jiān)，一。一!—一，一CLONINGSEQUENCEANALySISANDPROKARYOTICEXPRESSIONOFLOCALISOLATEOFAvIANINFLUENZAHAANDNAGENEAbstractAvianinflu

2、enza(AI)WaScausedbyAtypeavianinfluenzavirus，amemberofOnhomyxoVirdaeAvianinfluenzawithlowvirulenceWaScharacterizedbyrespiratorysymptomandlowproductirityAvianinfluenzaH9N2subtypehaSbeenamainsubtypeinChinasinceitwasreported

3、in1994，whichcaninfectflowandpigandhumanbeingSoitofgreatinteresttostudyAvianinfluenzaH9N2subtypefrommolecularlevelnotonlyintheoryofvirologyandveterinarysciencebutalsoinpublichygieneInthisstudyvlruSoftheloea]isolateA／Chick

4、en／Modanjian∥0823／00(H9N2)(MJ00)WaSpropagatedinSPFchickenembryoandnudeicacidWaSextractedAccordingtotheconservatiresequenceof3’terminusofeightgenefragmentfromAIvapieceofprimerwasdesignedtevelsetranscriptRNAAccordmgtotheeD

5、NAsequenceofHAgeneandNAgenefromH9N2AIVpublbhedinGenBank，twopairsofspecificprimersweredesignedtoamplifyHAgeneandNAgenebysoftwareOli9041andPrimer50andtheproductsofRTPCRwasclonedintopMDl8TvectorThesequencingresultsshowthatc

6、DNAofHAgenehasacompleteopenreadrngframeof1707bpwhichencodes560Aminoacidresiduesindudingsignalpeptideof18aminoacidresiduesandHAlof320aminoacidresiduesandHA2of222aminoacidresiduesBycomparingwith24strainsofthesamesubtypepub

7、lishedinGeneBankthehomologyofnucleotideofHAgeneisbetween815％and99％，andthehomologyofdeducedaminoacidresiduesisbetween87，5％and979％TheaminoacidsequenceofcleavageisRSSRIGLFaccordingwiththelowvirulenceAIVstraineDNAofNAgeneis1

8、448bpinlengthencoding469aminoacidresiduesBycomparingto24strainsofthesamesubtypepublishedinGenBankthehomologyofnucleotidesequenceofNAisbetween825％and989％andthehomologyofdeducedaminoacidresiduesisbetween857％and98％Comparing

9、withHongKongstrainanddomesticstrain，thereisnodeletionofthreeaminoacidresiduesinstackofMJ00NATheHAgenewassubclonedintoprokaryoticexpressionvectorpGEX6p—l，andthepositrverecombinantplasmidwastramsformedintocompetemocellBL21

10、(DE3)pLySThecolonycontainingthepositireplasinidpGEX6PHAWasscreenedandinducedby1mmol／LIPTGandWaSanalyzedbySDSPAGETheresultingelWaS88kufusedwithGST(26ku)，andthetargetproteinis62kuInthisstudyHAgeneandNAgeneofthelocalisolate