1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文馬皰疹病毒1型及4型gG基因的原核表達(dá)及間接ELISA方法的建立姓名：田志革申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：生物學(xué)；微生物學(xué)指導(dǎo)教師：曲娟娟20120620東北農(nóng)業(yè)大學(xué)理學(xué)碩士學(xué)位論文ExpressionofEquineHerpesvirusTypelandType4gGGenesinProkaryocyteandEstablishmentofIndirectELISADiagnosisAbstractEquinerhi

2、noDneumonitisisagenerictermforseveralhighlycontagiousEquusanimaldiseasesThepathogensaretwoalphaherpesviruswhicharecloselyrelated：Equineherpesvirustype1(EHVI)andEquineherpesvirustype4(EHV4)Exceptcausingrespiratorydisease，

3、EHV1iSalsoacommoncauseofpregnantmareabortionandcentralnervoussystemdisease；EHV04mainlycauserespiratorysymptomsandoccasionallycancauseabortionEHV1andEHV4antibodiesinpolyclonalserumcannotbedifierentiatedbecauseofextensivea

4、ntigeniccrossreactivitybetweenthetwovirusesForthisreason，thisstudywantstoestablishadiagnosticELISAassaytodistinguishhorsesinfectedbyEHV1and／orEHV4AccordingtoGenBank，comparingaminoacidssequencesofEHV1andEHV4gGgenes，wefind

5、thatCterminalregionshowsconsiderablymoredivergencethantheNterminalregionAminoacids1to287ofEHV1gGand1to286ofEHV4gGshowanidentityof75％，whereastheapparentlycorrespondingregionscomprisingaminoacids287to382ofEHV4gGand288350of

6、EHV1gGhavedivergedwidelyandshowonly21％aminoacididentitysotwopairsofspecificprimersaredesignedtoabtainEHV1andEHV4gG3’terminalgenesTheEHV1gGgeneisclonedintotheBamHlandSalIsitesofplasmidpGEX6p一1toconstructrecombinantexpress

7、ionplasmidpGEX—lgG；andtheEHV4gGgeneisclonedintotheEcoRIandHindIIIsitesofpET28a()toconstructpET一49G；ThepGEXlgGandpET一49GaretransformedintoBL21(DE3)andRosetta(DE3)respectivelyProteinsarehighlyexpressedbyoptimizingcondition

8、swiththesizesof35ku(EHV1gO)and17ku(EHV4gG)WesternblotconfirmsthatthepurifiedproteinshavegoodantigenicityandspecificityIndirectELISAsareestablishedtodistinguishhorsesinfectedbyEHV1and／orEHV4withpurifiedgGproteinsTheoptima

9、ldilutionofAntigenandserumtestshowedthattheantigencoatingconcentrationWas025ng／btLandthedilutionofserumwas1：400andtheoptimaltimeiS60minoptimaldilutionofantiHRPiS1：10000andtheoptimaltimeiS30rain，thecutoffvaluesare026and03

10、1respectivelyExcepthighagreementwithcommertialkit，thespecificitysensitivityandrepeatabilitywerealsosuitableforequinerhinopneumonitisantibodiesdetectionThestudynotonlyestablishessimpleandconvenientserologicaldetectionmeth