豬傳染性胃腸炎檢測試劑的研制及快速診斷方法的研究.pdf

1、東北農(nóng)業(yè)大學碩士學位論文豬傳染性胃腸炎檢測試劑的研制及快速診斷方法的研究姓名：姜騫申請學位級別：碩士專業(yè)：預(yù)防獸醫(yī)學指導教師：李一經(jīng)2003.6.1一。，，，，一一。：，—耋!!些查蘭鐾些蘭堅耋一。一。。。，：一。，一。PREPARATl0NFORTHEDETECTl0NREAGENTSANDDEVELOPM哐NTOFTHEM匝THODSFORRAPIDDIAGNOSISINTRANSMISSIBLEGASTROENTERITISABS

2、TRACTEcoliDH5aharbouringrecombinantplasmidpProExHTbNwasinducedwithIPTGtoexpresstherecombinantnucleoprotein(rNP)oftransmissiblegastroenteritisvirus(TGEV)TherNPcollectedaftereelIextractionwasappliedontothenickeInitrilotria

3、ceticacid(Ni，NTA)resinTheoptimizedprocedureofpurificationwaslysingtheEcoltbyBufferA(containing1mmol／LPMSF)washingtheresinwith2XBu行erA(pH80)、2BufferB(pH63)、2BufferC(pH59)andcollectingtheelutionofBu岱“C(80mmol幾imidazole)The

4、molecularweightofpurifiedrNPwas47kuanalyzedbySDSPAGETheresultofWestellaBlotshowedthattherNPcanacttosaraofanti—TGEVTheconcentrationoftheelutionincludingrNPwasidentified154799perml479799permlbyBradford’sassayatthesameratio

5、152mgrNPcanbe曲rainedpergramEcolisedimentDotELlSAandindirect—ELISAwereestablishedbythe小lP_TheoptimalamountoftherNPcoatedinDot—ELISAwas125pgperdotThepre—dotmembranecanbekept5weekslongat4℃ThespecificassayshowedthatrNPhaveno

6、crossreactionwithporcineepidemicdiarrheacoronavirus(PEDV)、porcinerotavirtts(PRV)Inindirect—ELISAtheoptimalamountofcoatedantigenwas051嶺perwell，theoptimalblockingsolutionwas5％drymilktheidealdilutionofserumwas200andthefitre

7、actiontimewas30min、theadequatablereacriontimeofsubstratewas10minThemonoclonalantibody(McAb)detectingTGEVwasperformedbyimmunizingtheBALB／cmicewithpurified肥CellsfusionwascarriedoutbystandardmethodandhybridmaswithrNPantibod

8、y—positivesupematantsweresubclonedthirdbythelimitingdilutionOnehybridomacelllinesecretingmonoelonalantibodyspecificagainstrPNwaspickedoutbyindirect—ELISAdesignatedElOFl0D5111eevennumberofchromosomesperhybridomawas8710pai

9、resTheantibodytiterofasciteswas1：12800determinedbyindirect，ELISATGEVwasapplicatedasantigeninWesternBlotandindirect—ELISAtoanalyzetheMcAbTheresultshowedthattheMcAbcouldindetifiedcertainantigenicepitopeofTGEVNproteinMethod

10、todisposeTGEVinintestinalsamplewasoptimizedTwomethodstodetectantibodiesofTGEVwereestablishedfirstlybyusingrNpasantigencoatedinDotELISAandindirectELISAindomestic，Onehybridomaeell1inesecretingmonoclonalantibodyrecognizingT

11、GEVNproteinwasproducedAlltheworkfoundedmaterialbaseforTGEdiagnosisaccuratelyandrapidlyPostgraduate：QianJiangMajor：PreventiveVeterinaryScienceSupervisor：ProfessorLiYiJingKeywordTGEvRecombinantNucleoprotein；ELISA；Monoelona