豬血清1型胸膜肺炎放線桿菌突變體庫的構(gòu)建及初步篩選.pdf

1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文豬血清1型胸膜肺炎放線桿菌突變體庫的構(gòu)建及初步篩選姓名：赫明雷申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：基礎(chǔ)獸醫(yī)學(xué)指導(dǎo)教師：劉思國20090620AbstractIICONSTUCTIONINITIALSCREENINGOFSIGNATURETAGGEDMUTAGENESISLIBRARIESOFActinobacilluspleuropneumoniaeSEROTYPE1AbstractPcinecontagiouspleuro

2、pneumonia(PCP)isahighlycontagiousdiseasecausedbyActinobacilluspleuropneumoniae(APP)acterizedbyhemrhagicpleuropneumoniaseverefibrinousnecrotizinginswines.ThisdiseaseiswidelyspeadinthewldcausedgreateconomiclossesinswinesIn

3、dustry.InthisstudythelibrariesofAPP1mutagenesiswereConstructedbyusingSignaturetaggedmutagenesis（STM）hopingtoobtainthegeneswhichaffectAPP1SurvivalintheanimalswhichprovidesessentialbasistoresearchthefunctiongeneofAPP1.Inth

4、isstudythetechniqueoftransposonmutagenesiswasusedtoidentifygenesinpathogensthatarerequiredfgrowthinswineofActinobacilluspleuropneumoniaeserotype1（APP1）byusingasuicideplaspUTwhichincludedMiniTn5containingsignaturetags.12s

5、uicideplasspUTMiniTn5Km2whichincludespecificsignaturetagswereconstructed，transfmedintoE.coliβ2155respectively.Filterhybridizationwasusedtoconstructoptimizetheconjugationsystem.Conjugationmutantswerescreenedgetbyusingcult

6、ureplateswhichhaveresistanceheterotrophia.ThoseconjugationmutantswereidentifiedbyPCRusinguniversalprimerofresistance12specifictagsprimerApxIVtoxinsidentificationprimerrespectivelythensequencing.TheresultsshowedE.coliβ215

7、5APP1canbetransposedconjugated12librariesoftransposonmutagenesisweregotwhichhad421mutagenesisinall.Theseresultsprovidedessentialbasistoresearchthepathogenicmechanism、discovervirulencedrugresistancegenedevelopattenuatedli

8、vevaccineofAPP1.SixlibrariesofAPP1mutagenesiswerechoosenromlyfromtwelvelibrariesofAPP1mutagenesisonemutantstrainwasedromlyfromeachofthesixlibrariesthemutantstrainswhichedfromthesixlibrariesfmedanewlibrarywhichnamed“Input

9、pool”(thesixmutantstrainswere132、718、811、1023、118、1222)sixmice(theexperimentalgroup)wereinfectedwiththesamedoseofthemixtureofthesixkindsofbacterialliquidthroughthenasalcavityTheinfectivedosewas1.2~1.5108cfumice3miceinthe