重組腐敗梭菌α毒素的免疫原性研究.pdf

1、獨(dú)創(chuàng)性聲明本人鄭重聲明：所呈交的學(xué)位論文，是本人在導(dǎo)師指導(dǎo)下，進(jìn)行研究工作所取得的成果。除文中已經(jīng)注明引用的內(nèi)容外，本學(xué)位論文的研究成果不包含任何他人創(chuàng)作的、已公開發(fā)表或者沒有公開發(fā)表的作品的內(nèi)容，也不包含為獲得中國(guó)獸醫(yī)藥品監(jiān)察所或其它教育機(jī)構(gòu)的學(xué)位或證書而使用過的材料。對(duì)本論文所涉及的研究工作做出貢獻(xiàn)的其他個(gè)人和集體，均已在文中以明確方式標(biāo)明。本學(xué)位論文原創(chuàng)性聲明的法律責(zé)任由本人承擔(dān)。學(xué)位論文作者簽名：彭I虱睇卅年廠月7日關(guān)于論文使用

2、授權(quán)的說明本人完全了解中國(guó)獸醫(yī)藥品監(jiān)察所有關(guān)保留、使用學(xué)位論文的規(guī)定，即：中監(jiān)所有權(quán)保留送交論文的復(fù)印件和磁盤，允許論文被查閱和借閱，可以采用影印、縮印或掃描等復(fù)制手段保存、匯編學(xué)位論文。同意中國(guó)獸醫(yī)藥品監(jiān)察所可以用不同方式在不同媒體上發(fā)表、傳播學(xué)位論文的全部或部分內(nèi)容。(保密的學(xué)位論文在解密后應(yīng)遵守此協(xié)議)研究生簽名導(dǎo)師簽名：虱嘩——廣一、文帆聊年廠月7日時(shí)間：勃／匕年咱擴(kuò)Et彭耘ABSTRACTClostridiumseptiuma

3、stheGram。positiveanaerobicbacilluscancausediseasestoanimalsandhumanbeingssuchasmalignantedema，musclenecrosis，gasgangrene，necrotizingenteritisandSOon，andClostridiumseptiumisalsothepathogenofsheepbraxyThebacteriumcansecret

4、emanykindsofexotoxinssuchasc【、D、Yand6，anditsmainpathogenicfactorandimmuneprotectiveantigenis0【‘toxinwhichhashemolysis，necrosisandlethalactivityAccordingtothegenesequencesofClostridiumsepticumcLtoxininGenBankPCRprimerswer

5、edesigned，and0【toxingene(csa)andtwotruncated0【一toxinsgenes(csan219andcsac439)wereamplifiedThesethreegenesequenceswereinsertedintopET28a()，andtheexpressionvectorswereconstructedandtransformedintoEcoliBL21(DE3)Therecombina

6、ntEcoliBL21(pET28a—csa)，BL21(pET28a219)andBL21(pET28a439)wereidentifiedbyPCR，doubleenzymedigestionandsequencingThestudyresultsconfirmedthattheseexpressionvectorswereconstructedsuccessfullySDSPAGEtestresultsshowedthatreco

7、mbinantEcolicaneffectivelyexpresstherecombinant0【toxinandtwotruncated0【一toxinsbytheinducerIPTGTheftexpressionproductionsweremainlyinclusionbodys，andalittleofexpressionproductionsweresolubleTheconditionsofEcoliBL21(pET28a

8、—csa)expressingrecombinant0【一toxinwereoptimizedThistestresultshowedtheoptimumconditionsofinducingandexpressingTheinducingtimepointwaswhentherecombinantEcoliwasculturedtothemiddleorendoflogarithmicgro叭hasnumericalvaluesof

9、bacteriaculturefluidOD600wasintherangeof0850～0950TheconcentrationofinducerIPTGwas040mmol／LTheamountofinducingtimewasfor56hAndtheinducingtemperaturewas37。CInthisoptimumcondition，therelativeexpressionquantityofrecombinanta

10、—toxincouldreachto25O％Inthesameconditions，recombinantEcoliBL21(pET28a219)couldexpress340％fortruncatedcttoxinCSAN219andrecombinantEcoliBL2l(pET28a439)couldexpress170％fortruncated0【toxinCSAC439，Biologicalcharacteristicsoft

11、herecombinant0【toxinandtruncated0【一toxinsweretreatedWestern—blotresultsshowedthattherecombinant0【toxinandthetruncatedatoxinscanreactwithantitoxinofClostridiumsepticumspecificallyThemousetoxicitytestindicatedthattherecomb

12、inant0【一toxinhadthelethalactivitytomouse，whichcanbeneutralizedbytheantitoxinofClostridiumsepticum，butthetruncatedatoxinsdidnothavelethalactivitytomouseAndthecytotoxicitytestshowedthattherecombinantc【toxinhadcytotoxicityb

13、utthetruncated0【一toxinsdidnotAndtherecombinant0【toxinandtnmcatedatoxinsdidnothavehemolyticactivityonsheepbloodTherabbitswereimmunizedwiththevaccinesofrecombinant0【一toxinandtruncateda—toxinsandthebloodofrabbitswascollecte

14、daftertwoweeksTheimmuneprotectiontestsresultsshowedthattherecombinant0【toxinhadthebestimmunogenicityAndtherecombinantatoxinofvariousexpressingformsallshowedhigherimmunogenicityandcouldprotecttheimmunizedrabbitsfromchalle