鴨肝炎病毒VP1基因的克隆與表達.pdf

1、獨創(chuàng)性聲明本人鄭重聲明：所呈交的學位論文，是本人在導師指導下，進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獸醫(yī)藥品監(jiān)察所或其它教育機構(gòu)的學位或證書而使用過的材料。對本論文所涉及的研究工作做出貢獻的其他個人和集體，均已在文中以明確方式標明。本學位論文原創(chuàng)性聲明的法律責任由本人承擔。學位論文作者虢穆、礎年多月膨日關(guān)于論文使用授權(quán)的說

2、明本人完全了解中國獸醫(yī)藥品監(jiān)察所有關(guān)保留、使用學位論文的規(guī)定，即：中監(jiān)所有權(quán)保留送交論文的復印件和磁盤，允許論文被查閱和借閱，可以采用影印、縮印或掃描等復制手段保存、匯編學位論文。同意中國獸醫(yī)藥品監(jiān)察所可以用不同方式在不同媒體上發(fā)表、傳播學位論文的全部或部分內(nèi)容。(保密的學位論文在解密后應遵守此協(xié)議)研究生簽名：導師簽名：時間：彤年石月伊日時間：剔／7年∥序喀日關(guān)號氟夥瓦中國獸醫(yī)藥品監(jiān)察所碩士學位論文ABSTRACTABSTRACTDu

3、ckviralhepatitis(DVH)causedbyduckhepatitisvirus(DHV)isanacuteandfatalinfectiousdiseasewhichspreadsrapidlyinducklingswithin3weeks。ThediseasehasashortdurationandahighmortalityOpisthotonoscanbeobservedinclinicalsymptomsandp

4、athologicalchangesaretheswelling，necrosisandhemorrhageoftheliverTheDVHcausedbyduckhepatitisvirustype1(DHV1)andduckhepatitisvirusnewserotype(N—DHV)hadbeenapprovedtospreadwildlyinChinaVPlproteinofDHVisthemostantigenicprote

5、inbeingresponsibleforinducingneutralizingantibodiesandinspiringaprotectiveimmunityProkaryoticexpressionsystemwasattemptedtoexpresstheprotein，butnoproteinwasobtainedowingtothegenestructureAfterremovaloftheInst135bp，recomb

6、inantexpressionplasmidswasobtainedDHVIVPlproteincouldbesuccessfullyexpressedButtheproteincannotreactwithDHVIantiserabyserologicaltests，whichindicatedthefailureofexpressionforDHVIVPlNoproteinofN—DHVVPlwasobtainedThenBacto

7、—BacexpressionsystemwasusedfortheexpressionofVPlgenesTwosetsofspecificprimersforVP1genesweredesignedandtheDNAfragmentsofVPIfromYB3andGDstrains，respectivelywereamplifiedbyRTPCRThegenesofinterestwereclonedintopFastBacHTAtr

8、ansfervectorandnamedaspFastBacHTA—YB3VP1andpFastBacHTAGDVPI，respectivelyTherecombinantvectorsweretransferredintoDH10BaccompetentcellscontainingashuttlevectorBacmidandtheVP1geneswereintegratedintoBacmidbysitespecifictrans

9、positionTherecombinantbacmidswerenamedasBacmidYB3一VPlandBacmidGD—VPI，respectivelySubsequentlySf9cellsweretransfectedwithBacmid—YB3VPlandBacmid—GDVPlandtherecombinantbaculovirusculturesnamedasvBac—YB3andvBac—GD，respective

10、lywereharvestedandthethirdgenerationbaculoviruscultureswereusedtoexpresstheVPIproteinsTheproteinbandsofabout30KDaweredetectedbySDS—PAGEelectrophoresisThetwoproteinshadagoodreactivitywithantiseraofDHVIandN—DHVrespectively