豬瘟病毒QuantiGene ViewRNA原位雜交方法的建立及初步應(yīng)用.pdf

1、獨創(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。對本論文所涉及的研究工作做出貢獻(xiàn)的其他個人和集體，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：赴舷20l嚕年6月l。Et關(guān)于論

2、文使用授權(quán)的說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(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：越燕、導(dǎo)師簽名：時間：鈿l印年5月jo目時間：如，9年多月，口目中國獸醫(yī)藥品監(jiān)察所碩士學(xué)位論文ABSTRACTC

3、lassicalSwineFever(CSF)causedbyClassicalSwineFeverVtrus(CSFV)isahighlycontagiousandhemorrhagicdiseaseofpigsandhascausedsignificanteconomicallossesinpigindustryworldwideInthisstudy，CSFVQuantiGeneViewRNAinsituhybridization

4、techniquewasestablished；thenresearchedontheproliferationdynamicsanddistrmutionregularityofCSFVRNAininfectedcellsinvitroanddetectionCSFVRNAinvivowiththeestablishedmethodInordertodetectandlocalizeCSFVaccuratelyandexploreth

5、eproliferationdynamosanddistrmutionregularityofCSFVRNAafterinfectingPKl5invitro，CSFVspecificprobesandswinehouse—keepinggenepactinprobesweredesignedandconductedresearchonCSFVmoderatestrainHeBHHl／95(TCID50equag10’4一／2009L)

6、culturedinPKl5cellsInconsiderationofdetectionresults，fluorescentintensityandrepeatabilitythehybridizationeffectscouldbebestwhentheProteaseKconcentrationwasl：1000andFormaldehydefixedtimeWas30minUnderthefluorescentconfocal

7、microscopythepositivecellscouldbedetectedwithbrightredfluorescenceComparedwithCSFVimmunohistochemicaltechniqueandCSFVmonoclonalantibodyimmunofluorescentmethod，CSFVQuantiGeneViewRNAinsituhybridizationtechniqueWasthemostse

8、nsitivewhosedetectionlimitCanbeupto10一dilution，higher4and3degrees，respectivelyOthervirusesandotherCSFVstrainswereusedtotestforspecificityCSFVspecificprobescouldbondtodifferentCSFVstrainsratherthanotherviruseswhichindicat

9、edthegoodspecificityInordertostudytheaccuratelocalizationandproliferationdynamicsofCSFVRNAininfectedcellsinvitroinfectedPKl5cellswithCSFVmoderatevirulentstrainHeBHHl／95andHCLVdetectedCSFVRNAwithestablishedCSFVQuantiGeneV

10、iewRNAinsituhybridizationtechniqueatdifferenttimessuch罄05hpi’1hpi’15hpi，2hpi，25hpi，3hpi，35hpi，4hpi，45hpi，5hpi’55hpi’6hpi，12hpi，18h以24hpi，36hpi，48hpi，72hpiand96hpiunderfluorescentconfocalmicroscopyTheresultsshowedthatafte

11、rinfectionbyHeBHHl／95andHCLVwiththetimegoing，virusreplicatedandproliferatedincellsuntil72hpi；CSFVRNAcouldbedetected05h毗andduringO5hpiand12hpi，CSFVRNAwasinnucleus，whileafter18hpi，CSFVRNAWasmainlyaroundthenucleusuntil96hpi

12、HeBHHl／95andHCLVhasthesimilarproliferationregularityInawordthisstudyclarifytheproliferationdynamicsanddistrmutionregularityofCSFVinvitrofirstlyInordertovisuallydetectCSFVRNAincellsinvivoaccuratelyCSFVinfectedanimalmodelW

13、asestablishedandtonsilperipheral咖phnodesandkidneyweresampledtomakeFormalinFixedParaffinEmbedded(FFPE)，thenusedQuantiGeneViewRNAtechniquetodetectCSFVRNAinvariousinfectedorgansandt孤getedcellsTheresultsshowedthatredfluoresc

14、entdotscouldbeseenunderthefluorescencemicroscopewhichindicatedthattheestablishedCSFVQuantiGeneViewRNAinsituhybridizationtechniquecouldvisuallydetectCSFVRNAinFFPEaccuratelyTherefore，thismethodcouldbehelpfultoexplorethedif