雞病毒性關節(jié)炎病毒分離株S1基因的克隆與序列分析及RT-PCR檢測方法的研究.pdf

1、內(nèi)蒙古農(nóng)業(yè)大學碩士學位論文雞病毒性關節(jié)炎病毒分離株S1基因的克隆與序列分析及RTPCR檢測方法的研究姓名：常繼濤申請學位級別：碩士專業(yè)：預防獸醫(yī)學指導教師：關平原申之義20060501CloningandSequenceAnalysisoftheS1GeneofAvianViralArthritisVirusIsolatesandDetectionofAVAVbyRT—PCRAbstractTheAvianViralArthritisV

2、iDIS(AVAV)isolatedinInnerMongoliaandTianjin(AVAVB一98，C一98，G98，T98)WaSpropagated，andthetotalRNAofviruswasextractedAccordingtothepublishedS1genesequenceofARVSl133straininGeneBank，twopairsofprimerweredesigned，oneissequencin

3、gprimer，theotherisdiagnosticprimerTheS1geneofviruswasamplificatedwithsequencingprimerbyRT—PCRandthenclonedintopGEMTEasyplasmidsandthensequencedEachoftheacquiredsequencescontains1643bp，andallcongtainthreeORFs(ORFl，ORF2and

4、ORF3)andthenontranslatedregionP10P17and03proteinwasencodedbyORFl，ORF2andORF3respectivelyTheresultofsequencecomparisonindicated：therearen’tdifferencesinP10proteinandP17proteinamongthefourisolatesofAVAV，andonlythreenucleot

5、idesdifferencein03gene；theirhomologyisabove95％comparedwiththefourisolatedAVAVstrainswithotherreferencestrainSexceptARV138andNelsonBayVirusmBV)ThisindicatedthattheS1gene’SdependablityishighbetweentheAVAVisolatedfromChinaa

6、ndreferencestrainsThevirusRNAwasamplificatedwithdiagnosticprimerbyonestepRT—PCI乙thenestablishedRT—PCRdiagnosticmethod，anddetectedthismethod’SspecificityandsensitivityTheresultsindicatedthattheonestepRTPCRmethodcandetectt

7、henucleicacidofstandardstrainvirus(ARV—S1133)andloealityisolatedstrains(B98，C98，G一98，T98)，whichwerepropagatedinchickembryoWegeta435bpDNAfragment，whichisthesame、Ⅳitllweexpected，whilethedetectionresultsofotherreferenceetio

8、logicalagentsfNDV，IBDV，IBV，ILTV，EDS76V)werenegative；theminimumARVRNA’SdetectionlevelofthisRTPCRmethodisO5ngthisillustratedthatthismethodisspecificandsensitiveforthedetectionofAvianreovirusKeywords：AvianHralArthritisVirus