1、V774863分類號：密綴：’弋目差遺犬等碩士學位論文單位代碼；10019學號：S02318火雞冠狀病毒的分離和初步鑒定Isolationandpreliminaryidentificationofturkeycoronaviruses研究生：指導教師：申請學位門類級別：專業(yè)名稱：研究方向：所在學院：楊仉生趙立紅副教授農(nóng)學碩士預防獸醫(yī)學實驗動物學動物醫(yī)學院2005年5月AbstractAnacute，highlyinfectiousdi

2、seasewasencounteredinturkeypoultsat15—28daysofageinaturkeyfarmin2003Clinicalsignsincludedwaterdroppingsand10％～20％mortalitymostly，Thelesionsarelimitedtothehypememiaandhemorrhageofduodenumandrectum，tumescenceofceeumandyell

3、owcontentsfilledinintestinesItwassuspectedasBluecombdiseaseSointhisstudytheviruseswereisolatedandidentifiedinfollowingmethods1Livers，spleensandintestinesobtainedfromtheinfectedturkeypourswerehomogenizedwith5volumesofOOlM

4、PBScontaining20000U／mlpenicillinand20000mg，mlstreptomycinThenthehomogenatewereincubatedat4“Cfor2hoursAftercentrifugedat5000。gfor20minutes，thesupernatantwereobtainedandinoculatedinto15layoldSPFchickenembryosviatheallantoi

5、ccavityTheintestinesandyolkswereharvestedseparatelyfromthesecondpassageschickenembryos，andtestedbyHAwithrabbiterythrocytes(titerwas20)2Afterfivesuccessivepassages，theyolkofchickenembryoswereharvested72hourspostinoculatio

6、n，degreasedby2％polyethyleneglycol(MW6000)，andconcentratedwith10％polyethrlaneglyc01Theintestinesofchickenembryoswereharvestedandhomogenizedwith5volumesof001MPBScontaining4000U／mlpenicillinand4000mg／mlstreptomycinTheintest

7、ineshomogenatewereultracentfifugedat9000xgfor20rainstemovethetissuedebrisTheviruseswereconcentratedwith10％polyethyleneglycolinthesamemethodsasaboveThentheconcentrationsofthevirusofyolkandintestinewereseparatelyaddedtheto

8、pofastepgradientconsisting35％～55％ofsucrosesolutions，andultracentrifugatedat91000。gfor2hoursTheopalescentbandswithdensityof1156to1260咖m3werecollectedandultracentrifugatedat96000。gfor2hourstemovethesucrosesolutionsThepelle

9、tscontainingviruseswereobtainedandstainedwithhosphotungsticacid，andexaminedbyelectionmicroscopySeveralsphericalorovalparticlessurroundedregularlybyspacedpetalorpear—shapedprojectionswereobservedTheparticlediameterisabout

10、120nm3ThetotalRNAofinfectedchickenembryoswasextractedTurkeyCoronavirusesS2genewasamplifiedbyreversetranscription—polymerasechainreaction(RTPCR)withdesignedprimerTheproductswereidenticalwithexpectation41dayoldBluecombsusc

11、eptibleturkeypoultswereinoculatedviaorallywithlmloftheintestineshomogenateoftheinfectedchickenembryos3dayspostinoculation，theinoculatedturkeysshowedweightlost，waterydroppingsandwith20％mortality，whichwerethesameclinicalsy