5株P(guān)RRSV ORF3，5，6基因的變異分析及ORF3-5串聯(lián)基因重組腺病毒的構(gòu)建.pdf

1、‘卜文摘要5株P(guān)RRSV分離株ORF356基因的變異分析及ORF35串聯(lián)基因重組腺病毒的構(gòu)建摘要本試驗(yàn)通過(guò)對(duì)PRRSV5株廣‘東流行株ORF3563個(gè)基因的分析，探討廣東省5株流行株之間及其與其他毒株間的遺傳變異關(guān)系，為探索)東省PRRSV的來(lái)源、分子流行病學(xué)特征和抗原變異規(guī)律提供依據(jù)，同時(shí)為研制基因工程疫苗奠定了基礎(chǔ)。另外將腺病毒表達(dá)系統(tǒng)與PRRSV的主要抗原多膚的基因有機(jī)地結(jié)合起來(lái)，利用腺病毒對(duì)動(dòng)物體細(xì)胞的廣泛感染性，改善PRRSV

2、的GP3GP5對(duì)動(dòng)物免疫系統(tǒng)的刺激效率和方式，模擬PRRSV病毒的自然感染過(guò)程，以期更好地發(fā)揮PRRSVGP3GP5的抗原活性，為研制PRRSV高效安全的基因工程疫苗開(kāi)辟新途徑。本試驗(yàn)的主要內(nèi)容及結(jié)果如下:1.5株P(guān)RRSVGD株ORF3ORF5ORF6基因的變異分析根據(jù)GeneBank中發(fā)表的序列分別設(shè)計(jì)合成了針對(duì)PRRSVORF3ORF5和ORF6基因的引物，利用RTPCR方法從廣東省送檢的5份可疑病料中均分別擴(kuò)增得到大小約7876

3、p6306p550bp的片段，克隆入pMD18T載體并測(cè)序。應(yīng)用DNAStar軟件分析所測(cè)的序列，與國(guó)內(nèi)外已發(fā)表毒株和疫苗株(RespPRRSReproRespPRRSMLV)及LV4.2.1等毒株的序列進(jìn)行比較，結(jié)果表明:廣東省的5個(gè)毒株在遺傳關(guān)系上接近HB1(sh)株，本研究中分離于不同豬場(chǎng)的5個(gè)毒株間的核普酸序列差異不顯著，可能有共同的來(lái)源。2.PRRSVORF35串聯(lián)基因重組腺病毒的構(gòu)建用WP3L和WP3R引物從pTORF3陽(yáng)性

4、質(zhì)粒上擴(kuò)增完整的ORF3片段，利用TA連接的原理將片段亞克隆入pMD18T載體EcoV處，按常規(guī)方法轉(zhuǎn)化DH5a篩選出正向重組質(zhì)粒并測(cè)序，命名為pMDORF30用WP5L和WP5R引物從pTORF5陽(yáng)性質(zhì)粒上擴(kuò)增ORF5基因，用SphIIHindIII雙酶切后回收目的片段，將重組質(zhì)粒pMDORF3也經(jīng)SphIHindIII雙酶切并去磷酸化，與目的片段于16℃連接，按常規(guī)方法篩選出重組陽(yáng)性質(zhì)粒并測(cè)序，命名為pMDORF35e用NotIHi

5、ndlII從重組質(zhì)粒載體pMDORF35上切取ORF35基因插入pAdTrackCMV穿梭載體，挑取卡那霉素(Kan)抗性的單菌落，按常規(guī)方法獲得陽(yáng)性重組子pAdTrackCMV35。將構(gòu)建的攜有PRRSVORF35串聯(lián)基因的重組穿梭載體PmeI線性化后與腺病毒骨架載體pAdEasy1共轉(zhuǎn)染E.coiiBJ5183，使之同源重組，經(jīng)PCR酶切鑒定獲得重組腺病毒質(zhì)粒pAdCMV35然后用PacI線性化，用脂質(zhì)體介導(dǎo)轉(zhuǎn)染入人胚腎細(xì)胞(293

6、細(xì)胞株)，獲得含】，RRSVORF35基因的重組腺病毒AdCMV3Sa以上研究為進(jìn)一步了解PRRS在廣東的流行情況，制定確實(shí)可行的防制措施提供英文摘要SEQUENCEANALYSISOFFIVEPRRSVISOLATESORF356GENESCONSTRUCTIONOFTHERECOMBINANTADENOVIRUSCONTAININGF35FUSIONGENEABSTRACTPorcinereproductiveandrespirat

7、orysyndromevirus(PRRSV)wasoneofthemosteconomicallyimportantpathogensofswinediease.ORF356geneofPPPSVGDstrainswasamplifiedbyRTPCRandthesequencewasanalysisedinthisstudy.BoththerelationshipamongPRRSVGDisolateseachotherandrel

8、ationshipbetweenGDisolatesandotherstrainswerediscussed.SothattoshowthebasisoforiginsearchingforPRRSVinGDprovicineandmanufacturingthegeneengineeringvaccine.ThemajorantigengeneORF356ofPRRSVwerecombinedtotherecombinantadeno

9、virus.expresssystem.BasedontheextensiveinfectionofadenovirustobodycellofanimaltheORF35fusiongeneweredevelopedandisertedintothegenomeoftheadenovirusexpressedsysteminordetoraisetheactivationofantigentothesystemofanimal.The

10、paperincludetwoparts:(1)GeneticvariationanalysisORF356genesofPRRSVGDisolatesAccordingtothecompletesequenceofAmericanPRRSV(theATCCVR2332)，threepairsofprimerswithspecialrestrictenzymesitesweredesigned.ORF356genesofPRRSVGDi

11、solateswereobtainedfrom5PRRSVinfectedswinesamplesbyRTPCRandsequencedafterdirectlycloningintothepMD18Tvector.ThesequencesofORF356genesofGDisolateswerecomparedwiththeotherPRRSVstrainsinGenebank.Theresultsshowedthattheseque

12、ncesofORF356oftheGDisolateswererelatedcloselywithATCCVR2332andfartherwithPRRSVLVstrain.AndthephylogenetictreesalsorevealedtheGDisolateswasrelatedcloselywithHB1(sh)(2)ConstructionofrecombinantadenoviruscarryingORF35fusion

13、geneAccordingtothesequenceofATCCVR2332geneinGenebanktwopairsofPCRprimersweredesigned.TheORF3geneafragmentof780bywasamplifiedfromthepTORF3byPCRwhileusingWP3LandWP3RthenclonedintopMD18Tvectorandidentifiedby&II，NotMindIIIdi

14、gestionandtherecombinantplasmidsnamedpMDORF3wereobtained.The011175genewereamplifiedfrompTORF5byPCR.ThePCRproductswereclonedintopMD18ORF3therecombinantplasmidsobtained.ThepAdTrackCMVvectorandtheinterestinggene(ORF35)werea