【硕士论文】APR834H1N1NA基因包装信号对H5N1流

摘要摘要历史上，全球范围内的流感病毒广泛流行，造成上千万人的死亡和巨大的经济损失，严重威胁人类的公共安全和社会经济。自1997年首例H5N1亚型高致病性禽流感病毒突破种间屏障感染人并致死事件以来，该病毒一直在整个东南亚地区肆虐，预示有可能出现流感大流行病毒株。疫苗免疫接种是流感防治的昀有效的手段。在全病毒灭活疫苗株的制备中，NIBRG-14是世界卫生组织(WHO)推荐使用的一株H5N1流感疫苗株，其表面抗原基因来自A/Vietnam/1194/2004(H5N1)(VN1194)，但研究表明实验室研究表明NIBRG-14的抗原性、免疫原性和鸡胚生长特性均可满足其作为疫苗株的要求，但该疫苗株在鸡胚中生长不佳，HA抗原产量通常较低。本研究以VN1194毒株的NA基因为研究对象，以提高疫苗株产量为目的，证明了包装信号的恢复能够有助于提高病毒产量。我们研究发现，在PR8背景下，VN1194NA基因被包装入重组病毒中的效率仅为正常包装量的38%-68%，因此推测有一部分重组病毒为不含有NAvRNA的缺陷型病毒粒子.我们在VN1194NA基因完整CDS的5'和3'两端嵌合PR8NA基因包装信号序列(vRNA3'末端41bp，5'末端67bp)，通过反向遗传操作成功拯救了不同HA和NA组合的4株重组病毒，分别命名为PR8HA-PR8(21/39)VNNA、PR8HA-VNNA、VNHA-PR8(21/39)VNNA、VNHA-VNNA。从包装效率分析，RNA凝胶电泳直观的表明，相较疫苗株，恢复了包装信号的病毒NA基因包装效率有了很大提高。通过荧光定量PCR，更加精确的从分子水平证明了包装信号的添加能使NA的包装效率从38%-68%增加到101%~103%，重组病毒中NAvRNA的包装效率得到完全恢复。我们以相同EID50接种鸡胚，每12h取样在MDCK进行滴度测定，绘制生长曲线，我们发现恢复包装信号的病毒在鸡胚的生长滴度提高了10倍。从HA抗原产量分析，以相同EID50接种鸡胚，通过纯化病毒的SDS-PAGE，用BandScan5.0分析HA的含量，结果表明恢复包装信号的病毒其HA抗原含量提高了约2.7倍。总之，本文就PR8NA基因包装信号序列对异源NA基因的包装效率、重组毒株的生长特性以及HA抗原含量的影响进行了探讨，为提高H5N1流感疫苗株I，为疫苗的研制提供了新的思路。关键词：H5N1流感疫苗包装信号病毒生长AbstractAbstractWorldwidepandemicinfluenzahascausedmillionsandmillionsdeathandhugeeconomiclossinhumanhistory,whichthreatenpublicsanitaryandsocialeconomy.SincethefirstreportabouthighpathogenicH5N1influenzavirustransmittedfromaviantohumanandresultedinfatality,theH5N1hasbeenravagingalloverthesoutheastAsiaregion.Itwasindicatedthattheremightbeapotentialoutbreakofflu.ThemostfeasibleapproachtopreventinfluenzaviruswouldrequiresafeandeffectivevaccineagainstH5N1.NIBRG-14wasoneoftheH5N1candidatevaccinevirusesrecommendbyWHO(WorldHealthOrganization)topreparewholevirusvaccine,developedwiththehemagglutinin(HA)andneuraminidase(NA)genesderivingfromA/Vietnam/1194/2004(H5N1,VN1194).Theantigenicityandimmunogenicityofthevirusiscapabletomeettherequirementsforvaccineproduction.However,NIBRG-14hadbeenreportedtoyieldlowtiterinembrynatedeggsandinsufficientamountsofHAantigen.OurstudyfoundthattheNAvRNAofVN1194waspoorlypackaged(38%-68%)intotherecombinantviruseswithabackboneofPR8genes,causingtheformationofdefectivevirionswithouttheNAvRNAinviralgenome.UsingrecombinantDNAtechniques,weconstructedachimericNAgenewiththecodingregionofVN1194NAflankedbythepackagingsignalsequence(vRNA3'end41bp,5'end67bp)ofPR8NA.WehaverescuedfourstrainsofreassortantvirusesindifferentcombinationwhichnamedPR8HA-PR8(21/39)VNNA、PR8HA-VNNA、VNHA-VNNA、VNHA-PR8(21/39)VNNArespectively.ThePolyacrylamidegelelectrophoresisofRNAsshowedthatthepackagingofNAvRNAwasenhancedgreatlyintherecombinantvirusescomparedwiththeformercandidatevaccinevirus.Besides,themorepreciseQ-PCRresultsindicatedthepackagingsignalincreasedthepackageefficiencyofNAgenefrom38%-68%to101%~103%.ThepackagingofNAvRNAwascompletelyrestoredintherecombinantviruseswiththechimericNAgene.Moreover,weinoculatedthefourvirusesintoembroynatedeggswithequalEID50andassayedvirustiterinMDCKcellper12h.FromthegrowthcurvewefoundtherecombinantvirusescontainedthechimericNAgenereplicatedbetterinchickenembroynatedeggsthanrecombinantviruseswithwildtypeNAgeneofVN1194,asindicatedbya10-foldincreaseinvirusIIIAbstracttiter.Intheend,weanalyzedtheHAantigencontentthroughSDS-PAGEwiththepurifiedvirusandtheresultsshowed2.7-foldincreasecomparedwiththeformervirus.Inconclusion,thestudiesexploredwhatspecialrolePR8NAgenepackagingsignalplayedonpackagingefficiency、growthcharacteristicsandHAantigencontent.ThesefindingssuggestedanovelstrategytoimprovetheyieldofNIBRG-14invaccinemanufacture.Keywords：H5N1influenza，vaccine，packagingsignal，virusgrowthIV目录目录第一章引言.............................................................................................1第二章绪论（禽流感疫苗的研究背景与进展）................................21.1流感病毒概述·································································································21.2流感病毒基因组结构与功能··········································································21.3流感病毒编码蛋白的结构与功能··································································31.3.1血凝素（Hemagglutinin，HA）···············································································31.3.2神经氨酸酶（neuraminidase，NA）········································································31.3.3基质蛋白（Matrixproteins,M）···············································································41.3.4核蛋白（Nucleoprotein,NP）···················································································41.3.5聚合酶（Polymerase）······························································································51.3.6非结构蛋白（Nonstructuralproteins,NS）·······························································51.4流感病毒的复制·····························································································61.4.1吸附····························································································································61.4.2膜融合和脱壳············································································································61.4.3病毒RNA的复制和转录··················································