h9n2禽流感病毒反向遗传系统转录表达载体的构建和验证.pdfVIP

维普资讯 第20卷第4期 中 国 病 毒 学 20(4)：388—392 2005年 8月 VIROLOGICA SINICA August2005 H9N2禽流感病毒反 向遗传系统转录 ／表达载体 的构建和验证 卢建红，邵卫星，龙进学，刘玉 良，石火英，刘秀梵” (扬州大学 农业部畜禽传染病学重点开放实验室 ，江苏扬州 225009) ConstructionandConfirmation ofPlasmidsTranscriptingandExpressingGenes from Avian InfluenzaVirusH9N2Subtype IU Jian—hong，SHAO Weixing，IONG Jin—xue，IIU Yu—liang，SHIHuo—ying，IIU Xiu—fan* (AnimalIn (tiousDiseaseLaboratory．SchoolofVeterinaryMedi~ine，YangzhouUniversity．Yangzhou225009．China) Abstract：Eightfull—length cDNAsofH9N2Avian influenzavirus (AIV)geneswereamplified andseparatelyclonedintothetranscription／expressionvector，pHW 2000．A totalofthreegenetic tagsofsilentmutationswereintroducedintoPB2，PB1andNA genes，respectively．Six3+ 5re— assortantsweregeneratedbyreversegenetics，eachcontainingthreegenes，HA ，NA andanyone oftheinternalgenes，from theH9N2virus，andtheremainingfiveinternalgenesfrom A／WSN／ 33．Thereby，thesixtransfectantswerealldesignedasH9N2subtypes．Theresultsshowedthat influenzaA viruscouldobtainoneormoregenesegmeWsfrom anotherviruswhichwasremotely related，andsuggestedthatthesurfacegenesandasingleinternalgenewerenotenoughtOexhib ithostrangerestrictionfortheH9N2virusonCOS一1mammaliancells．Theresultsalsoshowed thateachoftheeightconstructsworkedefficiently．Thisreversegeneticssystem wouldbeause fultOOlforfurtherstudiesonthestructureand functionofH9N2influenzavirusgenes．and the relationshipbetweenAIV anditshosts． Keywords：Avian influenzavirus；H9N2subtype；Reversegenetics；Reassortment 摘要 ：设计带有 BsmBI、BsaI或 AarI酶切位点的引物 ，用 RTPCR扩增 H9N2亚型禽流感病毒 (AIV)的 8个基 因 全长片段 。克隆人双向转录 ／表达载体 pHW2000，并在 PB2、PB1和 NA基因中共 引入了 3个沉默突变标签 。将其 2个表面基因