human cosavirus套式pcr检测方法的建立及基因组序列分析-establishment of nested pcr detection method for human cosa virus and analysis of its genome sequence.docx

性分析发现7份阳性样品中共有5株为HCoSVA种（HCoSV-A），表明HCoSV-A可能为上海地区的流行毒株。实验表明，建立的套式PCR方法可用于临床粪便样品的检测，具有特异、敏感、准确、简洁的特点，该方法的建立为HCoSV感染的临床快速诊断提供了科学依据。根据HCoSVGenBank已知序列信息共设计10对引物，结合RT-PCR和GenomeWalking等技术对其中一株病毒的全基因序列进行扩增，并对该株病毒的基因序列进行分析。结果显示：病毒的基因组长度约为7262nt,包括一个多聚蛋白为2125aa。将扩增出的VP3基因片段，克隆到原核表达载体pET-30a上，经酶切、测序鉴定后，将重组表达质粒转化至大肠杆菌BL21中，用终浓度lmmol?L-1IPTG诱导，表达产物以包涵体形式存在。超声波裂解菌体后对包涵体进行溶解，再用Ni-NTA柱对重组蛋白进行纯化，并对纯化的蛋白进行复性，全基因组序列的获得与VP3蛋白的表达为下一步蛋白功能基因组研究及ELISA检测方法的建立奠定了基础。关键词：HCoSV，套式PCR，序列分析，VP3蛋白，原核表达VESTABLISHMENTOFANESTEDPCRASSAYANDANALYSISOFTHEGENOMEOFHUMANCOSAVIRUSABSTRACTPicornavirusesarenon-enveloped,positive-strandedRNAviruseswithanicosahedralcapsid.HumancosavirusisanewlydescribedcandidatepicornavirusgenusconsistingoffivespeciesmakingupanewgenusinthePicornaviridaefamilythatmakethefamilyPicornaviridaeconsistsof14genera:Aphthovirus,Avihepatovirus,Cardiovirus,Enterovirus,Erbovirus,Hepatovirus,Kobuvirus,Parechovirus,Sapelovirus,Senecavirus,Teschovirus,Tremovirus,CosavirusandSealPicornavirus1.HumanCosavirusesaremostcloselyrelatedtomembersoftheCardiovirusandSenecavirusgenera,buttheylackaleaderpolypeptide.Althoughtherelationshipbetweenthehumancosavirusandclinicalsymptomisunclear,thehighdetectionrateofcosavirusinhumansacrosstheworldsuggestedthathumancosavirusmaybringpotentialharmtothehealthyofhumansormaybeafactorthatinducingsomediseasesincludingdiarrhea.So,itisimportanttodevelopamethodforhumancosavirusdetection.AboutthenestedPCRdetectionmethodofhumancosavirus,apairprimerswasdesignedtothe5’UTRandthebestconditionsofthemethodisasfollows:theoptimalconcentrationofMg2+was1.25mmol?L-1,theoptimalconcentrationofTaqenzymeis0.1U/ulandtheoptimalannealingtemperatureis55℃.Stoolspecimenswerecollectedfrom188hospitalizedchildrenwithdiarrheaand60healthycontrolsinShanghaiweredetectedbyusingthismethod,6(3.2%)specimensfromthe188hospitalizedchildrenand1(1.6%)fromthe60healthychildrenwerefoundtobepositiveforHCoSVandthetotalmasculineratiowas2.8%.WhencomparedwithavailablecosavirusstrainsinGenBankonthebasisofthesequence