猪乙型脑炎病毒SH0601株基因组序列分析及全长cDNA的构建

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1、文章编号：1002-2694(2008)02-0140-05猪乙型脑炎病毒SH0601株基因组序列分析及全长cDNA的构建郑浩，余丹丹，孙志，高飞，袁世山摘要：目的对務源乙型脑炎病♦SH0601株进行全序列测定与分析•以了解该株病毒的遗传特征•并构建全长cDNA克隆•方法将精源JEVSH0601株基因组RNA分5段进行RT-PCR并克隆入pCRII-Blunt-TOPO和pBluescript載体中.测定了全长cDNA序列.根据cDNA克隆障切图谱分析•将5个片段先后克隆入pBluescript中构

2、建JEV全长基因组cDNA.根据E爰白基因序列对SH0601株进行了遺传分型。结果序列分析表明，SH0601株基因组全长10977nt,与GenBank中的Beijing-1、P3、SA14和SA14-14-2株基因组全长相比•在3'NTR的10701nt处多一碱基“G”・与上述4株病聲的全长核昔酸的同源率都达97.2%以上•而与国外猪源分海株KV1899和JEV/sw/Mte/41/2002全长核昔酸的同源率仅达8&7%•但SH0601株与6株病毒的全长氨基酸同源率均达97.0%以上.以E畫白基因

3、序列对SH0601作进化分析•该分离株属基因型ID型.结论SH0601株属于乙型脑炎病毒基因皿型・在离拷贝质粒pBluescript中可构建稳定的乙型脑炎病毒全长cDNA克隆.关律词：乙型脑炎病廿$病序基因组；全长cDNA克隆中图分类号:R512.3文献标识码：AGenomicsequencingandcDNAcloningofJapaneseencephalitisvirusSH0601strainIsolatedfromporcineZHENGHao,YUDan-dan,SUNZhi,GAOFe

4、i,YUANShi-shan{DepartmentofAnimalInfectiousDisease«ShanghaiVeterinaryResearchInstitute♦ChinaAcademyofAgriculturalSciencetheKeyLaboratoryofAnimalParasitologyChineseMinistryofAgriculture

5、01strainisolatedfromporcinewereclonedintovectorsPCRI]-Blunt-TOPOandpBluescript*followedbydeterminationoffull-lengthgenomicsequence.ThecompletegenomicsequenceofSH0601strainwas10977nucleotidesinlength.ComparedwiththegenomesequencesofBeijing-1.P3.SA14andS

6、A14-14-2>nodeletionofSH0601sequencewasfound«whileaWGHwasinsertedin10701bpof3’NTRofSH0601♦and97.2%nucleotideidentitywasdetectedamongthem.However•thesimilarityofnucleotidesequencebetweenSH0601andKV1899

7、wasonly88.7%.butthesimilarityofaminoacidsequencebetweenSH0601andthesixstrainviruswas97.0%.GenotypewasdeterminedbasedonEgenesequence♦classifyingSH0601asamemberofGDIJEV.Basedontherestrictionendonuclasemapping,5individualcDNAclonesofthegenomicRNAwereasse

8、mbledintoapBluescriptvector•andthecompletecDNAofthegenomewasconstructed.KEYWORDS：Japaneseencephalitisvirus»full-lengthgenomic»cDNA乙型脑炎是一种重要的人畜共患病。自1931年日本爆发乙型脑炎并分离岀其病原体以来,东亚、南亚各国陆续发现乙型脑炎，近年澳洲也有该病发生的报道⑴。乙型脑炎病毒由蚊传播，感染人和马，主要呈现脑炎神经症状，而猪不分大小