尘螨变应原derf2基因克隆及序列分析

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1、尘螨变应原Derf2基因克隆及序列分析作者：崔玉宝,周鹏,彭明,周鹰,彭江龙【摘要】　　目的:获得尘螨变应原Derf2基因及其生物信息学资料.方法:提取粉尘螨总RNA，RTPCR合成Derf2的cDNA片段，回收PCR产物并连接至pMD19Tsimple，经测序验证后亚克隆入表达载体pET28a(+)，转化大肠杆菌感受态细胞后提取质粒，双酶切鉴定.用ExPaSy,EBI,NCBI网站的在线软件对测序结果进行分析.结果:RTPCR扩增获得了Derf2cDNA片段，酶切、电泳结果表明克隆和亚克

2、隆获得成功.与参考序列相比，测序结果多了87bp(77~163)，但Blastn发现中国广州和德国Reinbek报道的序列中也含此87bp.同源性、相似性、序列比对及分子进化分析提示，该序列与中国广州和德国Reinbek报道的序列亲缘关系较近.推测该变应原由176个氨基酸组成，与附睾分泌蛋白E1具有同源性，信号肽位于1~17aa处，在6~24aa处有一跨膜螺旋.二级结构由a螺旋(16.57%),延伸链(32.57%)和随机卷曲(50.86%)组成.结论:获得了粉尘螨变应原Derf2编码基因及其分

3、子特征，为进一步生产基因工程变应原用于临床诊治变态反应性疾病奠定了基础.【关键词】粉尘螨；Derf2基因；克隆；变应原；生物信息学11　　CloneandsequenceanalysisofamajorallergenDerf2fromhousedustmites【Abstract】AIM:Tocharacterizethegroup2allergenofthehousedustmiteDermatophagoidesfarinaefromHainanIsland,atropicalregioni

4、nSoutheasternChina.METHODS:AfterthetotalRNAofD.farinaewasisolated,cDNAencodingthegroup2allergenwassynthesizedbyRTPCR,andthePCRproductswereinsertedintothevectorpMD19Tsimple,whichweresubclonedintopET28a(+)andtransformedintoE.coliandidentifiedwithrest

5、rictionenzymeanalysis.SubsequentlythesequencingresultwasanalyzedbysoftwareinExPaSy,EBIandNCBIweb.RESULTS:ThecDNAcodingforDerf2wasclonedandsequencedsuccessfully,andwefoundanadditionalregionof87bp(from77to163bp)inourstrainbutitwasabsentinreferencesequen

6、ce(GenBankAB195580).ByBlastnanalysis,thesameadditionalnucleotideswerefoundinthestrainsreportedfromReinbek,GermanyandGuangzhou,China.Apolygenetictree,constructedusingthenucleotidessequencescodingforDerf2reportedfromdifferentregionsorcountriesshowedthat

7、oursequencewasclusteredwiththestrainsfromReinbekandGuangzhou.Analysisofthetranslatedaminoacidsequencesuggestedthattheencoded11peptidewashydrophobicandextracellularwithacleavagesitebetween17aaand18aaandastrongtransmembranehelicesfrom6aato24aa,indicatin

8、gaMD2relatedlipidrecognitiondomain.Thesecondarystructureoftheproproteinconsistedof16.57%alphahelix,32.57%extendedstrandand50.86%randomcoil.CONCLUSION:WehaveobtainedagenecodingforDerf2andthisgeneshowssignificantdifferencewiththosereportedpre