捻转血矛线虫雌雄成虫半乳糖结合凝集素基因的克隆鉴定及序列分析

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1、农业生物技术学报JournalofAgriculturalBiotechnology2005,13(1):86~91·研究论文·捻转血矛线虫雌、雄成虫galectin基因的克隆、鉴定及序列分析*李春华李祥瑞**魏晓锋徐立新（南京农业大学动物医学院，南京210095）摘要：根据GenBank中发表的捻转血矛线虫()雌、雄混合虫体的半乳糖结合凝集素（galectin）基因序列设计1对引物，分别以山羊捻转血矛线虫雌、雄成虫的总RNA为模板，采用RT-PCR技术，均扩增出约880bp的产物。将其分别克隆入pMD18-T载体，经双酶切鉴定、PCR鉴定及DNA序列测定，表明

2、扩增片段为捻转血矛线虫cDNA。经核苷酸序列、氨基酸序列和抗原性比较分析发现，雌、雄成虫的galectin基因存在一定差异，在开放阅读框内二者有3个核苷酸的差异，2个氨基酸的差异。与已发表的其它galectin相比，雌、雄成虫的galectin与Hco-gal-3b的同源性最高。二级结构及蛋白质特性预测分析发现，雌、雄成虫的galectin均具有琢螺旋、茁折叠和茁转角，雌虫的galectin蛋白比雄虫的分别多出一个琢螺旋和茁折叠区。二者的亲水性存在一定差异，但抗原性几乎无差异，且抗原表位比较集中。如同其它galectin一样，在雌、雄成虫的galectin蛋白的

3、N末端不存在信号肽序列。关键词：捻转血矛线虫；半乳糖结合凝集素；序列分析Cloning,CharacterizationandSequenceAnalysisofgalectincDNAofFemaleandMaleAdultWormsofLIChun-HuaLIXiang-Rui**WEIXiao-FengXULi-Xin()Apairofgene-specificprimersweredesignedaccordingtothesequencesofgalectingenesofmaleandfemalemixtureofacceptedinGenBank.

4、The880bpproductwasamplifiedbyreversetranscriptase-polymerasechainreaction(RT-PCR)withtotalRNAextractedfromfemaleormaleadultwormsofastemplaterespectively,andclonedintopMD18-Tvector.CharacterizationbyrestrictionenzymesR玉/玉digestion,PCRandsequencingconfirmedthatthesetwoamplifiedproducts

5、weregalectingenesof,whichweretermedasand.Comparisonandanalyseofnucleotideacidsequences,thededucedaminoacidsequencesorantigenicitiesofgalectinsofandothergalectinsshowedthatthereweredifferencesbetweengalectingeneoffemalewormsandthatofmaleworms,with3nucleotideand2aminoacidvariationsinth

6、eopenreadingframe(ORF).Andweresimilartowith99.2%and99.5%identity,respectively.Predictionandcomparisonofsecondarystructureorcharacteristicsofdeducedfemaleandmalegalectinproteinsshowedthat琢-helixes,茁-sheetsand茁-turnsexistedinthem,buttherewasonemore琢-helixand茁-sheetinfemalegalectinprote

7、inthaninmaleone.Therewereslightlydifferentinhydrophilicityandhardlyanydifferenceinantigenicitybetweenthem.Theirantigenicepitopeswereclustered.Similarasthatofothergalectinsofnematodeparasites,therewerenosignalpeptidesequencesintheN-terminalofHco-gal-fandHco-gal-m.;galectin;sequenceana

8、lysis捻转血矛线虫（