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	小菜蛾中肠氨肽酶基因PxAPN5的克隆、原核表达及蛋白质同源建模分析; Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera:Plutellidae)
	许炼 ; 高焕娟 ; 潘志针 ; 朱育菁 ; 陈清西 ; 刘波
	2015-01-20
关键词	小菜蛾 氨肽酶基因 基因分析 原核表达 酶活性 配体印迹 同源建模 Plutella xylostella aminopeptidase(APN) gene gene analysis prokaryotic expression enzyme activity ligand blot homology modeling
英文摘要	【目的】克隆和表达小菜蛾PluTEllA XylOSTEllA氨肽酶基因,并进行基因序列分析和同源建模分析。【方法】以小菜蛾中肠C dnA为模板克隆分析氨肽酶基因序列,原核表达氨肽酶蛋白并进行酶活性测定,应用配体印迹分析氨肽酶与Cry2Ab蛋白的结合,通过蛋白质建模对突变位点进行分析。【结果】从小菜蛾中肠C dnA扩增出氨肽酶基因,该基因全长2 853 bP,编码950个氨基酸,预测蛋白分子量为107.3871 k dA,等电点为5.24;进化树分析显示,克隆得到的氨肽酶基因属于APn家族5,将其命名为PX APn5(gEn bAnk登录号:kM034756)。PX APn5蛋白具有鳞翅目昆虫氨肽酶蛋白的保守性特征,即含有n-糖基化位点、O-糖基化位点和gPI锚定位点,具有“HEXXH“锌蛋白酶结构域和C端跨膜区域。在大肠杆菌ESCHErICHIA COlI中原核表达PX APn5,表达产物经SdS-PAgE电泳,在110 k dA附近出现特异性条带;酶活性测试显示菌体破碎上清液具有氨肽酶活性,比活力为1 047.2 u/g。配体印迹结果显示表达的PX APn5能与Cry2Ab蛋白特异性结合。多序列比对结果表明,与其他已报道的小菜蛾氨肽酶相比,PX APn5氨基酸序列有3个保守性位点发生了突变,并通过蛋白质建模的方式表征突变位点。【结论】本研究成功克隆和表达了具有氨肽酶活性的小菜蛾氨肽酶,并发现其能与Cry2Ab蛋白特异性结合;通过蛋白质建模对氨肽酶突变位点的特征及功能进行了预测。这些结果对小菜蛾氨肽酶的功能性研究提供了理论基础。; 【Aim】The aim of this study is to analyze the cloning,expression and homology modeling of midgut aminopeptidase gene Px APN5 in the diamondback moth,Plutella xylostella.【Methods 】 A aminopeptidase( APN) gene was cloned from the P.xylostella midgut,and bioinformatics analysis of the gene was performed.The APN protein was expressed using prokaryotic expression system,and its enzymatic activity was assayed.The interaction between APN and Cry2 Ab was determined by using ligand blot analysis.Homology modeling of APN gene was conducted for the prediction of characteristics and functions of mutation sites.【Results】The sequencing results showed that the cloned APN gene( NCBI accession no.: KM034756) is 2 853 bp in length and encodes 950 amino acids with the predicted molecular weight of 107.3871 k Da and isoelectric point of 5.24.Phylogenetic tree analysis indicated that this APN gene belongs to class 5 of APN family,and it was named as Px APN5.Px APN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and Oglycosylation sites,GPI anchor point,C-transmembrane domains and zinc-metalloprotease domain(361HEXXH365).A 110 k Da specific protein band appeared when APN protein was inducibly expressed in Escherichia coli.Aminopeptidase activity assay showed that the supernatant of broken bacteria possessedaminopeptidase activity and its specific activity was 1 047.2 U / g.The ligand blot result indicated that Px APN5 could bind to Cry2 Ab specially.Multiple alignment of amino acid sequences demonstrated that there are three mutations in Px APN5 when compared to other known APN proteins from P.xylostella.【Conclusion】The Px APN5 protein with aminopeptidase activity was successfully cloned and expressed,and it could bind to Cry2 Ab.Prediction of characteristics and functions of mutation sites in Px APN5 was carried out by homology modeling.These results laid the foundation for the functional research of Px APN.; 国家自然科学基金项目(31371999;31370059)
语种	zh_CN
内容类型	期刊论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/119283]
专题	生命科学－已发表论文
推荐引用方式 GB/T 7714	许炼,高焕娟,潘志针,等. 小菜蛾中肠氨肽酶基因PxAPN5的克隆、原核表达及蛋白质同源建模分析, Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera:Plutellidae)[J],2015.
APA	许炼,高焕娟,潘志针,朱育菁,陈清西,&刘波.(2015).小菜蛾中肠氨肽酶基因PxAPN5的克隆、原核表达及蛋白质同源建模分析..
MLA	许炼,et al."小菜蛾中肠氨肽酶基因PxAPN5的克隆、原核表达及蛋白质同源建模分析".(2015).

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