›› 2014, Vol. 57 ›› Issue (11): 1272-1280.

• 研究论文 • 上一篇    下一篇

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

许炼2, 高焕娟2, 潘志针1, 朱育菁1, 陈清西2,*, 刘波1,*   

  1. (1. 福建省农业科学院农业生物资源研究所, 福州 350003; 2. 厦门大学生命科学学院, 福建厦门 361005)
  • 出版日期:2014-11-20 发布日期:2014-11-20
  • 作者简介:许炼, 男, 1990年生, 福建漳州人, 硕士研究生, 主要从事微生物技术研究, E-mail: xulian2014@sina.com

Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera: Plutellidae)

XU Lian2, GAO Huan-Juan2, PAN Zhi-Zhen1, ZHU Yu-Jing1, CHEN Qing-Xi2,*, LIU Bo1,*   

  1. (1. Agricultural BioResources Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China; 2. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China)
  • Online:2014-11-20 Published:2014-11-20

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cDNA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cDNA 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 kDa, 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PxAPN5(GenBank登录号: KM034756)。PxAPN5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PxAPN5, 表达产物经SDS-PAGE电泳, 在110 kDa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PxAPN5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, PxAPN5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。

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

Abstract: 【Aim】 The aim of this study is to analyze the cloning, expression and homology modeling of midgut aminopeptidase gene PxAPN5 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 Cry2Ab 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 kDa 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 PxAPN5. PxAPN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and O-glycosylation sites, GPI anchor point, C-transmembrane domains and zinc-metalloprotease domain (361HEXXH365). A 110 kDa specific protein band appeared when APN protein was inducibly expressed in Escherichia coli. Aminopeptidase activity assay showed that the supernatant of broken bacteria possessed aminopeptidase activity and its specific activity was 1 047.2 U/g. The ligand blot result indicated that PxAPN5 could bind to Cry2Ab specially. Multiple alignment of amino acid sequences demonstrated that there are three mutations in PxAPN5 when compared to other known APN proteins from P. xylostella. 【Conclusion】 The PxAPN5 protein with aminopeptidase activity was successfully cloned and expressed, and it could bind to Cry2Ab. Prediction of characteristics and functions of mutation sites in PxAPN5 was carried out by homology modeling. These results laid the foundation for the functional research of PxAPN.

Key words:  Plutella xylostella, aminopeptidase (APN) gene, gene analysis, prokaryotic expression, enzyme activity, ligand blot, homology modeling