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【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。
【Objective】 Cloning and expression of Plutella xylostella aminopeptidase gene was carried out and gene sequence analysis and homology modeling analysis were performed. 【Method】 The amino acid sequence of the aminopeptidase was cloned by using the c DNA of the diamondback moth as a template. The aminopeptidase protein was expressed in prokaryotic cells for enzyme activity determination. The binding of aminopeptidase and Cry2Ab protein was analyzed by Ligand blotting. Analysis of the mutation site. 【Result】 Amino peptidase gene was amplified from c DNA of midgut of Plutella xylostella. The full length cDNA was 2 853 bp encoding a protein of 950 amino acids. The predicted molecular weight was 107.3871 kDa and the isoelectric point was 5.24. The cloned aminopeptidase gene belongs to the APN family 5, which is named Px APN5 (Gen Bank Accession No. KM034756). The Px APN5 protein has the conserved characteristics of the lepidopteran insect aminopeptidase protein, ie, contains an N-glycosylation site, an O-glycosylation site, and a GPI anchor site with a “HEXXH” zinc protease domain And C-terminal transmembrane region. The prokaryotic expression of Px APN5 was carried out in Escherichia coli. The expressed product showed specific band around 110 kDa by SDS-PAGE electrophoresis. The enzyme activity test showed that the supernatant of the bacterial cell had aminopeptidase activity with specific activity of 1 047.2 U / g. Ligand blotting results show that the expressed Px APN5 specifically binds to the Cry2Ab protein. Multiple sequence alignment showed that three conserved sites of Px APN5 amino acid sequence were mutated compared with other reported amino-peptidases, and the mutation sites were characterized by protein modeling. 【Conclusion】 The present study successfully cloned and expressed the aminopeptidase aminopeptidase, and found that it could specifically bind to Cry2Ab protein. The characteristics and functions of the mutant site of aminopeptidase were studied by protein modeling prediction. These results provide a theoretical basis for the functional study of aminopeptidase in Plutella xylostella.