草地贪夜蛾组织蛋白酶B的基因克隆、序列分析、三维结构预测及其分子对接模拟

doi:10.13560/j.cnki.biotech.bull.1985.2017-0818

摘要/Abstract

摘要： 昆虫组织蛋白酶B（Cathepsin B）在昆虫发育和代谢过程中发挥重要作用。本研究首先克隆了草地贪夜蛾Spodoptera frugiperda（J.E.Smmith）的组织蛋白酶B基因Cathepsin B（SCB）,获得其开放阅读框（ORF）序列（GenBank 登录号：HQ110064）。生物信息学分析表明,该基因的开放阅读框包含1 026 bp 的片段,编码341 个氨基酸,预测N-末端含有长度为20 个氨基酸残基的信号肽序列,去除信号肽序列后,预测成熟蛋白分子量为35.6 kD,等电点为6.59。氨基酸序列与15 种物种Cathepsin B比较有51.2%-96.2%的一致性,与甜菜夜蛾Spodoptera exigua（Hiibner）相似度最高,达96.2%。利用同源建模预测SCB的三维结构,经动力学的优化,SCB折叠成紧密而稳定的球状结构,含6 个对结构有稳定作用的二硫键,亲水性氨基酸主要包被在蛋白表面。同时分子对接模拟结果表明,SCB结合口袋比较浅,形状不规则,袋口较宽。ARG19、VAL23和ASN24为活性位点关键氨基酸残基,抑制剂CA与SCB活性位点3个关键氨基酸残基有较强的相互作用,且均主要表现为静电相互作用能,其中VAL23、ASN24与CA形成氢键。以期为进一步运用生物实验手段研究Cathepsin B的结构和功能,设计和开发昆虫组织蛋白酶类抑制剂类杀虫剂奠定基础。

关键词: Sf9细胞, Cathepsin B, 基因克隆, 同源建模, 分子对接, 抑制剂

Abstract: Cathepsin B plays an important role in development and metabolism of insect.In present study,Cathepsin B（SCB）gene was cloned from Spodoptera frugiperda（J.E.Smmith）（GenBank accession number：HQ110064）.Bioinformatics analysis showed that the open reading frame（ORF）of SCB was 1 026 bp,encoding 341 amino acids,and the predicted N-terminus hydrophobic region contained 20 amino acid residues displaying the characteristic features of a signal peptide. The predicted molecular weight（MW）and isoelectric point（pI）of SCB was 35.6 kD and 6.59 respectively while excluding signal peptide. The similarity of Cathepsin B amino acid between S. frugiperda and other 15 species ranged from 51.2%-96.2%,while at the highest of 96.2% with Spodoptera exigua（Hiibner）.The three dimension structure of SCB was predicted by homology modeling method,SCB was found to fold into a tight global structure with the dynamic optimization and contain 6 disulfide bonds responsible for the stability of the structure,and hydrophilic amino acids were mainly coated on the surface of the protein. Moreover,molecular docking simulation showed that the SCB binding pocket was relatively shallow,wide and irregular. ARG19,VAL23,and ASN24 were the key amino acid residues at active sites of SCB,and the inhibitor CA-074me（CA）interacted strongly with these three key amino acid residues by mainly electrostatic interaction energy. CA formed 2 hydrogen bonds with VAL23 and ASN24 respectively. This study lays a foundation for the further research on structure and function of SCB by using biological experimental methods,and for the development of cathepsin inhibitor pesticides.

Key words: Sf9 cell, Cathepsin B, gene cloning, homology modeling, molecular docking, inhibitor

程杏安, 叶静敏, 蒋旭红, 刘展眉, 胡美英. 草地贪夜蛾组织蛋白酶B的基因克隆、序列分析、三维结构预测及其分子对接模拟[J]. 生物技术通报, 2018, 34(1): 183-194.

CHENG Xing-an, YE Jing-min, JIANG Xu-hong, LIU Zhan-mei, Hu Mei-ying. Gene Cloning,Sequence Analysis,and Three Dimensional Structure Prediction of Cathepsin B in Spodoptera frugiperda and Its Molecular Docking Simulation[J]. Biotechnology Bulletin, 2018, 34(1): 183-194.

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