›› 2018, Vol. 61 ›› Issue (4): 410-422.doi: 10.16380/j.kcxb.2018.04.003

• 研究论文 • 上一篇    下一篇


程杏安1, 林贤伟1, 蒋旭红1, 刘展眉1, 钟国华2,* , 胡美英2,*   

  1. (1. 仲恺农业工程学院天然产物化学研究所, 广州 510225; 2. 华南农业大学昆虫毒理研究室, 广州 510642)
  • 出版日期:2018-04-20 发布日期:2018-04-20

cDNA cloning, prokaryotic expression and polyclonal antibody preparation of cathepsin L in Spodoptera frugiperda (Lepidoptera: Noctuidae)

CHENG Xing-An1, LIN Xian-Wei1, JIANG Xu-Hong1, LIU Zhan-Mei1, ZHONG Guo-Hua2,*, HU Mei-Ying2,*   

  1. (1. Institute of Natural Product Chemistry, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; 2. Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China)
  • Online:2018-04-20 Published:2018-04-20

摘要:  【目的】克隆草地贪夜蛾Spodoptera frugiperda的组织蛋白酶L(cathepsin L, CatL)基因,分析该基因的序列特征并制备该酶多克隆抗体,为探析其生理功能奠定基础。【方法】根据甜菜夜蛾Spodoptera exigua的组织蛋白酶L基因开放阅读框(ORF)序列两端直接设计引物克隆草地贪夜蛾组织蛋白酶L基因。利用生物信息学软件分析该基因的序列特征,利用ClustalX2软件进行同源比对和进化分析,利用同源建模预测该酶三维结构。通过原核表达重组蛋白,多次免疫新西兰大白兔制备该酶多克隆抗体。【结果】克隆获得了草地贪夜蛾的组织蛋白酶L基因SfCatL (GenBank登录号: HQ110065),ORF序列长1 035 bp,编码344个氨基酸,预测N-末端含有长度为16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36.8 kD, 等电点为6.69。SfCatL氨基酸序列与其他13个物种的组织蛋白酶L氨基酸序列比较有53.7%~96.8%的一致性,与甜菜夜蛾组织蛋白酶L氨基酸序列一致性最高,达96.8%。同源建模预测表明,SfCatL折叠成紧密而稳定的元宝状结构,含3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的表面。原核表达、纯化SfCatL蛋白制备抗血清,其效价超过1∶40 000,Western blot鉴定结果表明抗血清与草地贪夜蛾Sf9细胞SfCatL蛋白能够特异性结合。【结论】获得了草地贪夜蛾SfCatL完整ORF序列,分析了其特征,经原核表达、纯化获得高纯度的融合蛋白,成功获得多克隆抗体。本研究为进一步研究该基因的功能并开发组织蛋白酶抑制剂类杀虫剂提供理论依据。

关键词: 草地贪夜蛾, 组织蛋白酶L, 基因克隆, 原核表达, 抗体

Abstract:  【Aim】 The objective of this study is to clone cathepsin L gene from Sf9 cells of Spodoptera frugiperda, to analyze its sequence features and to obtain the polyclonal antibody, so as to provide a theoretical basis for further studying the function of the gene in this insect. 【Methods】 Cathepsin L gene was cloned directly by using the specific primers which were designed based on the open reading frame (ORF) sequence of cathepsin L gene of S. exigua. The sequence characteristics of this gene were analyzed by using bioinformatics, and the deduced amino acid sequences of the gene were aligned using the ClustalX2 program. The three-dimensional structure of cathepsin L was modeled by homology modeling method. Finally, the anti-rabbit polyclonal antibody was produced by immunizing rabbit based on the prokaryotic expression of the gene. 【Results】 A cathepsin L gene named SfCatL (GenBank accession no.: HQ110065) was cloned from Sf9 cells of S. frugiperda, with the ORF of 1 035 bp in length, encoding 344 amino acids with the predicted N-terminal hydrophobic region of 16 amino acid residues displaying the characteristic features of a signal peptide. The predicted molecular weight (MW) and isoelectric point (pI) of SfCatL without signal peptide are 36.8 kD and 6.69, respectively. SfCatL has 53.7%-96.8% amino acid sequence identity with the cathepsin L proteins from other 13 species, showing the highest amino acid sequence identity (96.8%) with the cathepsin L protein from S. exigua. The three-dimensional structure of SfCatL showed that SfCatL folds into a shape of fortune cookie and contains three disulfide bonds, which are responsible for the stability of the structure. Hydrophilic amino acids are mainly coated on the surface of protein. After prokaryotic expression, the SfCatL protein was purified to produce the antiserum of SfCatL, whose titer reached 1∶40 000 by ELISA assay. Western blot assay indicated that the antiserum could specifically bind with the SfCatL protein from Sf9 cells. 【Conclusion】 The complete ORF of SfCatL was cloned, and its sequence features were analyzed. The SfCatL recombinant protein was prepared and purified, and finally the polyclonal antibody was acquired. This study provides a theoretical basis for further study of the gene function and the development of cathepsin inhibitor insecticides.

Key words: Spodoptera frugiperda, cathepsin L, gene cloning, prokaryotic expression, antibody