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

• RESEARCH PAPERS • Previous Articles     Next Articles

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

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