ABSTRACT
Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.
Subject(s)
Fish Diseases , Flatfishes , HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins , Phylogeny , Animals , Flatfishes/immunology , Flatfishes/genetics , Flatfishes/microbiology , Flatfishes/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Fish Diseases/immunology , Fish Diseases/microbiology , Fish Diseases/genetics , Fish Diseases/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Fish Proteins/immunology , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/veterinary , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/genetics , Molecular Docking Simulation , Aeromonas salmonicida/immunology , Molecular Chaperones/metabolism , Molecular Chaperones/geneticsABSTRACT
Cypermethrin (CYP) is an insecticide in the pyrethroid family and is used widely in agriculture and for public health purposes. However, CYP has been shown to have negative impacts on reproduction, immunity and nerves in mammals. In this study, a monoclonal antibody (mAb) against CYP was prepared and used to establish an indirect competitive immunosorbent assay (ic-ELISA) and colloidal gold lateral flow immunoassay (LFIA) for the quantitative and qualitative determination of CYP residues in agricultural products. The half inhibition concentration of the ic-ELISA was 2.49 ng/mL, and the cut-off value and visual limit of detection of the LFIA were 0.6 and 0.3 µg/mL, respectively. The recovery rates of the ic-ELISA ranged from 78.8% to 87.6% in tomato, cabbage and romaine lettuce. The qualitative results of LFIA and quantitative results of ic-ELISA and HPLC were in good agreement in blind samples. Overall, the established ic-ELISA and LFIA proved to be accurate and rapid methods for the determination of CYP in agricultural products.