Antifungal Effects Of Bombyx Mori Antimicrobial Protein Attacin 1 On Beauveria Bassiana And Expression Characteristics Of Antimicrobial Peptide Cecropin A

Antimicrobial peptides are a class of important antimicrobial complex produced by host during immune process.Bombyx mori Attacin 1(BmAttacin 1)is one of the antimicrobial peptides in the silkworm.Its open reading frame(ORF)encodes a peptide of 214 amino acids.Our previous study indicated that BmAttacin 1 gene was significantly up-regulated in silkworm larvae against Beauveria bassiana infection.In this study,bioinformatics analysis revealed that the N-terminal sequence 1 to 17 amino acids is the signal peptide,and tertiary structures of the mature protein mainly forms anti-parallel β-sheet structure.Real-time fluorescence quantitative PCR(RT-qPCR)results further confirmed the significant up-regulation of BmAttacin 1 gene in the larvae infected with B.bassiana.The coding sequence of BmAttacin 1 mature protein was cloned from the cDNA of the third instar larvae of silkworm strain p50,and BmAttacin 1 recombinant protein was expressed with prokaryotic expression system.The expressed recombinant BmAttacin1 exhibited antimicrobial activities against Gram-negative bacteria Escherichia coli and entomopathogenic fungus B.bassiana in antimicrobial experiment,but not against Gram-positive bacteria Bacillus subtilis.Furthermore,after incubation of BmAttacin 1 with B.bassiana conidia,indirect immunofluorescence detection indicated that BmAttacin 1 could bind to the surface of conidia.Over-expression of BmAttacin 1 gene in BmN cell line up-regulated the expression of other seven antimicrobial peptides,among which BmCecropin A was up-regulated for 10-folds and BmCecropin B for 9-folds,implying that the expression of BmAttacin 1 could synergistically enhance the expression of other antimicrobial peptides.These results laid a good foundation for further study on the interaction between BmAttacin 1 and B.bassiana and the roles it plays in immune response of silkworm against B.bassiana infection.Bombyx mori Cecropin A(BmCecropin A)is also one of the antimicrobial peptides in the silkworm.Its ORF encodes 63 amino acids.Our previous study indicated that BmCecropin A has obvious antimicrobial activities against Beauveria bassiana both in vivo and in vitro,but the specific mechanism of its role is still unclear.In this study,bioinformatics analysis revealed that the C-terminal sequence 1 to 22 amino acids is the signal peptide,and antigen epitope amino acids sequence is AIAVIGQA.With the structure analysis and homologous sequences alignment of BmCecropin A mature peptide,the peptide sequence 23 to 63 and 51 to 63 amino acids of BmCecropin A were chemical synthesized and linked with KLH and BSA,respectively,then mixed as antigens to immunize Balb/c mouses.Western blot results showed that the serum of immunized mouse had specific antigen-antibody response to BmCecropin A.Two positive hybridoma cell lines were obtained by cell fusion and hybridoma cell line screening.Hemolymph of silkworm was detected by Western blotting with the supernatant of hybridoma cell line culture medium.The results showed that the monoclonal antibody secreted by one of the hybridoma cell lines could bind to the antigen and only a single band was detected by western blotting,which is the BmCecropin A.It indicated that the monoclonal antibody produced by the hybridoma cell line could recognize the linear epitope of BmCecropin A and does not depend on the specific conformation of the antigen.Furthermore,monoclonal antibodies were prepared and the specific expression of BmCecropin A in various tissues of silkworm was detected by Western blotting.The results showed that specific antigen-antibody reaction was detected only in fat body.Isopropanol precipitation method was used to extract hemolymph protein.Western blotting showed that antimicrobial peptide Cecropin A was up-regulated obviously in the larvae infected with B.bassiana at 32 h.These results laid a good foundation for further study on Cecropin A and the roles it plays in immune response of silkworm against B.bassiana infection.