Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.

This work is to clone and express the Musca domestica peptidoglycan recognition proteins-SA(PGRP-SA)gene and research the microbial binding activity of it. The PGRP-SA gene was isolated from M. domestica larvae cDNA library,then primers were designed using cDNA plasmid as the template,and the complete encoding sequence of PGRP-SA was acquired by PCR. The bioinformatics methods were employed to predict and analyze the gene and its encoded proteins. The recombinant plasmid pET-28a(+)-PGRP-SA was expressed in Escherichia coli for induced expression and protein purification. RT-PCR was used to test the varied transcription levels of PGRP-SA gene in different tissues. The microbial binding activity of PGRP-SA protein was studied by the microorganism binding assay. The results indicated that the ORF full-length of PGRP-SA gene was 615 bp,encoding 204 amino acids. The molecular weight was 22.8 kD and pI 9.11 and had the conserved PGRP domain. After the recombinant plasmid pET-28a(+)-PGRP-SA was successfully constructed,it was expressed in E. coli by IPTG. SDS-PAGE and Western blot analysis showed that the functional protein purified by Ni2+ affinity chromatography was in consistent as the predicted in size. PGRP-SA was expressed in three instars hemolymph,fat body,foregut,midgut,trachea,malpighian tube except hindgut,the highest in the hemolymph,indicating that the expression of PGRP-SA was tissue-specific. Recombinant PGRP-SA protein bound to Staphylococcus aureus and E. coli,but not Monilia albica. Conclusively,the M. domestica PGRP-SA protein was successfully expressed and purified,and also it was confirmed that PGRP-SA protein from M. domestica bound to S. aureus and E. coli.