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.

Candida albicans is a major fungal pathogen in humans. Novel antifungal agents are urgent demanded due to the challenges of the resistance. Antimicrobial peptides (AMPs) are critical components of the innate immune system against pathogenic microorganism infection. MAF-1A is a novel cationic AMP that comes from Musca domestica and is effective against C. albicans, but the antifungal mechanism remains unclear. In this study, we performed a transcriptomics analysis in C. albicans using RNA-seq technique under the treatment of MAF-1A. A total of 5654 genes were identified. Among these, 1032 were differentially expressed genes (DEGs), including 575 up-regulated genes and 457 down-regulated genes. In these DEGs, genes encoding ergosterol metabolism and fatty acid biosynthesis were identified to be significantly down-regulated, while genes associated with oxidative stress response and cell wall were identified to be significantly up-regulated. Using pathway enrichment analysis, 12 significant metabolic pathways were identified, and ribosome, oxidative phosphorylation, citrate cycle were mainly involved. The results revealed that MAF-1A induces complex responses in C. albicans. This study provides evidence that MAF-1A may inhibit the growth through affect multi-targets in C. albicans cells.

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.

目的探讨家蝇甘油醛-3-磷酸脱氢酶（GAPDH）基因在不同饲料饲养、不同发育期及不同组织的转录和表达情况,评价其作为家蝇功能基因研究内参的稳定性。方法运用PCR技术扩增GAPDH基因完整编码序列,生物信息学方法预测该基因及其编码蛋白功能。构建pET-28a（+）-GAPDH重组质粒,转化到大肠埃希菌Transetta（DE3）中进行诱导表达,纯化重组蛋白制备多克隆抗体。运用Real-time PCR和Western blot检测家蝇不同发育期、三龄幼虫不同组织和不同饲养物处理下GAPDH基因的表达情况。结果家蝇GAPDH基因ORF全长999bp,编码332个氨基酸,理论分子质量37.3ku,等电点为8.57,具有GAPDH家族的蛋白保守结构域;构建的重组质粒经PCR及测序鉴定正确;SDS-PAGE分析显示重组质粒在Transetta（DE3）中表达,纯化后的表达产物与目的蛋白大小相符,Western blot显示该蛋白可被相应血清识别;Real-time PCR分析GAPDH基因在不同饲料饲养3龄幼虫、不同发育期均保持较好的转录稳定性,同时,Western blot分析GAPDH分子在家蝇不同发育期及3龄幼虫不同组织中均有表达,且表达量无明显差别。结论 GAPDH基因在不同发育期、不同组织及不同饲养物饲养的家蝇表达稳定,可作为家蝇可靠性内参基因使用。

Objective To clone and express the gene named as Annexins B2 of Taenia saginata asitica so as to analyze the immunogenicity of its recombinant protein.Methods By online analysis of the gene at bioinfomatics websites to identify the gene from the Taenia saginata asiatica full-length cDNA plasmid library,the gene was cloned into prokaryotic expression vector pET-28a(+) and then transformed to E.coli BL21 with IPTG induction.Thereafter,the gene product was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography.In addition,the immunoreactivity of the purified recombinant proteins was analyzed by Western blotting.Results As demonstrated by PCR,double enzyme digestion and DNA sequencing indicated that the recombinant plasmid was successfully constructed and highly expressed.The recombinant protein tested with Western blotting analysis could react with Taenia asiatica and Taeniarhynchus saginatus infected patient serum.Conclusion The Annexins B2 of Taenia saginata asitica has been cloned and expressed in the prokaryotic expression system and the purified protein has been confirmed with immunogenicity.