| In comparison to higher animals,insects as the most widely distributed and numerous organisms in nature have their unique immune system and mechanisms of immunity that can be generally divided into two innate types: humoral immunity and cellular immunity.ENF peptide,as a widely distributed cell factor in insects,participates in various physiological functions,among which the main function is to participate in the immune response within the insect body.When insects are invaded by pathogens,ENF peptides in the body are activated by serine proteinases,cut from precursors into active forms,and then activate the immune response in the body.Currently,most studies on ENF peptides focus on how they activate the immune response,but it is not clear how the later immune response is terminated to maintain immune balance in the body and prevent excessive reactions.Interestingly,in many insects,there also exists a class of proteins that specifically bind to ENF peptides,called ENF peptide-binding proteins.For example,growth-inhibitory peptide binding protein can specifically bind to growth-inhibitory peptide and silence its activity.Therefore,ENF peptide-binding proteins play an important role in the immune response and autoregulation processes in insects,being an important part of the immune response response loop.In summary,during the process of ENF peptide-induced immune response,there is a feedback regulation mechanism where ENF peptide activates the immune response followed by termination by ENF peptidebinding proteins,but the most important part of this mechanism,ENF peptide receptor protein,is still unclear.The screening and identification of ENF peptide receptor protein will help us better understand how cell factors play physiological functions and maintain organism homeostasis in insects,which is also of great significance for studying the mechanism of immune response termination.The main research content of this article is the screening and identification of ENF peptide receptor protein,using immunoprecipitation and liquid chromatography-mass spectrometry techniques to fish out the receptor protein that interacts with ENF peptide PP from the fat body of silkworms.Further identification at the level of cells,tissues,and individuals has verified the importance of the ENF peptide receptor protein Mth2 in regulating the immune response.The main research results obtained are as follows:1、Screening of ENF peptide(PP)receptor proteinUsing immunoprecipitation technology,membrane receptor proteins that can bind to ENF peptide PP were fished out from the fat body tissue of silkworms.Through mass spectrometry identification of differential bands,a total of 191 proteins with transmembrane sequences were identified.Further bioinformatics analysis and functional annotation of these transmembrane proteins led to the selection of 10 possible receptor protein targets.Molecular cloning of these target genes was performed,and the coding DNA sequence(CDS)of nine of them was successfully cloned.Using homologous recombination molecular cloning technology,eight of the target genes were successfully constructed in a vector containing a red fluorescence tag.By expressing these receptor protein genes with red fluorescence tags on BmE cells and adding ENF peptide PP labeled with FITC,fluorescence colocalization was observed using confocal microscopy.Among them,neuropeptide F receptor(NFR)and G protein-coupled receptor Mth2 exhibited fluorescence colocalization with ENF peptide.In vitro Pull-down technology was used to verify that both NFR and Mth2 can bind to PP,indicating that they are preliminary identified as ENF peptide receptor proteins.2、Identification of ENF peptide(PP)receptor proteinTo verify whether the receptor proteins NFR and Mth2 mediate the immune response induced by ENF peptide,NFR and Mth2 were overexpressed in BmE cells.After successful overexpression,ENF peptide was added to the overexpressing cells for stimulation at 48 hours,and the downstream levels of antimicrobial peptide genes were detected.After overexpression of NFR,there was no significant change in antimicrobial peptide genes.However,after overexpression of Mth2,downstream antimicrobial peptides Cecropin A,Moricin,and early immune-activated peptide PGRP-S2 were significantly upregulated within 3 hours.Meanwhile,after successful interference with the Mth2 gene in BmE cells,adding ENF peptide for antimicrobial peptide gene detection showed significant downregulation of downstream genes such as Attacin,Moricin,and PGRP-S2 within 3 hours.Therefore,it can be seen that Mth2 is involved in the regulation of immune response induced by ENF peptide,while NFR is not involved.Bioinformatics analysis of Mth2 revealed that it belongs to the B-class Methuselah(Mth)proinsulin-like receptor protein in GPCR,which has a large extracellular domain at the N-terminus for receiving intracellular substances such as peptides.Mth2-Nter,the extracellular region of Mth2,was expressed using a prokaryotic expression system and mainly existed in the form of inclusion bodies.The Mth2-Nter protein was purified using inclusion body protein purification and multi-clonal antibodies were prepared.Furthermore,Mth2-Nter and full-length Mth2 proteins were expressed and purified using an insect cell baculovirus expression system to explore the immune function of Mth2 receptor protein.3、Functional Validation of Mth2 in the Immune System of SilkwormsTo validate the immune function of Mth2 as a receptor protein for ENF peptide,further verification of its immune function was conducted at the tissue level.Mth2 antibodies were incubated with isolated adipose tissue and stimulated with ENF peptide,or recombinant expressed Mth2 protein was simultaneously added to isolated adipose tissue and stimulated with ENF peptide to detect the expression levels of downstream antimicrobial peptide genes.It was found that both the addition of Mth2 antibodies and recombinant protein effectively downregulated the downstream humoral immune response induced by ENF peptide.In addition to upregulating the expression of antimicrobial peptide genes and inducing humoral immunity in adipose tissue,ENF peptide also induces the upregulation of phagocytosis-related genes in downstream blood cells,leading to cellular immunity.The addition of Mth2 antibodies to cultured hemocytes of silkworms followed by ENF peptide stimulation also resulted in the downregulation of downstream phagocytosis-related gene expression.These tissue-level results indicate that Mth2 participates in the immune response regulation of silkworms as a receptor protein for ENF peptide.At the same time,we also used CRISPR/Cas9 to create transgenic silkworm with Mth2 whole-body knockout.When Mth2-knockout transgenic silkworm was injected with ENF peptide PP at the individual level,the downstream antibacterial peptide genes showed significantly down-regulated expression at 3h compared with the control group,indicating that Mth2 was also involved in the immune regulation caused by ENF peptide at the individual level.The economic characters of transgenic bombyx mori were statistically analyzed,and there was no significant difference in cocoon weight,cocoon layer ratio and pupa weight compared with the control group.According to the statistics of the growth cycle,it was found that the growth cycle from larval stage to upper cluster stage was extended by 2 days compared with the control group,which indicated that Mth2 was related to the growth and development cycle and life span of silkworm and did not affect the economic effect of silkworm. |
