Cloning And Expression Analysis Of Macrophage Related Factors In Grass Carp

Inflammation is often accompanied by activation of the innate immune system,followed by the differentiation of monocytes into macrophages.Among immune cells,macrophages are a key cell type that not only clears microorganisms and debris through phagocytosis,but also controls processes such as wound healing.It therefore plays a crucial role in inflammation and processes that can lead to disease.The interleukin-1family（IL-1）is an important cytokine mainly secreted by macrophages and is one of the main components that mediate inflammatory signals.Arylhydrocarbon receptor（AHR）is an important transcriptional regulator expressed on the surface of macrophages that can control adaptive immune responses.In this study,grass carp（Ctenopharyngodon idellus,Ci）was used as a research model to clone a novel IL-1 family member il-1fmb and aryl hydrocarbon receptor homolog ahr1a,and to deeply explore the expression and function of IL-1Fmb and Ah R1a.A novel IL-1 family member,IL-Fmb,may function as a pro-inflammatory factor in the immune system of teleost fish,and Ah R1a may be regulated by inflammatory factors.The research contents are as follows:1.In this study,we cloned and identified grass carp il-1fmb and ahr1a by rapid amplification of c DNA ends（RACE）by aligning with the zebrafish genome library.The full-length c DNA of il-1fmb gene is 837 bp,including 37 bp of 5’untranslated region（UTR）,77 bp of 3’UTR,and 723 bp of open reading frame（ORF）encoding 240 amino acids（AA）,without the typical signal peptide domain.Multiple sequence alignment and cleavage site prediction showed that IL-1Fmb did not contain a typical inflammatory cysteine protease（Caspases）cleavage site,but there was neutrophil elastase in front of the IL-1 family domain（Neutrophil elastase）cleavage sites V⁶⁹、V⁷⁰、V⁷³、A⁷⁵、A⁷⁸、A⁸²、V⁸³.IL-1Fmb did not share high homology with other IL-1 family members,and the identity with zebrafish（Danio rerio）IL-1Fmb was 41.8%.IL-1Fmb means 6 exons and 5 introns.Phylogenetic tree and gene colinearity indicated that IL-1Fmb was conserved in teleost evolution.The full-length c DNA of grass carp ahr1a is 2 893 bp,including 5’UTR 292 bp and 3’UTR 57 bp,ORF 2 544 bp,encoding 847 amino acids.Grass carp Ah R1a had the highest similarity and consistency with the Yellow River carp（Cyprinus carpio）,81.5%and 74.7%,respectively,and the Ah R1a（Ah R）similarity and consistency with other species also reached more than 45.4%and 38.8%.2.In order to better study and determine the biological function of IL-1Fmb,this study cloned the predicted mature peptide fragment of CiIL-1Fmb(F⁷⁷-Y²⁴⁰)and CiIL-1β(F¹³⁰-K²⁷⁰)into p ET-21d prokaryotic expression vector,using isopropyl thiogalactoside（IPTG）to obtain the recombinant protein through the E.coli expression system,and use gel chromatography and affinity chromatography to purify it.Monoclonal antibodies were prepared using the recombinant proteins of CiIL-1Fmb and CiIL-1β,and two hybridoma cell lines were screened out by Western Blotting to purify and label them.The purified antibody could well detect EPC cells transfected with CiIL-1Fmb and CiIL-1β.3.The expression of Ciil-1fmb and Ciahr1a in healthy grass carp was analyzed by real-time quantitative PCR technology.The results showed that both Ciil-1fmb and Ciahr1a were constitutively expressed in various tissues,with the highest expression of Ciahr1a in the gill and spleen,and the highest expression of Ciahr1a in the intestine and gill.In order to explore the functions of IL-1Fmb and Ah R1a in immune response,this study used LPS to stimulate primary head kidney cells of grass carp,and found that il-1fmb mRNA expression peaked at 6 h and decreased at 12 and 24 h.The expression level of ahr1a was significantly decreased after 6 h of LPS stimulation,and returned to the background level after 12 and 24 h.Stimulation with CiIL-1βrecombinant protein at 6 h and 12 h reduced the expression level of ahr1a.Compared with single stimulation,LPS and r IL-1Fmb co-stimulation at 6 h significantly increased the expression levels of il-1β,il-6,il-8 and il-10,while the other two members of the IL-1 family,il-fma and il-1fmb have no effect.Costimulation of r IL-1βand r IL-1Fmb had no significant effect on the expression levels of il-1β,il-1fma,il-6,il-1fmb,il-8 and il-10.To explore whether IL-1Fmb has an effect on the NFкB pathway,the effect on the promoter was detected by stimulating grass carp ovary cells transfected with human NFкB promoter with r IL-1Fmb,r IL-1βand LPS.The results showed that LPS（4944.63±245.46）could significantly enhance the luciferase activity of human NFкB promoter,while r IL-1Fmb（6970.04±361.56）and r IL-1β（6804.21±241.22）could significantly enhance its luciferase activity.These results suggest that IL-1Fmb plays a role in the NFκB pathway.The above results suggest that grass carp IL-1Fmb is involved in pro-inflammatory regulation,and it is likely to conduct signal transduction through the NFκB pathway;and the expression level of Ah R1a is also regulated by pro-inflammatory factors,suggesting that it has a certain role in the inflammatory response.4.Ciahr1a was detected by infecting grass carp with Flavobacterium columnare and Reovirus of grass carp（GCRV）by intraperitoneal injection.In the head kidney,the expression of ahr1a was down-regulated 12 and 24 h after injection of Flavobacterium columnare,and returned to the background level after 48 h.In the spleen,the expression of ahr1a was down-regulated at 24 and 48 h,and the expression level at 72 h had no significant change compared with the control group;Except for the significant decrease of ahr1a mRNA level in head kidney and spleen after GCRV-II infection,other time changes were not significant.In this study,grass carp il-1fmb and ahr1a genes were identified,and the basic characteristics of genes and amino acids were analyzed by bioinformatics.IL-1Fmb was preliminarily explored by recombinant protein,real-time quantitative PCR and dual-luciferase reporter gene.At the same time,the expression profile of ahr1a after injection of Flavobacterium columnare and GCRV was analyzed.In conclusion,the above studies suggest that IL-1Fmb,a novel member of the teleost IL-1 family,may play an immunological function as a pro-inflammatory factor,and that Ah R1a may also be regulated in inflammation.