Molecular Cloning And The Immune Function Of TLR1 And TLR21 In Common Carp,Cyprinus Carpio L.

Toll-like receptors(TLRs)are important pattern recognition molecules that are linked to adaptive immune and natural immune bridges.TLRs receptors can be divided into six subfamilies,TLR1 family,TLR3 family,TLR4 family,TLR5 family,TLR7 family,TLR11 family.Fish is currently the most species of TLRs species,and 21 TLRs have been found in fish.The study of fish TLRs can provide basic data for evolutionary studies of natural immunity.In this paper,the full length of TLR1 and TLR21 genes was cloned by PCR and RACE.The full length of TLR1 cDNA was 2626 bp,the open reading frame(ORF)was 2166 bp,encoding721 amino acid residues,the full length of TLR21 was 3604 bp,ORF contained 2892 bp,encoding 963 amino acids,The domain was analyzed and found to have a typical TLRs receptor structure that contains an intracellular TIR domain,a transmembrane domain,and an extracellular leucine repeat sequence domain.Using BioEdit software to compare the amino acid sequences of different species,it was found that there were asparagine sites,suggesting that the formation of horseshoe-like structure and the intracellular TIR domain is highly conserved.The three-dimensional spatial structure of TLR1 and TLR21 was simulated by I-TASSER software,and confirmed the typical TLRs receptor family characteristics.Construction of phylogenetic tree of different fish and some mammals by MEGA6 software,it was found that TLR1 belongs to the family of TLR1 family,which is closest to grass carp,and TLR21 belongs to TLR11 family member,which is closest to zebrafish and Atlantic salmon.The expression of TLR1 and TLR21 in the liver,spleen,head kidney,foregut,hindgut,gill,gonadal,skin,muscle,oral epithelium and brain were detected by real-time fluorescence quantitative PCR.The expression of TLR1 in the brain,spleen,head kidney and gonad was higher,lower expression in the hindgut and oral epithelial cells,TLR21 was higher in the spleen and head kidney,and was lower in the skin and liver.The expression of TLR1 mRNA was significantly increased in the foregut,hindgut and liver after ascites injection of Aeromonas hydrophila,which increased 7 times in the hindgut and liver,The expression of TLR21 mRNA increased in the liver,foregut and hindgut.The expression of TLR1 and TLR21 in the liver,spleen,head kidney,foregut and hindgut was induced by intraperitoneal injection of poly(I: C),and the expression of TLR1 m RNA in the liver and foregut increased,Respectively,and the expression of TLR21 m RNA was up-regulated in the liver,spleen,foregut and hindgut,and the highest in the liver was higher than that in the control group.This study provides the basic data for the enrichment of natural immunity of fish,and also provides a theoretical basis for the research and prevention of pathogenesis of common carp diseases.