Identification And Expression Analysis Of Interleukin-10 Family In Spotted Sea Bass(Lateolabrax Maculatus)

Spotted sea bass(Lateolabrax maculatus)is an economically important fish found in coastal and estuarine areas of China.It becomes one of the main objects of cage culture and pond culture in the north and south because of its eurythermic and eurysalinity.The spotted sea bass tastes and is loved by people all over the world.Its breeding scale has been further expanded,but various bacterial diseases have plagued the development of L.maculatus breeding industry,which has brought certain economic losses.For the sustainable development of L.maculatus breeding,the innate immune response mechanism of L.maculatus was studied,and a more convenient and effective method for disease control of L.maculatus was found to be worthy of further study.Cytokine is a kind of small protein widely existing in vertebrates.After binding with the corresponding cytokine receptors,the signal is transmitted to the nucleus to play its biological role.Interleukin,as a kind of cytokine,plays an important role in immune response.IL-10 is an immunomodulatory factor with multiple effects and has multiple biological functions for different types of cells.Some cytokines related to IL-10 have been gradually discovered.They have similar gene structure and use similar receptor complex for IL-10,so these are classified as IL-10 cytokine family.They include IL-10,IL-19,IL-20,IL-22,IL-24 and IL-26.Currently,many studies have found that IL-10 family members can induce a variety of host defense mechanisms,maintain tissue homeostasis,promote innate immune response and accelerate the process of tissue healing after bacterial infection in vertebrates.They are cytokines closely related to inflammatory response in organisms.Specific biological functions of IL-10 family members of L.maculatus have not been thoroughly studied.This study aims to explore the role of IL-10 family members in innate immunity of L.maculatus by cloning the genes of IL-10 family and analyzing the m RNA expression level of immune tissue in L.maculatus after challenge.Then the prokaryotic recombinant proteins of the IL-10 family members were prepared in order to further study the function of the IL-10 family.At present,IL-10,IL-20 L,IL-22 and IL-26 members of the IL-10 family have been identified in fishes.In this study,LmIL-10,LmIL-20 L,LmIL-22 and LmIL-26 genes were obtained based on transcriptome database,homologous gene comparison and other methods.Bioinformatic analysis was carried out to study its role in immune response and immune regulation.The full-length of c DNA of four members were obtained by RACEPCR,which were 1126 bp,1075 bp,1093 bp and 1158 bp,encoding 187,175,191 and177 amino acids,respectively.Through multiple sequence alignment,it was found that the four genes of LmIL-10,LmIL-20 L,LmIL-22 and LmIL-26 all possessed the signature motifs of the IL-10 family.From mammals,birds,amphibians to fish,IL-10,IL-20 L and IL-22 had IL-26 cysteine residues.IL-26 is relatively less conserved during evolution.Further,phylogenetic tree analysis showed that the IL-10,IL-22,and IL-26 of spotted sea bass were well clustered with their counterparts of fish and mammals.While,fish IL-20 L were clustered with IL-19,IL-20 and IL-24 of higher vertebrates.Gene location showed that the four genes were strongly conserved in gene positions,LmIL-10 and LmIL-20 L were in the same locus,while LmIL-22 and LmIL-26 were in another locus.The linked genes of each family member from fish to mammals were consistent,and basically kept the same transcriptional direction.By qPCR technique,all IL-10 family members were found to be expressed in a variety of tissues,all members of the family had higher expression level in gill,and the level of LmIL-10 expression in all tissues except gill was greater than that of the other 3genes.After intraperitoneal injection of LPS and Edwardsiella tarda,it was found that LmIL-10,LmIL-20 L,LmIL-22 and LmIL-26 genes were up-regulated in the gill,head kidney,spleen and intestine immune groups to varying degrees.The results suggest that the IL-10 family is involved in host defence against bacterial infection and may play a significant role in the innate immune response of the spotted sea bass.The prokaryotic expression plasmids of pET-21d-IL-10,pET-21d-IL-20 L,pET-21dIL-22 and pET-21d-IL-26 were constructed,and the prokaryotic recombinant proteins of rLmIL-20 L and rLmIL-22 were obtained.This will lay the foundation for the subsequent study on the related biological functions of rLmIL-20 L and rLmIL-22 in the spotted sea bass.