Identification And Expression Analysis Of Immunoglobulin Superfamily Genes In Spotted Sea Bass(Lateolabrax Maculatus)

The immunoglobulin（Ig）superfamily is a molecular superfamily with domains similar to immunoglobulins in the molecular structure,which mainly exists on the cell surface in the form of membrane proteins.Immunoglobulins（Igs）,Major histocompatibility complex（MHC）and T cell receptor（TCR）are three important gene families in the immunoglobulin superfamily related to antigen recognition and presentation.It plays an important role in the adaptive immune response.Among them,Igs play a key role in the humoral immune response and can directly bind to free specific antigens.Among them,Igs play a key role in the humoral immune response and can directly bind to free specific antigens.There are currently five known immunoglobulins,and IgM is the first to appear in phylogeny and dominates in serum.MHC is a gene family closely related to antigen presentation of the immune system.It can bind and present antigens to T lymphocytes,stimulate the body’s immune response and play the role of antigen presentation in the biological immune response.It can combine endogenous and exogenous antigens and present them to T lymphocytes to stimulate the body to produce an immune response.TCR is a characteristic molecular marker on the surface of T cells.It can specifically recognize peptides that bind to MHC,present bound antigens,activate T cells,and ultimately rely on lymphocytes to release cytokines to eliminate antigens,and is mainly involved in cellular immunity.In the long-term evolution,most bony fish have an adaptive immune response mechanism.When they are threatened by various environmental microorganisms during their growth,development and reproduction,while the innate immune mechanism in the body plays a role,the adaptive immune response will also be induced by antigens to produce a corresponding mechanism of action.Spotted sea bass（Lateolabrax maculatus）is an important farming economic fish in my country.However,with the expansion of aquaculture scale and the development of high-density intensive farming methods,the frequent occurrence of diseases in the cultivation of seabass has become a bottleneck for the sustainable development of the industry.Therefore,this article cloned and identified the IgM,MHCⅡβand TCR genes related to the adaptive immune system of spotted sea bass,and tested their expression status and expression regulation at the mRNA level,and then prepare the corresponding prokaryotic expression recombinant protein.For the further exploration of the types,characteristics and functions of seabass B and T cells,to clarify the immune response/response laws and mechanisms of seabass to pathogenic microorganisms and environmental factors,and to provide theoretical support for the development of new disease resistance strategies.In this study,we cloned and identified the IgMH、MHCⅡβ和TCRα/β/γ/δgenes of spotted sea perch by RT-PCR and RACE approaches.The full-length cDNAs of IgMH,MHCⅡβ,TCRα,TCRβ,TCRγand Lm-TCRδwere 1992 bp,1212 bp,1210 bp,1235 bp,1323 bp and 1768 bp,encoding 590aa,249aa,252 aa,294 aa,313 aa and 271 aa,respectively.Using biosynthesis analysis software to analyze their structure respectively,it is found that except for IgMH,they all have signal peptide,Igdomain,connecting peptide,transmembrane region and intracellular region.IgMH has 1 variable region（IgV）and 4 constant regions（IgC）and does not have a transmembrane domain.It was a secreted IgMH protein.Phylogenetic tree analysis and Amino acid identity proved the evolutionary status of each gene and the distance of genetic relationship.The mRNA expression levels of IgMH、MHCⅡβ和TCRα/β/γ/δin various tissues of seabass were gained by Real-time fluorescence quantitative PCR（RT-q PCR）.The results show that the above-mentioned genes were have a higher expressionin tissues such as gills,spleen and head kidney which were interralated with immunity,but lower in liver and brain.Using artificial injection,LPS,Poly I:C stimulation and E.tard artificial infection experiments were carried out on spotted sea bass.The results showed that the mRNA expression levels of IgMH,MHCIIβand TCRα/β/γ/δgenes in the gills,spleen,head kidney,and intestines have changed significantly.The change trend of each gene of TCRα/β/γ/δwere basically the same,and the expression level of TCRα/β/γ/δwere significantly increased in the spleen and intestine after LPS stimulation.IgMH and MHCIIβwere significantly up-regulated in the gills and spleen after Poly I:C stimulation,while TCR was significantly down-regulated in the gills.The expression levels of IgMH,MHCIIβand TCRα/β/γ/δwere significantly up-regulated in the spleen and head kidney after infection with E.tarda,indicating that each gene produced a strong immune response to bacterial infection in the spleen and head kidney.In this study,pET-21d-IgMH,pET-21d-IgMH_C1C2and pET-21d-MHCⅡβrecombinant expression plasmids were constructed,and obtained recombinant proteins of IgMH and MHCⅡβ.The obtained pET-21d-IgMH protein was used to prepare polyclonal antibodies,and the characteristics of the antibodies were examined by immunoblotting.Western Blot results showed that the polyclonal antibody had an immunoreactive protein in the serum of spotted sea bass,and its molecular weight of about 90KDa was consistent with IgMH.The anti-IgM of spotted sea bass was successfully obtained,which laid the foundation for the research on the immune system and pathogen diagnosis of spotted sea bass.