The Study Of Zebrafish Thymosin In Vitro Expression And Innate Immune Function

The organism's living environment is full of various pathogenic factors,so the organism has evolved a series of defense mechanisms to adapt to the environment.Compared to adaptive immunity,innate immunity acts as an inherent protective mechanism and can be activated earlier and more rapidly by pathogens.Bony fish have the advantage of studying the immune system species in evolution.The immune system contains a large number of natural immune molecules such as antimicrobial peptides.They not only can resist the invasion of external pathogens effectively,but also maintain the body in a relatively balanced physiological state.Thymosin is a polypeptide that promotes the development of body and immune system,has a function of rapidly binding pathogens and initiating downstream pattern recognition of receptors or molecules for the immune defense mechanism.Members of the beta-thymosin family have a broad-spectrum direct bactericidal effect in addition to their ability to regulate and enhance immunity.At present,a variety of thymosins have been cloned and identified in bony fish,and they have also been studied to some extent in the development of the body and functions in immunity.In early studies,thymosin was found to be involved in the development of the body and cell differentiation.In invertebrates,thymosin has been reported to have the function of defense.The innate immune function of thymosin in vertebrate-bony fish is not well studied.In this study,model organism zebrafish was used as a research material to study the functional mechanism of ?-thymosin in antibacterial innate immunity.This study found that zebrafish thymosin contains 45 amino acids,and its spatial structure is predicted by modeling to include ?-helices and linear structures.Through analysis of the phylogenetic tree and amino acid sequence of a part of ?-thymosin family members,it was found that members of the thymosin ?-family are highly conserved and homologous,especially the actin binding domain(LKKTET).We cloned and identified zebrafish thymosin ORF;detected zearafish thymosin expression mode in normal individuals and found zebrafish thymosin in selected zebrafish tissues(brain,liver,foregut,hindgut,thymus,kidney,spleen,skin,muscle,etc.)are all expressed in the brain,liver,hindgut and muscle;the thymosin is stimulated by the soaking of the conditional pathogen(Aeromonas hydrophila).Increased expression levels in the skin,liver,kidneys,spleen,thymus,foregut,gills and midgut indicate that the thymosin gene may be involved in the innate immune response caused by bacterial stimulation.In this study,thymosin was expressed in prokaryotic cells and purified to obtain recombinant ?-thymosin(rT?).The in vitro antibacterial activity of rT? was measured.Bacterial inhibition experiments showed that the recombinant polypeptide can inhibit the growth of A.hydrophila;rT? has a significant agglutination activity against A.hydrophila and agglutination of S.aureus is weak;in the bacterial binding experiment,recombinant thymus can bind to A.hydrophila,K.pneumoniae,E.coli,S.aureus,B.thuringiensis,and B.subtilis to varying degrees;it is also observed by scanning electron microscope(SEM).It was found that rT? can destroy the surface of bacteria.The results show that zebrafish thymosin via binding to bacteria,agglutinating and damaging bacteria,to make bactericidal or antibacterial functions come ture.