Molecular Cloning, Evolution And Function Analyses Of IL-15 Subfamily And IL-15 Receptor Alpha Chain Family In Teleost Fish

Chapter 1 of this dissertation is an introduction of the recent researches on IL-15 and its specific receptor IL-15Rα.It was described in three parts.In the first part,we mainly introduce interleukin-15(IL-15) including its discovery,molecular cloning, gene structure,regulatory mechanisms of its expression,as well as its receptors and the corresponding signal pathways;and the second part introduces the molecular cloning and characterization of IL-15Rα(IL-15 receptor alpha chain) gene,including its expression and various types of alternative splicing.IL-15Rαspecifically recognizes IL-15 in an unusually high affinity,which could account for its co-secretion with IL-15,the various effects of soluble IL-15Rα,and trans-presentation of IL-15 signal.In the third part,we introduce the biological functions of IL-15 and IL-15Rαon different types of immune-related cells,including Natural Killer,TCRαβT lymphocytes and so on,as well as IL-15/IL-15Rαeffects in the pathologies of some immune diseases.Chapters 2-5 are the description of the main researches.In Chapter 2,we performed the molecular cloning and characterization of the IL-15's orthologue from Tetraodon nigroviridis,a teleost species,as well as its prokaryotic expression in E.coli and the purification of soluble recombinant protein.By comparative genomics and bioinformatics methods,we carried out the study on the evolution of vertebrates IL-15 genes,including a fish specific IL-15X gene,which thought to be a paralogue of IL-15 arisen from gene duplication.In Chapter 3,we gave our investigation into the molecular cloning,tissue expression and prokaryotic expression of Tetraodon IL-2 and IL-2L genes.IL-2L is a novel 4-αhelix cytokine which reported for the first time and it specifically exist in some bony fishes.As another copy of the common ancestral gene sharing with IL-2 and IL-2L,we perform the analysis into their evolutionary relationships and the molecular evolutions.Chapter 4 described the investigation of Tetraodon IL-15Rα,including its molecular cloning,prokaryotic expression,preparation of multiclonal antibody,and tissue expression by western blotting.We found two subtypes of Tetraodon IL-15Rα, containing one or two sushi domains respectively,which corresponding to cytokine binding.Thus,we named them as ssIL-15Rα(single sushi IL-15Rα) and dsIL-15Rα(double sushi IL-15Rα),and confirmed that dsIL-15Rαwas arisen from Fish Specific Genome Duplication and a following exon shuffling event.By comparative genomics analysis,it was approved that IL-2Rαgene of higher vertebrates was a copy of IL-15Rαtandem duplicated,and fish IL-15Rαmay played as an IL-2 receptor.By cytokine and receptor binding assay,both ssIL-15Rαand dsIL-15Rαwas found different capability in IL-2 or IL-15 binding,and dsIL-15Rαshowed a comparatively higher capability,which suggested that both sushi domain could contributed to cytokine binding.In this chapter,we also cloned and characterized IL-15Rαgene from Oncorhynchus mykiss,and testified that there were two copies of IL-15Rαin other teleost species.In Chapter 5,we mainly reconstructed the phylogeny of IL-15Rαand IL-2Rαgenes throughout vertebrate species,by multiple comparative genomics and bioinformatics means.Furthermore,both gene duplications and exon shuffling played important roles in the evolution of IL-15Rαand IL-2Rα,and the relative evolutionary rates were changed significantly after these events.On the other hand,there was a very possibility of the co-evolution between IL-15Rαand IL-15,as well as IL-2Rαand IL-2.Based on the above researches,we gave our hypothesis on the evolution of vertebrates IL-15Rαand IL-2Rαfamily.