Molecular And Functional Characterization Of IL-4 And IL-4Rα In Zebrafish

This article focused on the molecular cloning, functional characterization of Th2 immunity related molecules and preliminary identification of Th2 immunity in Danio rerio.Chapter 1 is an introduction of the recent researches on IL-4 and its specific receptor IL-4Rα. It was described in two parts. In the first part, we mainly introduce interleukin-4 (IL-4) and IL-4Ra (IL-4 receptor alpha chain) gene including its discovery, molecular cloning, gene structure, regulatory mechanisms of them expression, its receptors and the corresponding signal pathways, the biology functions of IL-4, as well as various types of alternative splicing of IL-4Rα. IL-4 and IL-4Ra was two reciprocal molecules with high affinity, they play the important function in Th2 immunity.Chapter 2 is the description of the main researches. It was described in three parts. In first part, we performed the molecular cloning and characterization of IL-4 from zebrafish, and its prokaryotic expression in E.coli and purification of soluble recombinant protein. By bioinformatics methods, we carried out the study on the evolution of vertebrates IL-4 genes. In the second part, we cloned IL-4Rα, and we also get a IL-4Rαlike (IL-4Rα-L) gene from zebrafish. IL-4Ra-L adjacent to IL-4Ra, they had high identity in cDNA sequences and gene structures, the most difference is that IL-4Rα-L laking the most cytoplasmic region. Both of them have its solubles from alternative splicing. By bioinformatics methods, we found that the gene structure, gene locus, protein structure and the main motifs for signaling were conserved across all species aligned. In the third part, we performed the function analysis on IL-4/IL-4Rα, provided the evidence on the two reciprocal molecules. And we found that, IL-4 could stimulate the proliferation of B cell, up-regulate the production of antibody, and promote the expression of Th2 cytokines and transcription factors. All the evidence showed that a possible Th2-mediated regulatory mechanism had been developed in the adaptive immunity during early vertebrate evolution.