Cloning, Identification And Biological Function Of BAFF, APRIL And TWEAK Genes In Chinese Wild Elk

The tumor necrosis factor (TNF) superfamily is a group of 29 receptors and 19 ligands. The Tumor Necrosis Factor (TNF) superfamily of cytokines activate signaling pathways for cell survival, death, and differentiation that orchestrate the development, organization and homeostasis of lymphoid, mammary, neuronal and ectodermal tissues.Among the different members of this family, an increased interest has been shown recently for the subset of B cell activating factor of the TNF Family (BAPF), a proliferation inducing ligand (APRIL), and TNF-like weak inducer of apoptosis (TWEAK). This article cloning and recombinant expression this three gene, study these biological activity.The results were summarized as follows:1. Molecular cloning and characterization of BAFF、APRIL and TWEAK cDNA from Pere David’s DeerIn the present study, the cDNA of Pere David’s Deer BAFF(miBAFF)、APRIL (miAPRIL)、TWEAK(miTWEAK)was successfully amplified from peripheral blood cells by RT-PCR. The length of this three gene ORF were 843,753,747bp, respectively.The deduced amino acid sequence of these three protein included a transmembrane domain, a putative furin protease cleavage site, a conserved basic TNF motif, then all of this three proteins were type-II transmembrane protein. Phylogenetic analysis exhibited this animal has high homology with the even-toed ungulates (artiodactyla) such as cow and sheep, but the genetic relationship with fishes was far.2. The expression and analysis of soluble BAFF、APRIL、TWEAKThe soluble fraction of the miBAFF (misBAFF) gene was cloned into the prokaryotic expression vector SUMO and transformed into Escherichia coli BL21 (DE3) for recombinant expression. In vitro MTT analysis revealed that purified misBAFF was able to increase the survival rate of Pere David’s deer lymphocytes in a dose-dependent manner, and it also can co-activing mouse lymphocytes with mouse anti-IgM.The soluble fraction of the miAPRIL (misAPRIL) gene was cloned into the prokaryotic expression vector pET43.1a and transformed into Escherichia coli BL21 (DE3) for recombinant expression. Observation by confocal microscopy demonstrated that misAPRIL has the ability to bind to the surface of Pere David’s deer lymphocytes. In vitro MTT and flow cytometry analysis revealed that purified misAPRIL was able to increase the survival rate of these lymphocytes in a dose-dependent manner. At last, we construction SUMO-misTWEAK expression vector and obtain purified protein in order to study this biological activity.