Cloning, Expression And Biological Activity Identification Of Tilapia BAFF And Human TNFRSF13B 2 -Fc

Tumor necrosis factor superfamily and their receptors play an important role in lymphatic organs,autoimmune diseases and anti-viral immunity.In order to obtain high expression and high activity of BAFF and TNFRSF13B2-Fc protein,In order to obtain active high-purity protein at a lower cost,we constructed a prokaryotic expression system,providing support for the development of future disease drugs.Section one.Research about BAFF of Nile TilapiaB cell activating factor(BAFF),a member of the tumor necrosis factor(TNF)family,play key roles in the regulation of inflammation,immune responses and tissue homeostasis.In the present study,the full-length cDNA of BAFF from the Nile Tilapia(Oreochromis niloticus,designated tBAFF)was amplified from spleen by reverse transcription-PCR(RT-PCR).The cDNA contains an open reading frame(ORF)of 786 nucleotides that are translated into a predicted 261 amino acid protein bearing typical TNF homology domain.Sequence comparison indicated that the amino acid of tBAFF possessed the TNF signature,including a transmembrane domain,a putative fruin protease cleavage site(R-R-R-R).Real-time quantitative PCR(qPCR)analysis revealed that tBAFF could be detected in various tissues and predominantly expressed in lymphoid tissue spleen.Recombinant tsBAFF fused to a pET28a vector was efficiently expressed in Escherichia coli BL21(DE3)and its expression was confirmed by SDS-PAGE,Western blotting analysis and Mass spectrometry analysis.After purification,WST-8 and Flow Cytometry assays and laser scanning confocal microscopy analysis revealed that tBAFF could bind to its receptors on B cells,and tsBAFF is able to stimulate survival of mouse splenic B cells in vitro.Therefore,our results suggest that tsBAFF may be a potential immunologic factor for enhancing immunological efficacy in Nile Tilapia.Section two.Research about human TNFRSF13B2-FcTNFRSF 13B is an important member of the tumor necrosis factor receptor superfamily(TNFRF).In vivo,TNFRSF 13B is considered as a negative regulation receptor of BAFF,and play the key role in regulating the number of B cells.TNFRSF 13B extracellular domain contains two CRD,the two CRD in N terminal can be selectively cut off one and change into another form,but the ability of ligand binding and induce signal without loss.In this study,extracted mRNA from human peripheral blood,TNFRSF13B cDNA was amplified by RT-PCR,Then primers were designed to amplify the extracellular region contains only one CRD structure(hsTNFRSF13B2).Construction of prokaryotic expression vector-pET28a-hsTNFRSF 13B2-Fc,and protein's optimized expression.Obtaining soluble protein of hsTNFRSF13B2-Fc by Protein A one step purification.Confirmed by SDS-PAGE and Western Blot analysis.The ELISA assay indicated that in vitro hsTNFRSF13B2-Fc protein can bind to the ligand BAFF in a dose dependent manner.