Cloning, In Vitro Expression And Biological Activity Identification Of Silver Carp BAFF And GILT

The present study,using cellular and molecular biology techniques and methods including PCR,prokaryotic protein expression,protein purification,cell culture,cell transfection,immunofluorescence,CCK-8 assay,laser confocal,flow cytometry and Western blotting,we successfully completed the cloning,expression and biological activity analysis of the B cell activating factor(ScBAFF)and Gamma-interferon-inducible lysosomal thiol reductase(GILT)from silver carp(Hypophthalmichthys molitrix).The detailed results are summarized below:1.The cloning,expression and biological activity analysis of ScBAFF.B cell activating factor(BAFF),which plays fundamental role in maturation,activiation and survival of B cell,is an important member of the the tumor necrosis factor(TNF)superfamily.In this study,a BAFF homologue,which was named ScBAFF has cloned from the spleen of silver carp for the first time.Results revealed that the ORF of ScBAFF was 804 bp,encoding 267 amino acids,with a 157 aa mature peptide.Amino acid sequence analysis showed that the protein structure has a transmembrane domain,which exhibited a classic type-? transmembrane protein structure similarly to other BAFFs.Amino acid sequence alignment and protein structure prediction results show that ScBAFF shared high homology and similarity of spatial structure with other BAFFs.Organizational expression analysis indicated that ScBAFF was predominantly expressed in the spleen and the head kidney,which are the immune organs of the silver carp.By constructing prokaryotic expression vector,we expressed His6-ScGILT recombinant protein in Escherichia coli BL21(DE3),SDS-PAGE and Western Blot analysis were proformed after purification.In vitro,the fusion protein His6-ScGILT could bind to the BAFF receptors in human peripheral blood B lymphocytes cells and Raji cells and promote the survival of the two cells.These results suggested that ScBAFF may play an important role in the immune system and provide a theoretical basis for the study of the immune system of silver carp.2.The cloning,expression and biological activity analysis of ScGILT.Gamma-interferon-inducible lysosomal thiol reductase(GILT)plays an important role in the processing and presentation of major histocompatibility complex(MHC)class II-restricted antigens by catalysing disulfide bond reduction.In this study,we identified a GILT homolog,designated as ScGILT,from silver carp.Results revealed that the ORF of ScGILT was 771 bp,encoding 256 amino acids that possesses the characteristic GILT signature sequence CQHGX2ECX2NX4C,the active-site CXXC motif,the potential N-linked glycosylation site and six conserved cysteines in the C-terminus.Organizational expression analysis indicated that ScGILT mRNA was expressed in a tissue-specific manner was markedly upregulated in the spleen and head kidney after induction with LPS.The recombinant protein His6-ScGILT was efficient expressed in Escherichia coli BL21(DE3)and identified by the SDS-PAGE and western blot.Biological activity tests showed that the ScGILT has thiol reductase activity which could restore the disulfide bond of human IgG.Finally,we analyzed the subcellular localization of ScGILT cells through the cell transfection and immunofluorescence technique,ScGILT was present in the same intracellular compartment as lysosomal-associated membrane protein 1(LAMP-1),a lysosomal and phagosomal marker.These results suggested that ScGILT may be involved in the immune response to bacterial challenge in silver carp.