Molecular Cloning,Expression Pattern And Functional Analysis Of BAFF In Chinese Sucker (Myxocyprinus Asiaticus)

Chinese sucker（Myxocyprinus asiaticus）belongs to the family Catostomidae and the genus Myxocyprinus is endemic to China.Chinese sucker is a freshwater species of high commercial value,which is an ornamental fish widely cultured because of its delicious meat and beautiful body color.Its natural habitat spans the upper reaches of the Yangtze River.Because of over-fishing,water pollution,dam construction,and severe infectious diseases,the species has been designated a class II state-protected animal since 1990s.Through scientific artificial breeding,its number has steadily increased.Artificial breeding has become an important means of protecting this species,but this fish is frequently suffered from infectious diseases.To establish effective measures against disease and enhance immune efficacy in Chinese sucker,identification of critical immunological components and understanding their functions are necessary.Cytokine can be defined as small proteins that usually act in an autocrine or paracrine manner and they are extremely potent.The production of these potent regulatory molecules is usually transient and tightly regulated.Thus cytokines are part of an extracellular signaling network,and the network plays a regulatory role in inflammation,defense against virus infection,proliferation of specific T-and B-cell clones and regulation of their differentiated function.Members of tumor necrosis factor superfamily（TNFSF）play a critical role in regulating the immune responses.The majority of these ligands are type II membrane proteins that can act in a membrane-bound form,or as a soluble cytokine retain biological activity after proteolytically cleaved by a protease.B-cell activating factor（BAFF）of the TNF family is such a class TNFSF cytokine for B-cell survival,proliferation and differentiation.Structurally,BAFF protein contains an intracellular N-terminal transmembrane domain（TMD）,and extracellular C-terminal TNF homology domain（THD）conserved between TNFSF ligands（20–30%）.In addition,there is an unusual long D-E loop（Flap region）in its soluble crystal structure compared to other TNF superfamily members,and this flap plays an important role in the formation of trimer or capsid-like multimer（i.e.a60-mer）and receptor binding.Three BAFF receptor family members have been identified:BAFF receptor（BR3,known as TNFSF-13C）,B cell maturation antigen（BCMA,also known as TNFSF-17）and tansmembrane activator and calcium modulator cyclophilin ligand interactor（TACI,known as TNFSF-17）.Recent data suggests that BR3 may be the primary receptor responsible for BAFF-mediated function.In addition to BAFF,BCMA and TACI also bind to APRIL,a TNF family member that shares a high level of sequence similarity with BAFF.BAFF is mainly expressed on the innate immune cells,such as monocytes,dendritic cells（DCs）,macrophages,activated T cells,neutrophils,and some non-hematopoietic cells.The Chinese sucker BAFF（designated MaBAFF）gene was cloned using RT-PCR and RACE（rapid amplification of cDNA ends）techniques.The full-length MaBAFF cDNA was 1382 bp,including 110 bp and 563 bp for the 5’non-coding region and 3’non-coding region.The open reading frame（ORF）of MaBAFF consists of 819bp and translates into a 272-aa protein with a transmembrane domain and a TNF family signature.The putative amino acid residues have an estimated molecular mass of 30.384kDa and an isoelectric point of 5.82.Amino acids sequence comparison indicated that the MaBAFF sequence possessed the TNF signatures,a transmembrane domain and a D-E loop like other reported BAFFs and shared the highest identity（81.07%）to Labeo rohita,80.71%identity to grass carp,and 76.43%identity to Danio rerio.The predicted three-dimensional（3D）structure of the Chinese sucker soluble BAFF（MasBAFF）monomer determined by SWISS-MODEL revealed that it was very similar to its human counterpart.The phylogenetic tree constructed using the N-J method showed that it was comprised two clearly distinct branches and MaBAFF was clustered with one containing all teleost fish sequence being separated from other higher vertebrates BAFF protein.The distribution pattern of MaBAFF mRNA in healthy Chinese sucker tissues was examined by relative and absolute qRT-PCR assay.In the current study,we used the geNorm program to evaluate the gene expression stability of four gene（GAPDH,β-actin,18s rRNA and L18a）in healthy Chinese sucker tissues.The order of the stability is:L18a=18s rRNA>β-actin>GAPDH.Therefore,L18a and 18S rRNA genes are more suitable as a reference gene used for normalization of real-time quantitative PCR experiments data.Absolute qRT-PCR analysis showed that MaBAFF was expressed in all tissues examined,with highest mRNA levels observed in the spleen.MaBAFF expression was at detectable level as early as the period of fertilized egg,and thereafter it was stably observed in all embryonic developmental stages.In Aeromonas hydrophila infection,MaBAFF and IgM gene expression were induced across the organs.The results showed the expression level of MaBAFF were significantly up-regulated in spleen at 48h and the expression of IgM in the head kidney also showed the same trend.For MaBAFF protein expression,the recombinant plasmid was transformed into E.coli BL21（DE3）.The His₆-tagged recombined protein was then expressed after IPTG induction and purified using Ni-nitrilotriacetic acid resin under optimal conditions.The samples collected during protein purification were subjected to SDS-PAGE analysis.On SDS-PAGE analysis,band of 20kDa was consistent with the expected sizes of the respective fusion products.Western blotting analysis indicated that purified protein showed a single band with the estimated molecular weight about 20kDa.The binding activity of MasBAFF to Chinese sucker peripheral blood leucocytes were confirmed by using indirect immunofluorescence staining.In vitro,the CCK-8 assay indicated that the MasBAFF recombinant protein（200ng/ml）could prolong the survival of peripheral blood leukocytes.In this study,we cloned the Chinese sucker BAFF gene,conducted the phylogenetic analysis,analyzed its protein structure and investigated gene expression patterns in various tissues of normal fish,and after challenge with bacteria.Better understanding of the immune factor may contribute to the development of management strategies for disease control.The results raise the possibility that MaBAFF may be useful to enhance immune efficacy in Chinese sucker disease defense.