cDNA cloning, expression and bioactivity of porcine BAFF.
B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805bp with a 702bp open reading frame, encoding 233 amino acids. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site corresponding to other identified BAFF homologues. The amino acid similarity between the functional soluble parts of pBAFF and human BAFF (hBAFF) or chicken BAFF (cBAFF) is 93% and 85%, respectively, with identity at the amino acid level was 88% and 76%, respectively. The characteristic of the three-cysteine residues of BAFF is conserved in pBAFF. RT-PCR showed that BAFF is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, kidney, thymus and PBLs. Recombinant soluble pBAFF (psBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. In vitro, purified psBAFF co-stimulates the proliferation of not only porcine B cells but also human B cells. In addition, hsBAFF binds to porcine B cells and has a positive effect on their proliferation. These findings indicate pBAFF plays an important role in proliferation of porcine B cells and functional cross-reactivity occurs between porcine and human BAFF. In vitro expression of bioactive psBAFF provides the basis for further investigation of its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic in pig. It also provides the basis for investigations on the role of BAFF in this important domestic species and an animal model for human diseases.
Guan
Zheng-Bing
ZB
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210097, PR China.
Dan
Wen-Bing
WB
Shui
Yan
Y
Ye
Ji-Lin
JL
Zhang
Shuang-Quan
SQ
D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D049109:Cell Proliferation; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D053264:B-Cell Activating Factor; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins