Molecular cloning, in vitro expression and bioactivity of quail BAFF.
B cell activating factor (BAFF), belonging to the TNF (tumor necrosis factor) family, is critical for B cell survival and maturation. In the present study, a quail BAFF cDNA, named qBAFF, was amplified from quail spleen by RT-PCR and RACE (rapid amplification of cDNA ends) strategies. The open reading frame (ORF) of qBAFF cDNA encodes a protein consisting of 288-amino acid. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site like other identified BAFF homologues. The qBAFF shows 96, 93, 93, 53 and 51% amino acid sequence identity with chicken (cBAFF), goose (gBAFF), duck (dBAFF), human (hBAFF) and mouse BAFF (mBAFF), respectively, with the functional soluble parts of qBAFF is 98, 99, 98, 78 and 71%, respectively. RT-PCR showed that BAFF is expressed in many tissues in the quail, including bursa, spleen, liver, brain, heart, intestine, kidney, thymus and muscle. Recombinant soluble qBAFF (qsBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its molecular weight of approximately 19kDa was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified qsBAFF was able to promote the survival of quail bursa B cells. Our results suggest that qBAFF plays an important role in survival of quail B cells cultured in vitro.
Chen
Chuan-mei
CM
Jiangsu Key Laboratory for Bioresource Technology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
Ren
Wen-hua
WH
Yang
Guang
G
Zhang
Chuan-song
CS
Zhang
Shuang-quan
SQ
D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011784:Quail / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D016415:Sequence Alignment
D053264:B-Cell Activating Factor; D012333:RNA, Messenger