cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF.
B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210046, China.
Cui
C
Min
Yangyang
Y
Han
Hongyan
H
Liu
Yuqing
Y
Chen
Shuangquan
S
Zhang
Zhigang
Z
Yao
Yuhua
Y
Ding
D000818:Animals; D053264:B-Cell Activating Factor / Q000235:genetics* / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003670:Deer / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism; D048069:Tumor Necrosis Factors / Q000235:genetics
D053264:B-Cell Activating Factor; D011993:Recombinant Fusion Proteins; D048069:Tumor Necrosis Factors