Molecular cloning, expression, and bioactivity of dove B lymphocyte stimulator (doBAFF).
B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is a novel member of the tumor necrosis factor ligand family and plays an important role in B lymphocyte maturation and survival. cDNA of dove B lymphocyte stimulator (doBAFF) was amplified from total RNA of dove spleen by RT-PCR (reverse transcription PCR). The open reading frame of doBAFF consists 867 bases encoding a protein of 288 amino acids. Sequence comparison indicated the amino acid sequence of doBAFF showed high identity to hBAFF (50.66%) and cBAFF (91.32%). The result of RT-PCR showed that doBAFF was highly expressed in the spleen and bursa of fabricius. To enhance the soluble expression of doBAFF in Escherichia coli, we fused the extracellular region of doBAFF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fused protein SUMO-sdoBAFF was highly expressed in DE3(BL21) with a molecular weight of 35kDa. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease, Ulp1. The sdoBAFF protein was further purified by Ni-NTA affinity chromatography. In vitro, the MTT assays indicated that the purified doBAFF as well as SUMO-sdoBAFF proteins were able to promote bursa lymphocyte survival in dose-dependent manner.
Lu
Wuguang
W
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, China.
Cao
Peng
P
Cai
Xueting
X
Yu
Jiemiao
J
Hu
Chunping
C
Cao
Meng
M
Zhang
Shuangquan
S
D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D002060:Bursa of Fabricius / Q000276:immunology; D003001:Cloning, Molecular; D010856:Columbidae / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis / Q000235:genetics; D013154:Spleen / Q000276:immunology
D053264:B-Cell Activating Factor; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins