1. Molecular structure, bioinformatics analysis, expression and bioactivity of BAFF (TNF13B) in dog (Canis familiaris)
- Author
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Shen, Yuefen, Wang, Shule, Ai, Hongxin, Min, Cui, Chen, Yuqing, and Zhang, Shuangquan
- Subjects
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MOLECULAR structure , *BIOINFORMATICS , *TUMOR necrosis factors , *DOG diseases , *CONFOCAL microscopy , *REVERSE transcriptase polymerase chain reaction , *LIGAND exchange chromatography , *LYMPHOCYTES - Abstract
Abstract: B cell activating factor (BAFF), belonging to the tumor necrosis factor (TNF) family, plays an important role in B cell survival, proliferation and differentiation. In the present study, we amplified the cDNA of dog (Canis familiaris) BAFF (designated doBAFF) from spleen by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) strategies. The open reading frame (ORF) of doBAFF covers 879bp encoding 292 amino acids, with a 152-aa mature peptide. The soluble mature part of doBAFF (dosBAFF) shares 91.5%, 72.1%, 96.7%, 94.0% and 91.5% identity with the human, mouse, cattle, pig and rabbit counterparts, respectively. The predicted three-dimensional (3D) structural analysis of dosBAFF analyzed by “comparative protein modeling” revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis revealed that doBAFF was mainly expressed in spleen. Two fusion proteins SUMO-dosBAFF and GFP/dosBAFF were efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy analysis showed that GFP/dosBAFF could bind to the mouse splenic B-cell. In vitro, SUMO-dosBAFF and GFP/dosBAFF were able to promote the survival/proliferation of dog lymphocytes or mouse splenic B cells with/without anti-IgM. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in dog. [Copyright &y& Elsevier]
- Published
- 2011
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