Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Fish Shellfish Immunol. 2011 Jun;30(6):1265-74. doi: 10.1016/j.fsi.2011.03.020. Epub 2011 Apr 2.
B-cell activating factor of the TNF family (BAFF) induces B-cell survival, proliferation, immunoglobulin secretion and plays a role in enhancing immune responses. In the present study, a BAFF homolog has been identified in mefugu Takifugu obscurus, and its biological activities have been characterized. The mefugu BAFF (fBAFF) cDNA is 789 bp in length and translates into a 262-aa protein. The fBAFF genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size. Amino acid sequence comparison indicated that fBAFF possessed the TNF signatures, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the fsBAFF monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR (qPCR) analysis revealed that fBAFF was predominantly expressed in mefugu lymphoid tissue spleen. The SUMO-fsBAFF and GFP/fsBAFF efficiently expressed in Escherichia coli Rosetta (DE3) were confirmed by SDS-PAGE and Western blotting analysis. After purification, the GFP/fsBAFF fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsBAFF could bind to its receptors. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-fsBAFF could promote the proliferation of mefugu splenocytes or mouse splenic B cells together with/without anti-mouse IgM. These findings indicate that SUMO-fsBAFF plays an important role in proliferation of mefugu B cells and has functional cross-reactivity among mefugu and other mammalians. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in fish.
B 细胞激活因子家族成员（BAFF）可诱导 B 细胞存活、增殖、免疫球蛋白分泌，并在增强免疫反应中发挥作用。本研究在红鳍东方鲀中鉴定到一个 BAFF 同源物，并对其生物学活性进行了研究。红鳍东方鲀 BAFF（fBAFF）cDNA 长 789bp，编码 262 个氨基酸。fBAFF 基因组序列由 6 个外显子和 5 个内含子组成，大小约为 1.8kb。氨基酸序列比较表明，fBAFF 具有 TNF 特征、跨膜结构域、潜在的弗林蛋白酶裂解位点和三个半胱氨酸残基，这是哺乳动物和鸟类 TNF 基因的典型特征。通过比较蛋白建模分析预测的 fsBAFF 单体的三维（3D）结构表明，它与人类的同源物非常相似。实时定量 PCR（qPCR）分析显示，fBAFF 在红鳍东方鲀淋巴组织脾脏中表达丰度最高。通过 SDS-PAGE 和 Western blot 分析证实，SUMO-fsBAFF 和 GFP/fsBAFF 在大肠杆菌 Rosetta（DE3）中有效表达。经过纯化，获得的 GFP/fsBAFF 融合蛋白获得的荧光光谱与 GFP 相似。激光扫描共聚焦显微镜分析表明 GFP/fsBAFF 可以与受体结合。体外 MTT 测定和流式细胞术分析表明，SUMO-fsBAFF 可以与抗小鼠 IgM 一起或不一起促进红鳍东方鲀脾细胞或小鼠脾 B 细胞的增殖。这些发现表明 SUMO-fsBAFF 在红鳍东方鲀 B 细胞的增殖中发挥重要作用，并在红鳍东方鲀和其他哺乳动物之间具有功能交叉反应性。因此，BAFF 可能是增强鱼类免疫效力的潜在免疫因子。
