Liu Hongzhen, Zhang Jiaxin, Li Jianfeng, Song Jinyun, Zhang Shuangquan
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210046, China.
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210046, China; Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, 210037, China.
Fish Shellfish Immunol. 2016 Apr;51:240-250. doi: 10.1016/j.fsi.2016.02.026. Epub 2016 Feb 22.
B cell-activating factor (BAFF)is a member of the tumor necrosis factor (TNF) family and plays roles in B cell survival and maturation. In this study, the full-length cDNA of Nile tilapia (Oreochromis niloticus) BAFF (tBAFF) was amplified from the spleen by reverse transcription PCR (RT-PCR). The open reading frame of this cDNA encodes a protein of 261 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFF. Real-time quantitative PCR (qPCR) analysis revealed that tBAFF is present in various tissues and is predominantly expressed in the spleen. The predicted three-dimensional (3D) structure of the Nile tilapia (Oreochromis niloticus) soluble BAFF (tsBAFF) monomer was determined by (3D) structure modeling monomeranalyzed by (3D) structure mouse counterpart. Both tsBAFF and EGFP/tsBAFF were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by SDS-PAGE and Western blot analysis. After purification, the EGFP/tsBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Laser scanning confocal microscopy showed that EGFP/tsBAFF bound to its receptor. In vitro, tsBAFF promoted the proliferation of Nile tilapia and mouse splenic B cells together with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, tsBAFF showed a similar proliferation-stimulating effect on mouse B cells compared to msBAFF. These findings indicate that tsBAFF plays an important role in the proliferation of Nile tilapia B cells and has functional cross-reactivity among Nile tilapia and mammals. Therefore, BAFF may represent a useful factor for enhancing immunological efficacy in animals.
B细胞激活因子（BAFF）是肿瘤坏死因子（TNF）家族的成员，在B细胞存活和成熟过程中发挥作用。在本研究中，通过逆转录PCR（RT-PCR）从尼罗罗非鱼（Oreochromis niloticus）脾脏中扩增出尼罗罗非鱼BAFF（tBAFF）的全长cDNA。该cDNA的开放阅读框编码一个含261个氨基酸的蛋白质，其含有一个预测的跨膜结构域和一个弗林蛋白酶切割位点，与哺乳动物、鸟类和爬行动物的BAFF相似。实时定量PCR（qPCR）分析显示，tBAFF存在于各种组织中，且在脾脏中表达量最高。通过三维（3D）结构建模确定了尼罗罗非鱼（Oreochromis niloticus）可溶性BAFF（tsBAFF）单体的预测三维结构，并与小鼠对应物的三维结构进行了分析比较。经SDS-PAGE和蛋白质免疫印迹分析证实，tsBAFF和EGFP/tsBAFF均在大肠杆菌BL21（DE3）中高效表达。纯化后，EGFP/tsBAFF融合蛋白显示出与EGFP相似的荧光光谱。激光扫描共聚焦显微镜显示EGFP/tsBAFF与其受体结合。在体外，无论有无启动剂（金黄色葡萄球菌Cowan 1，SAC）或抗小鼠IgM，tsBAFF均可促进尼罗罗非鱼和小鼠脾脏B细胞的增殖。此外，与小鼠BAFF（msBAFF）相比，tsBAFF对小鼠B细胞也显示出类似的增殖刺激作用。这些发现表明，tsBAFF在尼罗罗非鱼B细胞增殖中起重要作用，并且在尼罗罗非鱼和哺乳动物之间具有功能交叉反应性。因此，BAFF可能是增强动物免疫效力的一个有用因子。
