Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China.
Int Immunopharmacol. 2012 Feb;12(2):433-40. doi: 10.1016/j.intimp.2011.12.018. Epub 2012 Jan 9.
B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat (Vespertilio superans Thomas) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of bBAFF consists of 986 bases including an 873 bp open reading frame encoding 290 amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the bsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO (Small Ubiquitin-like Modifier)-bsBAFF was efficiently expressed in Escherichia coliBL21 (DE3) and confirmed by SDS-PAGE and Western blotting analysis. Laser scanning confocal microscopy analysis showed that bsBAFF could bind to its receptors on B cells. In vitro, the MTT assays indicated that SUMO-bsBAFF was not only able to promote survival/proliferation of bat lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These findings indicate that bsBAFF plays an important role in the survival/proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.
B 细胞激活因子（BAFF）属于肿瘤坏死因子（TNF）家族，对 B 细胞的存活、增殖、成熟、免疫球蛋白分泌以及 T 细胞的激活至关重要。本研究采用 RT-PCR 和快速扩增 cDNA 末端（RACE）技术，从蝙蝠（Vespertilio superans Thomas）中克隆了 BAFF 的全长 cDNA（命名为 bBAFF）。bBAFF 的全长 cDNA 由 986 个碱基组成，包括一个编码 290 个氨基酸的 873bp 开放阅读框。序列比较表明，bBAFF 的氨基酸具有 TNF 特征、跨膜结构域、潜在的弗林蛋白酶切割位点和三个半胱氨酸残基。通过比较蛋白建模分析预测的 bsBAFF 单体三维（3D）结构表明，它与同类物非常相似。实时定量 PCR（qPCR）分析表明，bBAFF mRNA 在蝙蝠淋巴组织脾脏中表达丰度最高。SUMO（Small Ubiquitin-like Modifier）-bsBAFF 在大肠杆菌 BL21（DE3）中高效表达，并通过 SDS-PAGE 和 Western blotting 分析得到验证。激光扫描共聚焦显微镜分析表明，bsBAFF 能够与 B 细胞上的受体结合。体外实验表明，SUMO-bsBAFF 不仅能够促进蝙蝠淋巴细胞的存活/增殖，还能够刺激小鼠 B 细胞的存活/增殖。这些发现表明 bsBAFF 在 B 细胞的存活/增殖中发挥重要作用，并在哺乳动物中具有功能交叉反应性。本研究结果可为蝙蝠免疫系统的研究提供有价值的信息。
