Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Dev Comp Immunol. 2012 Jan;36(1):191-8. doi: 10.1016/j.dci.2011.07.006. Epub 2011 Jul 27.
B-cell activating factor of the TNF family (BAFF) induces B cell survival, proliferation, immunoglobulin secretion and has a role in enhancing immune responses. In the present study, we amplified the cDNA of goat (Capra hircus) BAFF (designated gBAFF) from spleen by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of gBAFF covers 843 bp encoding 280 amino acids, with a 152-aa mature peptide. Sequence comparison indicated that the amino acid of gBAFF 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 soluble part of gBAFF (gsBAFF) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that gBAFF mRNA was predominantly expressed in goat lymphoid tissue spleen. Recombinant gsBAFF was fused with a small ubiquitin-related modifier (SUMO) gene to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-gsBAFF was efficiently expressed and purified using metal chelate affinity chromatography (Ni-NTA), then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-gsBAFF as well as gsBAFF could promote the survival/proliferation of goat splenic B cells or mouse splenic B cells. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in goat.
B 细胞激活因子肿瘤坏死因子家族（BAFF）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。在本研究中，我们通过反转录 - 聚合酶链反应（RT-PCR）从脾脏中扩增了山羊（Capra hircus）BAFF 的 cDNA（命名为 gBAFF）。gBAFF 的开放阅读框（ORF）覆盖 843bp，编码 280 个氨基酸，具有 152 个氨基酸的成熟肽。序列比较表明，gBAFF 的氨基酸具有 TNF 特征、跨膜结构域、假定的弗林蛋白酶切割位点和三个半胱氨酸残基。通过“比较蛋白质建模”分析 gBAFF 可溶性部分（gsBAFF）的预测三维（3D）结构表明，它与其他结构非常相似。实时定量 PCR（qPCR）分析表明，gBAFF mRNA 主要在山羊淋巴组织脾脏中表达。重组 gsBAFF 与小泛素相关修饰物（SUMO）基因融合，以提高大肠杆菌 BL21（DE3）中的可溶性表达水平。通过金属螯合亲和层析（Ni-NTA）高效表达和纯化所得融合蛋白 SUMO-gsBAFF，然后通过 SDS-PAGE 和 Western blot 分析进行确认。体外实验中，MTT 测定和流式细胞术分析表明，SUMO-gsBAFF 和 gsBAFF 均可促进山羊脾脏 B 细胞或小鼠脾脏 B 细胞的存活/增殖。因此，BAFF 可能是增强山羊免疫效力的潜在免疫因子。
