Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio)
Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio)
复制标题
DOI：
10.1016/j.fsi.2010.03.006
复制
发表时间：
2010-08-01
影响因子：
4.7
通讯作者：
Zhang, Shuangquan
中科院分区：
文献类型：
--
作者：
Liang, Zhenning;Kong, Yi;Zhang, Shuangquan
B细胞活化因子（BAFF）属于TNF家族，是B细胞存活、增殖、成熟和分化的关键细胞因子。本研究利用RT-PCR技术克隆了斑马鱼BAFF基因的cDNA（命名为zBAFF）。zBAFF的开放阅读框架（ORF）由807个碱基组成，编码268个氨基酸的蛋白质。其cDNA序列具有TNF家族特征、跨膜结构域和3个半胱氨酸残基，是哺乳动物和鸟类TNF基因的典型特征。系统发育分析显示，最高的同一性得分为67.6，61.4和66.9%的虹鳟鱼，四齿鱼和鲑鱼的对应物，分别。与鸟类和哺乳动物BAFF的同一性范围为49.7%至53.8%。将重组可溶性zBAFF（zsBAFF）与泛素相关修饰基因（SUMO）融合，在大肠杆菌BL 21（DE 3）中进行可溶性表达。SUMO-zsBAFF融合蛋白在大肠杆菌BL 21（DE 3）中得到高效表达，分子量约为38 kDa。用金属螯合亲和层析（Ni-NTA）纯化融合蛋白，用SUMO特异性蛋白酶切割融合蛋白，SDS-PAGE和Western blotting检测融合蛋白的表达。在体外，MIT测定表明，纯化的zsBAFF以及SUMO-zsBAFF蛋白能够以剂量依赖性方式促进脾淋巴细胞存活，也能够与抗IgM共刺激哺乳动物B细胞的增殖。因此，融合蛋白代表了一种容易获得的生物活性zsBAFF的来源，可以证明在斑马鱼BAFF及其受体的进一步研究中有用。(C)2010爱思唯尔有限公司版权所有。
B-cell activating factor (BAFF), belonging to the TNF family, is a critical cytokine for B-cell survival, proliferation, maturation and differentiation. In the present study we cloned the cDNA of zebrafish (Danio rerio) BAFF (designated zBAFF) by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of zBAFF consists of 807 bases encoding a protein of 268 amino acids. The deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, and three cysteine residues, which are the typical characteristics of TNF gene in mammals and birds. Phylogenetic analysis exhibits the highest identity score 67.6, 61.4 and 66.9% with the rainbow trout, tetraodon and salmon counterparts, respectively. The identity to avian and mammalian BAFFs ranges from 49.7 to 53.8%. Recombinant soluble zBAFF (zsBAFF) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-zsBAFF was highly expressed in BL21 (DE3) with a molecular weight of 38 kDa. The fusing protein was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MIT assay indicated that the purified zsBAFF as well as SUMO-zsBAFF proteins were able to promote spleen lymphocyte survival in a dose-dependent manner also to co-stimulate the proliferation of mammalian B-cells with anti-IgM. Thus, the fusion protein represents a readily obtainable source of biologically active zsBAFF that may prove useful in further studies on zebrafish BAFF and its receptors. (C) 2010 Elsevier Ltd. All rights reserved.