cDNA cloning, expression and bioactivity of porcine BAFF
cDNA cloning, expression and bioactivity of porcine BAFF
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DOI：
10.1016/j.dci.2007.03.006
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发表时间：
2007-01-01
影响因子：
2.9
通讯作者：
Zhang, Shuang-Quan
中科院分区：
文献类型：
--
作者：
Guan, Zheng-Bing;Dan, Wen-Bing;Zhang, Shuang-Quan
肿瘤坏死因子家族的B细胞激活因子通过其受体BAFF-R、BCMA和TACI对B细胞的存活、成熟和T细胞活化起关键作用。本研究采用RT-PCR和RACE技术克隆了猪BAFF基因，命名为pBAFF。PBAFF全长由805个氨基酸组成，702个开放阅读框，编码233个氨基酸。推导出的氨基酸序列包含一个预测的跨膜结构域和一个与其他已鉴定的BAFF同源物相对应的可能的Furin蛋白酶切割位点。PBAFF与人BAFF(HBAFF)和鸡BAFF(CBAFF)的氨基酸同源性分别为93%和85%，氨基酸水平上的同源性分别为88%和76%。BAFF的三个半胱氨酸残基的特性在pBAFF中是保守的。RT-PCR结果表明，BAFF在猪的脾、肝、肺、心、肠、肾、胸腺和外周血淋巴细胞等多种组织中均有表达。融合了His(6)标签的重组可溶性pBAFF(PsBAFF)在大肠杆菌BL21(DE3)中得到了高效表达，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)和Western blotting证实了其表达。在体外，纯化的psBAFF不仅能刺激猪B细胞的增殖，还能刺激人B细胞的增殖。此外，hsBAFF还能与猪B细胞结合，对猪B细胞的增殖有积极作用。这些结果表明，pBAFF在猪B细胞增殖中起重要作用，猪和人BAFF之间存在功能性交叉反应。体外表达具有生物活性的psBAFF，为进一步研究其作为免疫佐剂增强疫苗效力和用于猪的免疫治疗奠定了基础。这也为研究BAFF在这一重要的家养物种中的作用和人类疾病的动物模型提供了基础。(C)2007爱思唯尔有限公司。保留所有权利。
B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805 by with a 702 by open reading frame, encoding 233 amino acids. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site corresponding to other identified BAFF homologues. The amino acid similarity between the functional soluble parts of pBAFF and human BAFF (hBAFF) or chicken BAFF (cBAFF) is 93% and 85%, respectively, with identity at the amino acid level was 88% and 76%, respectively. The characteristic of the three-cysteine residues of BAFF is conserved in pBAFF. RT-PCR showed that BAFF is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, kidney, thymus and PBLs. Recombinant soluble pBAFF (psBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. In vitro, purified psBAFF co-stimulates the proliferation of not only porcine B cells but also human B cells. In addition, hsBAFF binds to porcine B cells and has a positive effect on their proliferation. These findings indicate pBAFF plays an important role in proliferation of porcine B cells and functional cross-reactivity occurs between porcine and human BAFF. In vitro expression of bioactive psBAFF provides the basis for further investigation of its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic in pig. It also provides the basis for investigations on the role of BAFF in this important domestic species and an animal model for human diseases. (c) 2007 Elsevier Ltd. All rights reserved.