Biochemical characterization of recombinant and circulating human Spα
Biochemical characterization of recombinant and circulating human Spα
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DOI：
10.1111/j.0001-2815.2004.00193.x
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发表时间：
2004-04-01
期刊：
影响因子：
--
通讯作者：
Lozano, F
中科院分区：
文献类型：
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作者：
Sarrias, MR;Padilla, O;Lozano, F
人Spalpha是存在于淋巴组织（脾、淋巴结、胸腺和骨髓）的巨噬细胞表达的一种可溶性蛋白，其功能和结构信息很少。它属于富含半胱氨酸的清道夫受体超家族（SRCR-SF）的B组，包括淋巴细胞表面受体CD5和CD6等。Spalpha能够与免疫系统的不同细胞（单核细胞和淋巴细胞）结合，这表明它可能在该系统的调节中发挥重要作用。为了研究Spalpha，利用哺乳动物外生表达系统（psep - pu /HEK 293-EBNA）产生重组形式（rSpalpha），用于生化研究和特异性杂交瘤的产生。通过Western blot、免疫沉淀和酶联免疫吸附试验，选择4种单克隆抗体对rSpalpha具有反应性。单克隆抗体识别Spalpha上的三个不同的表位。单克隆抗体显示，由于唾液酸含量不同，存在38和40 kDa的两种Spalpha亚型。他们还表明，Spalpha是一种相对丰富的血清蛋白（60马克杯/毫升），主要与其他血清蛋白一起循环。因此，rSpalpha允许亲和色谱分离多克隆和单克隆免疫球蛋白M （IgM）。这些数据表明Spalpha是一种循环蛋白，可能在IgM抗体的稳态中发挥作用。
Human Spalpha is a soluble protein expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), for which little functional and structural information is available. It belongs to the group B of the scavenger receptor cysteine-rich superfamily (SRCR-SF) that includes the lymphocyte surface receptors CD5 and CD6 among others. Spalpha is able to bind to different cells of the immune system (monocytes and lymphocytes), which suggests that it may play an important role in the regulation of this system. To study Spalpha, an episomal mammalian expression system (pCEP-Pu/HEK 293-EBNA) was used to produce a recombinant form (rSpalpha) that was utilized for biochemical studies and for the generation of specific hybridomas. Four monoclonal antibodies were selected for their reactivity against rSpalpha by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assays. The monoclonal antibodies recognized three different epitopes on Spalpha. The monoclonal antibodies revealed the existence of two Spalpha isoforms of 38 and 40 kDa, resulting from different sialic acid content. They also showed that Spalpha is a relatively abundant serum protein (60 mug/ml) that mostly circulates in association with other serum proteins. Accordingly, rSpalpha allowed affinity chromatography isolation of polyclonal and monoclonal immunoglobulin M (IgM). These data indicate that Spalpha is a circulating protein that may play a role in the homeostasis of IgM antibodies.