Isolation and comparison of natural and recombinant human CENP-A autoantigen.
Isolation and comparison of natural and recombinant human CENP-A autoantigen.
复制标题
天然和重组人 CENP-A 自身抗原的分离和比较。
DOI：
10.1006/jaut.1998.0249
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发表时间：
1998
影响因子：
12.8
通讯作者：
Hoch,SO
中科院分区：
文献类型：
--
作者：
Martinez,A;Sun,D;Billings,PB;Swiderek,KM;Sullivan,KF;Hoch,SO
抗着丝粒抗体（ACA）与表现出更良性或所谓的局限性疾病表现（lSSc）的系统性硬化症（硬皮病）患者相关。 ACA 反应性针对多个多肽靶标，其中最小的被指定为 CENP-A。 CENP-A 不是一种丰富的细胞成分；因此，为了最大限度地提高回收率，我们开发了一种以最少步骤从人类细胞系中分离 CENP-A 的方案。这种蛋白质的痕量细胞量清楚地决定了其重组对应物的产生，以促进确定 CENP-A 抗原在硬皮病发病机制中的作用。在这里，我们描述了使用杆状病毒介导的昆虫细胞感染来真核表达 CENP-A cDNA。非融合重组蛋白跨越人类CENP-A蛋白的天然残基，并且rCENP-A遵循与天然来源相同的色谱序列进行纯化。善意抗原的可用性提供了记录重组多肽真实性的关键标准。该抗原的两种形式已被比较并显示出相似的物理和抗原特性。
Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of thebona fideantigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.