Bloch D B, Rabkina D, Quertermous T, Bloch K D
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Clin Immunol Immunopathol. 1994 Sep;72(3):380-9. doi: 10.1006/clin.1994.1156.
Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens. Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides. In the present study, serum from a patient with Sjögren's syndrome was used to identify a novel autoantigen which was designated Ge-1. Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified. Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins. Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein. This immunoreactive region contained the protein's putative nuclear localization sequence (NLS). To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase. After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus. The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies.
自身免疫性疾病患者血清中的抗体已被用于鉴定人类自身抗原。由于自身抗体通常识别相应蛋白质抗原内的活性位点，自身抗体有助于这些多肽的功能表征。在本研究中，来自干燥综合征患者的血清被用于鉴定一种新型自身抗原，命名为Ge-1。利用患者血清，鉴定出一个编码Ge-1的4.8-kb cDNA。将该cDNA片段连接到原核表达载体中，在大肠杆菌中表达，并用于产生重组Ge-1融合蛋白。含有Ge-1不同部分的融合蛋白被用于鉴定该蛋白内一个58个氨基酸的免疫反应区域。这个免疫反应区域包含该蛋白假定的核定位序列（NLS）。为了证明该免疫反应区域能够作为一个NLS发挥作用，构建了一个真核表达质粒，以编码与细胞质蛋白鸡肌肉丙酮酸激酶融合的免疫反应区域。将该质粒转染到COS-1细胞后，在细胞核中检测到了融合蛋白。Ge-1和其他核自身抗原的免疫反应区域内存在NLS基序，表明NLS可能是人类自身抗体的一个靶点。
