Molecular biological study＼ies in a novel peripheral nerve myelin protein and its application to clinical neurology

我们试图鉴定一例恶性淋巴瘤和周围神经病患者的血清免疫球蛋白所识别的35kd抗原。免疫印迹显示，血清中的免疫球蛋白与人、牛和小鼠周围神经(PN)中的35kd抗原反应，而与其他神经和非神经组织不反应。免疫组织化学分析显示PN致密髓鞘中的免疫球蛋白免疫反应阳性。我们构建了人坐骨神经cDNA文库，并用患者的免疫球蛋白对其进行了筛选。我们鉴定了三个独立的克隆人。对这些克隆的插入片段进行同源性搜索，发现这些插入片段与P0基因同源。然而，所有的插入片段都对应于P0cDNA3‘非翻译区。然后，制备人和牛坐骨神经髓鞘组分，对35kd抗原进行生化分析。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法，从髓鞘粗提物中提纯了35kd抗原。当比较35-kd抗原对患者的免疫球蛋白Ig G和抗P0单抗与P0蛋白的免疫反应性时，35-kd抗原与这两种抗体均发生反应，而P0只与单抗P0发生反应。这些结果表明，35-kd抗原可能是P0的异构体。然而，这些针对35-kd抗原的自身抗体的存在似乎没有什么病理意义，因为在没有相关周围神经病变的恶性淋巴瘤患者的血清中也发现了针对该抗原的循环自身抗体。

We tried to characterize a 35-kd antigen recognized by the serum IgG of a patient with malignant lymphoma and peripheral neuropathy. On western blotting, the serum IgG reacted with a 35-kd antigen in human, bovine and mouse peripheral nerves (PN) but not with other neural and non-neural tissues. Immunohistochemical analysis showed immunoreactivity for the IgG in the compact myelin of PN. We constructed a human sciatic nerve cDNA library and screened it using the patient's IgG. We identified three independent clones. A homology search of the inserts of these clones revealed that the inserts were homologous to P0 cDNA. However, all the inserts corresponded to the 3'-untranslational region of P0 cDNA. Then, to analyze the 35-kd antigen biochmically, myelin fractions of human and bovine sciatic nerve were prepared. Using SDS-polyacrylamide gel electrophresis, the 35-kd antigen was purified from the crude myelin fraction. When the immunoreactivities of the 35-kd antigen for the patient's IgG and monoclonal anti-P0 antibody were compared with those of protein P0 for these antibodies, the 35-kd antigen reacted with both the antibodies, but P0 reacted with only monoclonal anti-P0 antibody. These results indicate the possibility of the 35-kd antigen being an isoform of P0. However, the presence of these autoantibodies against the 35-kd antigen seems to be of little pathological significance, because circulating autoantibodies against the antigen were also found in the sera of patients with malignant lymphoma without associated peripheral neuropathy.