Yamamoto K, Miura H, Moroi Y, Yoshinoya S, Goto M, Nishioka K, Miyamoto T
Department of Internal Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Japan.
J Immunol. 1988 Jan 1;140(1):311-7.
A cloned complementary DNA, termed pS2, was isolated from a human fibroblast cDNA library in the bacteriophage expression vector lambda gt11 after screening with a patient's serum containing a high titer of anti-ribonucleoprotein (RNP) antibodies. A reasonable amount of cro-beta-galactosidase fusion protein (pS2EX) was obtained through subcloning of the pS2 insert into a plasmid expression vector pEX-2. Antibody against pS2EX (anti-pS2EX) was purified from this patient's serum by Sepharose 4B conjugated with pS2EX. Immunofluorescent staining of HeLa cells with anti-pS2EX antibody exhibited a typical speckled pattern in the interphase nuclei. In the immunoblot analysis, the anti-pS2EX antibody recognized the 22 kDa protein. Using immunoprecipitation of cell lysate and subsequent RNA analysis, anti-pS2EX antibody was shown to precipitate U1 RNP only. The reactivities of various anti-RNP sera to pS2EX correlated well with the positive reaction to C polypeptide in the immunoblot. These findings indicate that pS2 is a cDNA for C polypeptide of U1 snRNP. In the Northern blot using human RNA and radiolabeled pS2, a single band about 800 base was observed. The nucleotide sequence of pS2 showed no significant homologies to known proteins.
从噬菌体表达载体λgt11中的人成纤维细胞cDNA文库中，用含有高滴度抗核糖核蛋白（RNP）抗体的患者血清进行筛选，分离出一个克隆的互补DNA，称为pS2。通过将pS2插入片段亚克隆到质粒表达载体pEX - 2中，获得了适量的cro-β-半乳糖苷酶融合蛋白（pS2EX）。用与pS2EX偶联的琼脂糖4B从该患者血清中纯化出抗pS2EX抗体。用抗pS2EX抗体对HeLa细胞进行免疫荧光染色，在间期核中呈现典型的斑点状模式。在免疫印迹分析中，抗pS2EX抗体识别22 kDa的蛋白质。通过对细胞裂解物进行免疫沉淀并随后进行RNA分析，表明抗pS2EX抗体仅沉淀U1 RNP。各种抗RNP血清对pS2EX的反应性与免疫印迹中对C多肽的阳性反应相关性良好。这些发现表明pS2是U1 snRNP的C多肽的cDNA。在用人RNA和放射性标记的pS2进行的Northern印迹中，观察到一条约800个碱基的单带。pS2的核苷酸序列与已知蛋白质没有明显的同源性。
