Yaneva M, Ochs R, McRorie D K, Zweig S, Busch H
Biochim Biophys Acta. 1985 Jul 26;841(1):22-9. doi: 10.1016/0304-4165(85)90270-3.
In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.
在对核仁抗原的研究过程中，开发了单克隆抗体，其中一种抗体识别出一种86 kDa的抗原，这是通过对来自HeLa或Namalwa细胞的核提取物进行分析得出的。对HeLa细胞的免疫荧光研究显示，单克隆抗体在核质中以及在核仁中的定位呈阶段依赖性，经DNA酶I消化后其定位减少，但经RNA酶A消化后则不然。为了进行纯化，通过用DNA酶I消化从细胞核中释放出抗原，然后通过在DEAE纤维素、磷酸纤维素和抗体-琼脂糖亲和色谱上进行色谱分离来纯化。有趣的是，免疫亲和纯化产物包含两条多肽链；具有免疫反应性的多肽的Mr为86000，pI为6.0。该复合物还含有一条70 kDa、pI 6.5的非抗原性多肽，二者比例为1:1。该复合物的总体纯化倍数为5700倍。两条多肽均含有约15 mol%的谷氨酸，70 kDa的多肽含有约15 mol%的丝氨酸。
