Ogata K, Ogata Y, Nakamura R M, Tan E M
J Immunol. 1985 Oct;135(4):2623-7.
Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.
增殖细胞核抗原（PCNA），也称为细胞周期蛋白，通过硫酸铵分级分离、DEAE-葡聚糖凝胶、高效液相色谱离子交换和高效液相色谱凝胶过滤柱色谱相结合的方法，从兔胸腺的磷酸盐缓冲盐水提取物中纯化得到。PCNA被纯化了600多倍，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹进行分析。SDS-PAGE显示，在该纯化过程中选择性分离出一种36kD的蛋白质，通过免疫印迹鉴定该蛋白质为PCNA。通过对流免疫电泳和酶联免疫吸附测定（ELISA）在该制剂中未检测到其他先前鉴定的核抗原，如Sm、核核糖核蛋白、SS-A/Ro、SS-B/La、组蛋白和DNA。纯化的PCNA用作抗原，开发ELISA用于快速、特异性检测人血清中的抗PCNA。为了进一步纯化，从SDS凝胶切片中电泳洗脱36kD条带。使用电洗脱的PCNA测定该蛋白质的氨基酸组成和N端的前25个残基。发现该氨基酸序列是独特的，并且在蛋白质鉴定资源数据库中与现有蛋白质的序列同源性很小。
