Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit
Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit
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Epstein-Barr 病毒确定的核抗原和具有 53,000 道尔顿亚基的相关蛋白的纯化和生化表征
DOI：
10.1128/jvi.35.3.592-602.1980
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发表时间：
1980
影响因子：
5.4
通讯作者：
G. Klein
中科院分区：
文献类型：
--
作者：
J. Luka;H. Jörnvall;G. Klein
通过热处理、dna -纤维素层析和羟基磷灰石层析三步程序，从Raji和Namalwa细胞提取物中纯化出Epstein-Barr病毒确定的核抗原（EBNA），纯度达到700倍，具有明显的同质性。采用酸固定核结合和补体固定监测抗原特异性。纯化的EBNA还能够特异性抑制EBNA对Raji靶细胞的常规抗补体免疫荧光反应。纯化后的抗原分子量为17万~ 20万。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，得到一个48000道尔顿（48K）单体。从相同的细胞提取物中也纯化了eba相关蛋白。其分子量约为20万，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳得到单个53K蛋白带。在eb病毒阴性的b细胞淋巴瘤株中也发现了相同的蛋白。通过氨基酸组成和肽图谱对两种蛋白质进行了鉴定。结果表明，53K和48K蛋白组分无长区共通点；这就排除了较小的产品是由较大的产品分解而产生的。残基分布不同，但在两种蛋白质中都发现了过量的亲水性残基，这表明它们在性质上有一定的总体相似性。不同细胞系的53K成分存在一定差异。爱泼斯坦-巴尔病毒阳性株携带两种53K成分，其中一种可能是53K稍微改良的产物。免疫络合试验显示，48K蛋白而不是53K蛋白携带EBNA特异性。在混合物中，53K蛋白与48K蛋白共沉淀。这些数据表明EBNA可能在细胞内与53K蛋白形成复合物。
The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700-fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-step procedure involving heat treatment, DNA-cellulose chromatography, and hydroxyapatite chromatography. Acid-fixed nuclear binding and complement fixation were used to monitor antigenic specificity. Purified EBNA was also capable of specifically inhibiting the regular anticomplement immunofluorescence reaction for EBNA against Raji target cells. The purified antigen had a molecular weight of 170,000 to 200,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it yielded a single 48,000-dalton (48K) monomer. An EBNA-associated protein was also purified from the same cell extract. It had a molecular weight of about 200,000 and yielded a single 53K protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same protein was also found in Epstein-Barr virus negative B-cell lymphoma lines. The two types of protein were characterized by amino acid composition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common; this excludes that the smaller product arises by breakdown of the larger product. Residue distributions were different, but an excess of hydrophilic residues was found in both proteins, suggesting a certain overall similarity in properties. 53K components from different cell lines appeared to differ somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a slightly modified 53K product. Immunocomplexing assay showed that the 48K, but not the 53K, protein carries EBNA specificity. In mixtures, the 53K protein is co-precipitated with the 48K protein. The data suggest that EBNA may form a complex with the 53K proten within the cell.