Dillner J, Wendel-Hansen V, Kjellström G, Kallin B, Rosén A
Department of Medical Cell Genetics, Karolinska Institute, Stockholm, Sweden.
Int J Cancer. 1988 Nov 15;42(5):721-7. doi: 10.1002/ijc.2910420516.
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is the only one of the EBNA proteins to have been implicated as an EBV-encoded transforming protein. More detailed studies of this protein have been hampered by the lack of EBNA-2-specific monoclonal antibodies (MAbs) and of purified protein. To overcome these problems, we isolated 5 hybridomas producing MAbs reactive with an 18 residue synthetic peptide corresponding to the carboxyterminus of EBNA-2. Four of the 5 MAbs were specifically reactive with EBNA-2 in its denatured form on immunoblots. The 5th antibody (115E) was reactive with the native form of EBNA-2. By using a one-step immunoaffinity purification method with 115E cross-linked to protein-A-Sepharose, we purified EBNA-2 to homogeneity, i.e., more than 1,200-fold, from Burkitt lymphoma cell extracts. A major 32-kDa associated protein and a less abundant 17-kDa protein were co-purified with EBNA-2. Immunoprecipitation with 115E from 35S-methionine-labelled cell extracts showed that the 32-kDa protein co-precipitated with EBNA-2 from EBV-positive cells, but was not detectable in immunoprecipitates of EBV-negative cells. When the immunoprecipitates or the purified proteins were immunoblotted with EBV-immune sera, only EBNA-2 was reactive, indicating that the associated proteins are of cellular origin. Immunoprecipitation of cells labelled with 32P-orthophosphate showed that EBNA-2, but not the associated proteins, is a phosphoprotein. The expression level of EBNA-2 varied between different EBV-carrying cell lines, as measured by a 2-site ELISA based on antibody 115E. In indirect immunofluorescence, the 115E MAb gave an EBNA-2-specific characteristic granular staining pattern. These characteristics of EBNA-2 resemble those of other viral transforming proteins.
爱泼斯坦-巴尔病毒（EBV）核抗原2（EBNA-2）是EBNA蛋白中唯一被认为是EBV编码的转化蛋白。由于缺乏EBNA-2特异性单克隆抗体（MAb）和纯化蛋白，对该蛋白的更详细研究受到了阻碍。为克服这些问题，我们分离出5株杂交瘤，它们产生的单克隆抗体可与对应于EBNA-2羧基末端的18个氨基酸的合成肽发生反应。5株单克隆抗体中有4株在免疫印迹中能与变性形式的EBNA-2特异性反应。第5种抗体（115E）能与天然形式的EBNA-2发生反应。通过使用与蛋白A-琼脂糖交联的115E进行一步免疫亲和纯化方法，我们从伯基特淋巴瘤细胞提取物中纯化出了均一的EBNA-2，纯化倍数超过1200倍。一种主要的32 kDa相关蛋白和一种含量较少的17 kDa蛋白与EBNA-2一起被共纯化。用115E对35S-甲硫氨酸标记的细胞提取物进行免疫沉淀表明，32 kDa蛋白在EBV阳性细胞中与EBNA-2共沉淀，但在EBV阴性细胞的免疫沉淀物中未检测到。当用EBV免疫血清对免疫沉淀物或纯化蛋白进行免疫印迹时，只有EBNA-2有反应，这表明相关蛋白是细胞来源的。用32P-正磷酸盐标记细胞的免疫沉淀表明，EBNA-2是一种磷蛋白，而相关蛋白不是。通过基于抗体115E的双位点ELISA检测，EBNA-2的表达水平在不同的携带EBV的细胞系之间有所不同。在间接免疫荧光中，115E单克隆抗体给出了EBNA-2特异性的特征性颗粒染色模式。EBNA-2的这些特征与其他病毒转化蛋白的特征相似。
