A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein.
A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein.
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BamHI 片段 M 中的第二个 Epstein-Barr 病毒早期抗原基因编码 48 至 50 千道尔顿的核蛋白。
DOI：
10.1128/jvi.56.3.860-866.1985
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发表时间：
1985
影响因子：
5.4
通讯作者：
Hayward,SD
中科院分区：
文献类型：
--
作者：
Cho,MS;Milman,G;Hayward,SD
我们使用针对EB病毒(EBV)BamHI片段M中一个开放阅读框的细菌合成产物而制备的抗血清来证明该阅读框(BMRF1)编码一个弥漫性早期抗原(EA)类核蛋白。在间接免疫荧光试验中，兔抗BMRF1抗血清在丁酸钠处理的Raji细胞中有约5%的细胞出现核染色，在丙酮固定和甲醇固定的细胞中都观察到阳性荧光。无论使用间接免疫荧光还是抗补体免疫荧光，未诱导Raji培养的阳性细胞都不到0.1%。在免疫印迹分析中，兔血清在B95-8细胞或丁酸诱导的Raji细胞的总蛋白提取物中鉴定出一组46至55千道尔顿(KDa)的多肽。在这两种细胞类型中，优势多肽是48 kDa和50 kDa的物种。当免疫印迹与R3单抗反应时，同样的多肽家族也被鉴定出来，我们认为该抗体也识别BMRF1开放阅读框的产物。将BamHI-M基因与新霉素抗性基因共转染新生仓鼠肾细胞，建立了含EBV BamHI片段M的成纤维细胞系。选择免疫荧光法检测到EBV抗原表达的氨基糖苷G418抗性克隆，建立克隆细胞系。传代3~4个月后，免疫荧光和免疫印迹分析均检测不到EA的合成。然而，在所检测的一个细胞系中，丁酸钠处理培养物可诱导48-50 kDa EA的合成。因此，这种EA在转基因成纤维细胞中的表达调节与在Raji淋巴母细胞中看到的相似。我们先前证明了BamHI片段M还包含一个60 kDa核EA的编码序列，因此BamHI-M编码弥漫性EA复合体的两个独立成分。
We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.
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