Molecular dissection of Rauscher virus gp70 by using monoclonal antibodies: localization of acquired sequences of related envelope gene recombinants.
Molecular dissection of Rauscher virus gp70 by using monoclonal antibodies: localization of acquired sequences of related envelope gene recombinants.
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使用单克隆抗体对 Rauscher 病毒 gp70 进行分子解剖：相关包膜基因重组体的获得序列的定位。
DOI：
10.1073/pnas.77.8.4524
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发表时间：
1980
影响因子：
11.1
通讯作者：
Elder,JH
中科院分区：
文献类型：
--
作者：
Niman,HL;Elder,JH
使用 Rauscher 病毒 gp70 片段的杂交瘤特异性免疫沉淀，结合肽模式（指纹图谱）和部分氨基酸序列分析，我们生成了 Rauscher gp70 的线性图。我们使用了一组 56 个杂交瘤，这些杂交瘤源自药物选择的 SP-2 骨髓瘤系与来自用纯化的 Rauscher 病毒 gp70 免疫的 129 GIX+ 或 GIX- 小鼠的脾细胞的融合。结果表明，通过“自然”分解，gp70 分裂成主要片段，Mr 45,000 (P45)、34,000 (P34) 和 32,000 (P32)。这些以及重叠片段的肽指纹分析与部分氨基酸序列分析相结合，使我们能够将片段排列成线性排列 NH2-P45-P32-COOH，其中 P34 是 P45 的 NH2 末端降解产物。在 56 个杂交瘤中，20 个同时免疫沉淀 P45 和 P34； 18 仅免疫沉淀 P45；和 18 仅免疫沉淀 P32。因此，杂交瘤将分子的三个结构域定义为NH2-P45/P34、仅P45和P32-COOH。让这些杂交瘤与两种 Rauscher 衍生的包膜基因重组病毒反应，产生了以下结果：(i) 所有 20 个 P45/34 反应器均与两种 Rauscher 重组体结合； (ii) 18 个仅含 P45 的杂交瘤中，10 个发生反应； (iii) 18 个 P32 反应器中只有 1 个与 Rauscher 重组体结合。最后的杂交瘤与多种鼠逆转录病毒发生反应，表明它针对 gp70 的保守决定簇。 R-gp70、重组gp70及其各自的分解产物的肽指纹图谱证实了杂交瘤所定义的同源性和非同源性。此外，肽模式显示重组gp70s COOH末端部分上的这些获得的序列与异源病毒gp70s相关。
Using hybridoma-specific immune precipitations of fragments derived from Rauscher virus gp70, coupled with peptide patterns (fingerprinting) and partial amino acid sequence analyses, we have generated a linear map of Rauscher gp70. We used a panel of 56 hybridomas derived from the fusion of the drug-selected SP-2 myeloma line with spleen cells from either a 129 GIX+ or a GIX- mouse immunized with purified Rauscher virus gp70. The results showed that by "natural" breakdown, gp70 splits into predominant fragments, with Mr 45,000 (P45), 34,000 (P34), and 32,000 (P32). Peptide fingerprinting of these as well as overlapping fragments coupled with partial amino acid sequence analyses allowed us to align the fragments into the linear arrangement NH2-P45-P32-COOH, with P34 being an NH2-terminal degradation product of P45. Of the 56 hybridomas, 20 immunoprecipitated both P45 and P34; 18 immunoprecipitated only P45; and 18 immunoprecipitated only P32. The hybridomas thus define three domains of the molecule as NH2-P45/P34, P45 only, and P32-COOH. Allowing these hybridomas to react with two Rauscher-derived envelope gene recombinant viruses yielded the following results: (i) all 20 P45/34 reactors bound to the two Rauscher recombinants; (ii) of 18 P45-only hybridomas, 10 reacted; and (iii) only 1 of 18 P32 reactors bound to the Rauscher recombinants. This last hybridoma reacted with various murine retroviruses, indicating that it was directed at conserved determinants of gp70. Peptide fingerprinting of R-gp70, the recombinant gp70s, and their respective breakdown products confirmed the homologies and nonhomologies defined by the hybridomas. Furthermore, peptide patterns showed that these acquired sequences on the COOH-terminal portion of the recombinant gp70s are related to xenotropic virus gp70s.