A MONOCLONAL-ANTIBODY AGAINST 2,2,7-TRIMETHYLGUANOSINE THAT REACTS WITH INTACT, CLASS-U, SMALL NUCLEAR RIBONUCLEOPROTEINS AS WELL AS WITH 7-METHYLGUANOSINE-CAPPED RNAS
A MONOCLONAL-ANTIBODY AGAINST 2,2,7-TRIMETHYLGUANOSINE THAT REACTS WITH INTACT, CLASS-U, SMALL NUCLEAR RIBONUCLEOPROTEINS AS WELL AS WITH 7-METHYLGUANOSINE-CAPPED RNAS
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DOI：
10.1111/j.1432-1033.1987.tb13439.x
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发表时间：
1987-10-15
期刊：
影响因子：
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通讯作者：
LUHRMANN, R
中科院分区：
文献类型：
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作者：
BOCHNIG, P;REUTER, R;LUHRMANN, R
分泌识别U snRNA的含2，2，7-三甲基鸟苷（m3 G）的帽结构的单克隆抗体（H-20）的杂交瘤来源于用含m3 G的人血清白蛋白缀合物免疫的小鼠。该抗体与完整的小核核糖核蛋白颗粒U snRNP特异性反应，并允许snRNP U1至U6通过亲和色谱法在制备规模上从真核细胞的核提取物中一步分离。 抗体结合的snRNP通过用过量的交叉反应性核苷7-甲基鸟苷（m7 G）洗脱而从亲和柱解吸，这保证了它们的天然结构的维持。亲和柱还允许通过在不同盐浓度下用m7 G顺序洗脱颗粒来将snRNP U1、U2和U 5与U4/U6 RNP分离。如通过竞争性放射免疫测定和蛋白质-A-琼脂糖免疫沉淀所确定的，mAb H-20与完整的m7 G帽结构交叉反应。特别地，我们可以显示非变性m7 G-加帽的SP 6/β-珠蛋白RNA被抗体有效地沉淀，而GpppG加帽或未加帽的RNA不反应。因此，单克隆抗体H-20显示出具有广泛的应用，不仅用于研究来自不同生物体的U snRNP的分子生物学和免疫学，而且用于表征和分离m7 G加帽的转录物。
A hybridoma secreting a monoclonal antibody (H-20) that recognizes the 2,2,7-trimethylguanosine(m3G)-containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G-containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess of the cross-reactive nucleoside 7-methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein-A-Sepharose immunoprecipitation, mAb H-20 crossreacts with intact m7G cap structures. In particular we could show that non-denatured m7G-capped SP6/.beta.-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RNAs did not react. Thus the monoclonal antibody H-20 showed have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G-capped transcripts.