Immunochemical isolation of prolactin messenger RNA.
Immunochemical isolation of prolactin messenger RNA.
复制标题
催乳素信使 RNA 的免疫化学分离。
DOI：
10.1016/s0021-9258(19)86109-4
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
R. Maurer
中科院分区：
文献类型：
--
作者：
R. Maurer
催乳素mRNA已分离使用免疫化学技术。初步实验表明，125 I标记的催乳素抗体能够与垂体多聚体结合，但不能与肝多聚体结合，表明结合是特异性的。催乳素合成的多聚核糖体通过与抗催乳素然后与抗抗体反应进行免疫沉淀。免疫沉淀的多聚核糖体RNA在oligo（dT）-纤维素上层析，并通过蔗糖梯度沉淀多聚（A）RNA。该程序导致催乳素mRNA的320倍纯化，如通过mRNA依赖性网织红细胞裂解物测定中的翻译所确定的。翻译分析还表明，分离的催乳素mRNA纯度大于95%。通过在含有10 mM氢氧化汞的琼脂糖凝胶上电泳测定的催乳素mRNA的分子量为350，000。纯化的催乳素mRNA用于通过禽成髓细胞瘤逆转录酶合成全长cDNA。使用该cDNA作为杂交探针表明，雌激素能够增加垂体中催乳素mRNA序列的浓度。
Prolactin mRNA has been isolated using immunochemical techniques. Initial experiments demonstrated that 125I-labeled prolactin antibody was able to bind to pituitary polysomes but not to liver polysomes, suggesting that the binding is specific. Prolactin-synthesizing polysomes were immunoprecipitated by reaction with antiprolactin followed by anti-antibody. Immunoprecipitated polysomal RNA was chromatographed on oligo(dT)-cellulose, and the poly(A) RNA was sedimented through a sucrose gradient. This procedure resulted in a 320-fold purification of prolactin mRNA as determined by translation in a mRNA-dependent reticulocyte lysate assay. Translation analysis also suggested that the isolated prolactin mRNA is greater than 95% pure. The molecular weight of prolactin mRNA determined by electrophoresis on agarose gels containing 10 mM mercury hydroxide was 350,000. Purified prolactin mRNA was used to synthesize full-length cDNA by means of avian myeloblastosis reverse transcriptase. Use of this cDNA as a hybridization probe demonstrated that estrogen is able to increase the concentration of prolactin mRNA sequences in the pituitary.