采用免疫吸附法从大鼠脑中纯化γ-烯醇化酶信使核糖核酸

Purification of gamma-enolase messenger ribonucleic acid from rat brain by an immunoadsorption method.

作者信息

Nybroe O, Hansen O C, Bock E

出版信息

Neurosci Lett. 1985 Jan 7;53(1):115-9. doi: 10.1016/0304-3940(85)90106-5.

Abstract

Messenger ribonucleic acid (mRNA) coding for the brain-specific protein gamma-enolase was isolated by an immunopurification procedure. Rat brain polysomes including nascent polypeptide chains were reacted with specific gamma-enolase antibody. The polysome-antibody complexes were subsequently adsorbed to protein A-Sepharose. After extensive washing, RNA was eluted and applied to an oligo(dT)-cellulose column. Purified mRNA was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system. The synthesized product was identical to gamma-enolase synthesized by free polysomes from rat brain. Immunoisolated gamma-enolase mRNA was enriched 380-fold compared to total mRNA extracted from free polysomes. This result indicates that low-abundance mRNAs may conveniently be isolated from brain tissue by immunoadsorption of polysomes.

摘要

通过免疫纯化程序分离出编码脑特异性蛋白γ-烯醇化酶的信使核糖核酸（mRNA）。将包括新生多肽链的大鼠脑多核糖体与特异性γ-烯醇化酶抗体反应。随后将多核糖体-抗体复合物吸附到蛋白A-琼脂糖上。经过大量洗涤后，洗脱RNA并将其应用于寡聚（dT）-纤维素柱。纯化的mRNA在依赖mRNA的兔网织红细胞裂解物系统中进行体外翻译。合成产物与大鼠脑游离多核糖体合成的γ-烯醇化酶相同。与从游离多核糖体中提取的总mRNA相比，免疫分离的γ-烯醇化酶mRNA富集了380倍。该结果表明，通过多核糖体的免疫吸附可以方便地从脑组织中分离低丰度mRNA。