Groner B, Hynes N E, Sippel A E, Jeep S, Chi-Nguyen-Huu M, Schütz G
J Biol Chem. 1977 Oct 10;252(19):6666-74.
The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.
通过对合成这些蛋白质的多核糖体进行免疫吸附,分离出了编码蛋清蛋白卵清蛋白、卵类粘蛋白和溶菌酶的信使核糖核酸。用兔制备的抗卵清蛋白、抗卵类粘蛋白和抗溶菌酶的单特异性抗体与鸡输卵管多核糖体反应。通过免疫吸附到偶联于不溶性基质的羊抗兔抗体上,分离出抗体 - 多核糖体复合物。洗脱特异性结合的多核糖体,并通过聚(U) - 琼脂糖凝胶柱层析获得信使核糖核酸。通过制备性凝胶电泳进一步纯化这三种特异性核糖核酸。信使核糖核酸制剂的纯度通过分析性凝胶电泳、在麦胚无细胞系统中指导合成特异性蛋白质产物的能力以及与从信使核糖核酸卵清蛋白(mRNAoa)和信使核糖核酸卵类粘蛋白(mRNAomu)转录的互补脱氧核糖核酸(cDNA)杂交来证明。纯化的信使核糖核酸卵清蛋白显示含有少于0.1%的信使核糖核酸卵类粘蛋白,而纯化的信使核糖核酸卵类粘蛋白相对于信使核糖核酸卵清蛋白约99%纯。纯化的信使核糖核酸溶菌酶(mRNAly)被信使核糖核酸卵清蛋白污染至0.34%以及被信使核糖核酸卵类粘蛋白污染至2.9%。