Hitzeman R A, Clarke L, Carbon J
J Biol Chem. 1980 Dec 25;255(24):12073-80.
An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.
一种免疫筛选技术已被用于在含有杂交质粒的细菌菌落库中检测产生特定抗原的克隆。该技术包括将抗血清共价连接到溴化氰活化的纸盘上，使该纸与琼脂平板上裂解的菌落接触，最后用125I标记的抗体检测结合的抗原。使用这种方法，我们鉴定出一个大肠杆菌菌落，其在质粒ColE1中含有酵母DNA插入片段，该菌落产生的抗原能与针对纯化的酵母3-磷酸甘油酸激酶的抗体结合。通过该程序获得的杂交质粒（pYe57E2）已通过生化和遗传方法证明含有酵母3-磷酸甘油酸激酶的结构基因PGK。通过对携带含有原始酵母DNA插入片段各种重组的质粒的大肠杆菌菌落进行免疫筛选，确定了pYe56E2上PGK结构基因的位置。对克隆的酵母PGK基因在大肠杆菌和酵母中的表达研究表明，功能性酶在酵母中由克隆基因合成，但在大肠杆菌中则不然。
