金标银染技术在表达文库筛选中的应用

THE APPLICATION OF GLSS TECHNIQUE TO SCREENING OF EXPRESSION LIBRARY

以胶体金标记钙调素作为探针再辅以银染法 (金标银染技术 ) ,自玉米 c DNA表达文库中筛选钙调素结合蛋白 .该探针保持钙调素的活性 ,特异性地与钙调素结合蛋白相结合 ,在胶体金标记的基础上进行银染 ,灵敏度获得显著提高 (达 ng级 ) .应用这一方法对玉米 c DNA表达文库进行筛选获得了一个与探针特异结合的阳性克隆 .以生物素标记的钙调素对该克隆表达的融合蛋白进行结合实验 ,结果表明该克隆编码的蛋白为一个钙调素结合蛋白 .本文首次报道了以金标银染技术运用于表达文库的筛选并得到阳性克隆 ,这一方法的建立为 c DNA表达文库的筛选提供了简便、快速的方法 .

The technique of Gold Label Silver Stain (GLSS) was used in screening Calmodulin Binding Protein (CaM BP) in Maize cDNA expression library. The calmodulin was labelled with colloidal gold solution to make the gold probe. The CaM BP 10 was binded with the probe to detect the specificity and sensitivity of this method. The probe could specifically bind with the CaM BP 10 and did not bind with other proteins. The sensitivity of GLSS could reach ng level after Silver Stain enhancement. The Maize cDNA expression library was screened by using GLSS technique and one positive clone which specifically binds with the probe was acquired. The mono positive clone was acquired after purification. The fusion protein binded with the biotinylated calmodulin after electrophoretical transferring to the nitrocellulose membrane. The result proved that this clone codes a CaM BP. The GLSS technique is a fast and convenient way to screen the expression library.