用T7噬菌体展示系统筛选与晚期糖基化终产物受体胞内段相结合的蛋白

Screening and identification of proteins that interact with receptor for advanced glycation end products (RAGE) via T7 select phage display system

目的 用T7噬菌体筛选系统筛选在细胞内与晚期糖基化终产物受体 (RAGE)结合的蛋白。方法 以重组RAGE为靶蛋白 ,用谷胱甘肽亲和树脂为介质 ,筛选T7人肺cDNA文库。结果 筛选 6轮后选择 6 4个噬菌体单个噬斑克隆 ,经EDTA处理后利用特异性PCR引物扩增插入片段 ,回收PCR产物并进行测序 ,将所得到的序列用BLAST软件搜索GenBank中的同源序列 ,得到了 2 5个编码蛋白的序列。结论 T7噬菌体展示筛选系统是筛选新的结合蛋白的一种简单、快速和有效的手段 ,所筛选的与RAGE胞内段相互作用的蛋白对进一步研究其信号转导通路的复杂功能和调节机制提供了很好的靶点。

Objective To screen and identify proteins that interact with receptor for advanced glycation end products (RAGE) with T7 select phage display system. Method GST-tagged fusion protein of RAGE was coated on glutathione suporflow resin and binding screens were performed with human lung T7 phage library. Results After 6 rounds of biopanning, 64 independent plaques were scraped and processed with EDTA. The inserted fragments in these plaques were amplified by PCR and the PCR products were purified by a gel recovery method. The sequences of the insertions were identified and analyzed with BLAST program in GeneBank. Twenty-five clones of encoding proteins were found. Conclusions T7 select phage display system is a convenient, rapid and effective method for screening binding proteins. This study provides an approach to explore complicated functions and novel mechanisms of RAGE-related signal transduction pathways.