应用改进的多聚组氨酸标签T7表达载体构建肺癌cDNA文库

应用噬菌体C端展示系统构建的cDNA文库缺乏开放阅读框筛选机制，文库中多数噬菌体克隆展示框外非天然短肽，给后期蛋白质的筛选带来了不便. 为实现噬菌体的ORF筛选功能，利用PCR技术对已有载体T7Select10-3b进行改造，在MCS处外源cDNA插入位点的3′端引入6聚组氨酸筛选标签，经包装后挑取成功表达的单克隆构建肺癌cDNA文库. 经镍柱亲和层析后，收集文库中表达组氨酸的克隆，利用化学发光免疫试验进行筛选效果鉴定. 结果显示，改造的新型载体可成功表达组氨酸标签，以此构建的肺癌cDNA文库经筛选后，含ORF插入的克隆由筛前的6 %提高至70 %，本研究为提高cDNA文库的质量提供了一种简便可行的方法.

Construction of Lung Cancer cDNA T7 Phage Library Using Modified Polyhistidine Tagged Expression Vector

The commercially available cDNA T7 phage library is constructed using C terminal display mechanism, which contains inadequate open reading frame (ORF) expressed protein epitopes. To improve the selection of ORF proteins, we modified the existing phage vector T7Select10 3b by inserting 6×His tag genes into the multiple cloning sites (MCS) at the 3′ terminus by PCR. The obtained phage clone expressing His tag was used for construction of a display library. The cDNA was extracted from the human lung cancer tissues and inserted into the His tag modified vector. The packaged phages with ORF inserts were enriched by Ni chelating affinity chromatography, and then screened by chemiluminescence immunoassay. The result showed that the 6×His tag allowed the enrichment of expressed peptides with an increased from 6 % to 70 % as compared to unselected the library. Our His taged phage library provided a convenient and practical method to improve the ORF selection.