应用改进的多聚组氨酸标签T7表达载体构建肺癌cDNA文库  

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

作　　者：董晓民[1] 钟理[1,2] 樊江平[1] 张旭朏[1] 

机构地区：[1]河北大学生命科学学院生物芯片研究室,保定071002 [2]Western University of Health Sciences, California 91766 ,USA

出　　处：《中国生物化学与分子生物学报》2013年第5期475-481,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81272444,No.81071795)~~

摘　　要：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.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 T7Selectl0-3b by inserting 6 x His-tag genes into the multiple cloning sites （MCS） at the 5＇ 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 x His-tag allowed the enrichment of expressed peptides with an increased from 6 % to 70 % as compared to unselected the library. Our His-raged phage library provided a convenient and practical method to improve the ORF selection.

关 键 词：组氨酸标签 T7噬菌体展示文库 开放阅读框 

分 类 号：Q78[生物学—分子生物学]