大肠癌噬菌体展示肽库的构建及大肠癌早期检测分子的筛选

Screening of molecular markers for early diagnosis of colorectal cancer by phage-peptide library

目的构建大肠癌噬菌体展示肽库,筛选用于大肠癌早期检测的分子标志。方法选取30例第二军医大学长海医院肛肠外科手术后立刻冻存的大肠癌组织标本构建T7噬菌体展示肽库;用结合protein-A/G的琼脂糖珠分别富集大肠癌组及非肿瘤对照组血清中的抗体进行5轮亲和筛选,富集大肠癌相关肽;随机挑取5轮筛选后的2000个噬菌体克隆,用ELISA方法进一步筛选与大肠癌患者血清和对照血清反应性存在差异的大肠癌相关标志物克隆,进行基因序列测定。应用通过Chilibot文献挖掘方法预测各克隆的蛋白功能,反证筛选结果。结果 (1)所建大肠癌噬菌体展示肽库的滴度为3.0×106pfu,经PCR鉴定其重组率为60%,库容为1.8×106pfu。(2)共筛选出18个有意义的噬菌体,测序后,预测其蛋白功能,其中12个与肿瘤的发生相关。结论应用ELISA对肿瘤重组抗原的噬菌体展示肽库进行筛选的方法 ,可以用来发现差异表达的抗原。所筛选出的噬菌体克隆所表达的抗原可用于早期筛检大肠癌。

Objective To establish a colorectal cancer phage-peptide library and to screen for biomarkers for early detection of colorectal cancer.Methods A T7 phage display peptide library was constructed using 30 surgical colorectal cancer specimens from Changhai Hospital,Second Military Medical University.Protein-A/G was used to enrich IgG from control sera as well as colorectal cancer sera.Five biopanning protocols were carried out for enrichment of colorectal cancer-specific phage clones,and 2 000 phage clones were randomly selected.ELISA was used for further screening of clones of different reactivities between the cancer serum and control serum;and the selected clones were subjected to DNA sequencing and the cloned protein function was forecasted by Chilibot for validation.Results （1） The titer of the colorectal cancer phage display peptide library was 3.0 × 106pfu,with a recombination rate of 60% as showed by PCR identification and a storage capacity of 1.8 × 106pfu.（2） Of the 18 phage clones selected by ELISA,12 were cancer-related genes.Conclusion ELISA for screening the recombinant tumor antigen phage display peptide library can be used to discover new differentially expressed antigens;and the selected phage clones expressing antigen might be used for early detection of colorectal cancer.