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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 construct phage display peptide library of colorectal cancer and screen the molecular markers for the early detection of colorectal cancer. Methods T7 phage display peptide library was constructed from 30 specimens of colorectal cancer cryopreserved immediately after anorectal surgery in Changhai Hospital of Second Military Medical University. Colorectal cancer group and non-tumor control group were enriched with protein-A / G agarose beads The antibodies in the serum were screened by 5 rounds of affinity screening and enriched in the peptide of colorectal cancer. 2000 phage clones after 5 rounds of screening were randomly selected, and their serum reactivity with colorectal cancer patients was further screened by ELISA Colorectal cancer related marker clones were sequenced. Application Through Chilibot literature mining method to predict the protein function of each clone, anti-screening results. Results (1) The titer of phage displayed peptide library of colorectal cancer was 3.0 × 106pfu. The recombination rate was 60% and the capacity was 1.8 × 106pfu by PCR. (2) Eighteen meaningful phage were screened out and sequenced to predict the function of the protein. Twelve of them were related to tumorigenesis. Conclusion ELISA screening of recombinant phage display peptide library of tumor antigen can be used to find differentially expressed antigens. The selected phage clones expressed the antigen can be used for early screening of colorectal cancer.