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目的以蛋白质组学相关技术进行初步筛选人源性大肠癌Fab段噬菌体抗体库,建立一种寻找特异性大肠癌抗原蛋白及相应的噬菌体抗体的方法。方法以二维凝胶电泳分离大肠癌组织和正常大肠粘膜组织蛋白组并将获得的2-DE凝胶电转至PVDF膜。然后以大肠癌组织匀浆为靶抗原对我们构建的人源性大肠癌Fab段噬菌体初级抗体库进行三轮淘洗,再用富集抗体库和PVDF膜进行Western-Blot印记实验。结果经Western-Blot印记实验我们发现了至少八个大肠癌表达而正常大肠粘膜不表达的有噬菌体抗体特异性结合活性的抗原蛋白。结论成功建立了双盲,双向,高通量筛选大肠癌特异性抗原及其相应噬菌体抗体的方法。
OBJECTIVE: To screen the Fab phage antibody library of human colorectal cancer by proteomics-related techniques and to establish a method to find specific colorectal cancer antigen protein and corresponding phage antibody. Methods Two-dimensional gel electrophoresis was used to separate colorectal cancer tissue and normal colorectal mucosal tissue proteome and the resulting 2-DE gel was electrotransferred to PVDF membrane. Then, the colorectal cancer tissue homogenate was used as the target antigen to perform three rounds of panning on the Fab phage primary antibody library of the human colorectal cancer, and then the Western blot assay was performed on the enriched antibody pool and the PVDF membrane. Results Western blotting experiments revealed that there were at least eight colorectal cancers but not normal colonic mucosa with phage antibody specific binding activity. Conclusion Double-blind, bi-directional, high-throughput screening of colorectal cancer-specific antigens and their corresponding phage antibodies has been successfully established.