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目的:寻找人源性大肠癌相关抗原基因.方法:构建3个以λ.Trip1 Ex2噬菌体作载体的大肠癌抗原cDNA表达文库,用自体或异体大肠癌患者血清应用SEREX方法进行免疫筛选.用平板法扩增阳性克隆噬菌体,提取纯化 DNA,用 Sfi I酶切和 PCR鉴定插入片段的大小.结果:构建成3个大肠癌抗原cDNA噬菌体表达文库,滴度分别为 2.39×10~6nfu/L,2.07×10~6nfu/L和 1.86×10~6nfu/L,插入片段长度为 0.5—4 kb,平均分别为 1.4 kb,1.6kb和1.3 kb.筛选发现4个阳性克隆,插入cDNA片段大小分别为 2.4 kb,1.8 kb,2.3 kb和 2.2 kb.结论:用SEREX方法筛选大肠癌抗原cDNA表达文库,可获得有价值的大肠癌的重组抗原基因克隆,将有利于大肠癌的早期诊断和重组疫苗的研究.
Objective: To search for human colorectal cancer related antigen gene.Methods: Three cDNA expression libraries of colorectal cancer antigen with λ.Trip1 Ex2 phage were constructed, and the serum of patients with autologous or allogeneic colorectal cancer was screened by SEREX method, Positive phage clones were amplified by PCR, and the purified DNA was digested with Sfi I and identified by PCR.Results: The cDNA phage expression libraries were constructed with 3 titers of 2.39 × 10 ~ 6 nfu / L, 2.07 × 10 ~ 6nfu / L and 1.86 × 10 ~ 6nfu / L respectively.The length of inserted fragment was 0.5-4 kb, with an average of 1.4 kb, 1.6 kb and 1.3 kb, respectively.The four positive clones were screened and the size of the inserted cDNA fragments were 2.4 kb, 1.8 kb, 2.3 kb and 2.2 kb.Conclusion: Screening cDNA library of colorectal cancer antigen by SEREX method can obtain valuable colony of recombinant antigen of colorectal cancer, which is good for early diagnosis of colorectal cancer and recombinant vaccine the study.