大肠癌抗原基因的筛选与初步分析

Screening and preliminary analysis of colorectal carcinoma-associated antigen genes

目的筛选鉴定大肠癌抗原基因,并进行生物信息学的初步分析。方法采用自体或异体大肠癌患者血清,应用 SEREX方法对大肠癌cDNA噬菌体表达文库进行免疫筛选。阳性克隆噬菌体在扩增、提取、纯化DNA后,用PCR方法和EcoR Ⅰ、HindⅢ双酶切鉴定cDNA片段的大小。阳性克隆cDNA捕入pUCm-T载体测序。序列生物信息学分析采用NCBI的All GenBank+EMBL+DDBJ+PDB Sequences Database进行BLAST基因比对分析。结果筛选出11个阳性克隆,cDNA片段大小分别为1100、1300、1000、2000、1200、1200、700、900、600、1200和1000 bp,代表9个抗原基因,7 个与已知基因同源。有3个阳性克隆为同1个基因片段,与干扰素诱导的跨膜蛋白-1基因同源,具有抗增殖作用;另6 个阳性克隆分别与不同的基因同源,分别是:BAC clone RP11-453E17“肿瘤解剖计划”基因,功能尚不清楚;软骨-毛发发育不良区基因,发生软骨-毛发发育不良综合征;人5号染色体克隆的序列,有插入突变,功能尚不清楚;类似抗肿瘤坏死因子α抗体的轻链Fab段基因,与IgGl抗体的γ链(重链)和κ链(轻链)的编码和调控其生物功能有关,可能与肿瘤的生长有关;B2微球蛋白的mRNA,与肿瘤细胞的增殖有关;醛缩酶A基因,异常表达可能促进肿瘤细胞的增殖; 尚有2个阳性克隆的cDNA序列没有同源性,功能有待进一步研究。结论用SEREX方法筛选大肠癌cDNA表达文库,可直接获得有价值的大肠癌抗原基因,对大肠癌发病机制的研究以及探讨早期诊断方法有重要意义。

Objective To screen and identify the genes coding for eolorectal carcinoma-associated antigen and analyze the bioinformation of their eDNA sequences. Methods Immunoscreening of the cDNA phage-display library derived from human colorectal carcinoma was performed with autologous or allogeneic serum antibody from patients with colorectal cancer through SEREX approach. After amplification of the positive phage clones, the phage DNA was extracted and purified with Qiagen kit, and the fragment sizes of the cDNA of positive clones were identified by PCR and EcoR Ⅰ and Hind Ⅲ restriction endonucleases. The cDNAs of the positive clones were ligated into pUCm-T vector and sequenced. The bioinformation of cDNA sequences were analyzed against GenBank＋EMBL＋DDBJ＋PDB Sequences Database. Results and Conclusion Eleven positive clones were obtained after immunoscreening, and the sizes of the eDNA fragments were 1100, 1300, 1000, 2000, 1200, 1200, 700, 900, 600, 1200 and 1000 bp, respectively, representing 9 antigen genes, including 7 with homology with the known genes. Among the 11 obtained positive clones, 3 were the same eDNA having homology with interferon-induced transmembrance protein-1 and possessing anti-proliferation effect; another 6 represented different genes, namely human BAC clone RP11-453E17 whose function have not been cleared, human cartilage-hair hypoplasia region gene responsible for cartilage-hair hypoplasia, human chromosome 5 clone CTD-2030B 15 with insertion mutation, human gene similar to anti tumor necrosis factor-alpha antibody light-chain Fab fragment associated with tumor growth, mRNA of human beta-2-microglobulin in relation to tumor cell proliferation, and human aldolase A gene promoting tumor cell proliferation. The other two cDNA sequences were not identified for homology with currently known genes in GenBank, and their functions awaits further investigation.