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目的:用SEREX方法鉴定HCC表达的肿瘤抗原,检测hcct-19的表达谱．方法:将HCC表达文库铺板,IPTG诱导蛋白质表达,BSA封闭．同1:1 000稀释的预吸收 HCC患者血清反应后,与1:5 000稀释的羊抗人抗体反应,在BCIP／NBT的作用下显色,挑取阳性克隆噬菌斑块,重复筛选和铺板3次,直至得到一致的单克隆免疫阳性重组噬菌体．挑取阳性克隆噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,PCR产物纯化测序, 序列结果用BLAST软件同GenBank中的已知基因进行对比分析．检测阳性克隆抗原基因 hcct-19在部分正常组织、肿瘤组织及肿瘤细胞株中的表达,半定量RT-PCR方法检测阳性克隆抗原基因在肝癌及癌旁组织中的表达．结果:共得到31个阳性克隆,代表14个不同的 cDNA序列,其中10个为已知功能的基因,4 个为未知功能的基因．在已知功能的基因中, RFC2、NDUFA4及MCART1首次被发现与 HCC有关．hcct-19在部分正常组织、肿瘤组织及肿瘤细胞株中表达,在肝癌组织中的表达强度明显高于癌旁组织(13．2±2．7vs2．9±0．3, P<0．05)．结论:本实验筛选出的HCC抗原有助于进一步阐明HCC的形成过程．hcct-19可能作为一个过量表达的基因参与了HCC的发病过程．
Objective: To identify HCC-expressed tumor antigens by SEREX method and detect hcct-19 expression profile. Methods: The HCC expression library was plated and IPTG induced protein expression. BSA was blocked. After reacting with 1:1 000 dilution of pre-absorbed HCC patient serum, react with 1:5000 dilution of goat anti-human antibody, develop color under the effect of BCIP/NBT, pick positive clone phage plaques, repeat screening and Plate 3 times until obtaining a consistent monoclonal immunoreactive recombinant phage. Positive colonies were picked and plaques were amplified by universal primers at both ends of the cloning site of the vector. PCR products were purified and sequenced. The sequence results were compared with known genes in GenBank using BLAST software. The expression of positive clone antigen gene hcct-19 was detected in some normal tissues, tumor tissues and tumor cell lines. Semi-quantitative RT-PCR method was used to detect the expression of positive clone antigen gene in hepatocellular carcinoma and adjacent tissues. Results: A total of 31 positive clones were obtained, representing 14 different cDNA sequences, of which 10 were known genes and 4 were genes with unknown functions. Of the known genes, RFC2, NDUFA4 and MCART1 were found to be related to HCC for the first time. Hcct-19 was expressed in some normal tissues, tumor tissues, and tumor cell lines. The expression intensity of hcct-19 in hepatocellular carcinoma was significantly higher than that in adjacent tissues (13.2±2.7 vs. 2.9±0.3, P<0.05). ). Conclusion: The HCC antigens screened out in this experiment will help further elucidate the formation of HCC. Hcct-19 may be involved in the pathogenesis of HCC as an overexpressed gene.