原发型肝癌组织cDNA文库的构建及抗原基因的筛选  

Construction of cDNA library from hepatocellular carcinoma tissue and screening of antigenic genes

作　　者：刘辉[1] 潘泽亚[1] 周伟平[1] 李爱军[1] 傅思源[1] 尹磊[1] 吴孟超[1] 

机构地区：[1]第二军医大学东方肝胆外科医院,上海200438

出　　处：《中国现代医学杂志》2008年第6期691-693,698,共4页China Journal of Modern Medicine

摘　　要：目的构建人原发型肝癌组织cDNA文库,以SEREX方法从cDNA文库中筛选肝癌抗原基因。方法采用确诊的原发型肝癌患者新鲜手术切除癌组织构建cDNA文库,测定原始文库滴度,进行蓝白筛选以确定文库的重组率。以肝癌患者的血清,采用血清学方法筛选所构建的cDNA文库,阳性克隆经PCR检测鉴定后进行序列分析。结果成功构建了混合原发型肝癌cDNA文库经测定原始文库滴度为7.42×106pfu/mL,含3.96×106个重组子,重组率为93%,扩增文库滴度为3.92×109pfu/mL,cDNA插入片段大小在0.5～3.0kb之间。文库经3轮血清学筛选共获得31个阳性克隆,分别代表了24个独立的cDNA插入片段(抗原基因)。其中17个与已知基因高度同源,另外7个基因与GenBank中已知基因的部分同源,其中有3个是新基因。利用SMART技术构建了高质量的人原发型肝癌组织cDNA表达文库,有利于以cDNA文库为基础的进一步的实验研究。结论应用SEREX技术初步筛选原发型肝癌组织cDNA文库,共得到17个原发型肝癌相关抗原基因,其中有3个是新基因,可能为原发型肝癌的免疫学研究提供新的研究分子。[Objective] To obtain the HCC-associated antigens, a powerful new method, SEREX （serological identification of antigen by recombinant cDNA expression library）, was used for identifying the antigens eliciting humoral immune response. Before performrlng serological analysis, a high quality cDNA library derived from human hepatocellular carcinoma （HCC） tissue was constructed. The primary library consisted of 3.96 ×10^6 recombinants and the recombinant rate was 93%. For better preserving the cDNA library, it was amplified. As a result, the Liter of the amplified cDNA library was 3.92×10^9 pfu/ml. With SEREX method, immunoscreening for the detection of reactive clones in the human NPC tissue cDNA library was performed with autologous serum. As a result, 31positive clones encoding antigenic genes were obtained after immunosereening, and the nucleotide sequences of cDNA inserts were determined and analyzed with BLAST software in GenBank. Results showed that the 31 reactive clones were derived from 24 different genes. 17 of 24 genes had high homologous to the genes known in GenBank, However, there were also 7 genes with low homology to the genes known in GenBank. Furthermore, 3 of 7 genes may be novel genes. The associations of these genes to HCC and the roles that they played in the occurrence and development of HCC should be revealed by further research.

关 键 词：原发型肝癌 CDNA文库 重组cDNA表达文库的血清学分析 抗原基因 

分 类 号：R735.7[医药卫生—肿瘤]