鼻咽癌组织cDNA文库的构建及抗原基因的筛选

Construction of cDNA Library from NPC Tissue and Screening of Antigenic Genes

构建人鼻咽癌组织cDNA文库 ,以SEREX方法从cDNA文库中筛选鼻咽癌抗原基因。采用确诊鼻咽癌患者新鲜活检癌组织构建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。以建库组织来源患者的自身血清 ,采用“一对一”的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。经测定原始文库滴度为 7 2 8× 10 6 pfu mL ,含 3 6 4× 10 6 个重组子 ,重组率为 94 % ,扩增文库滴度为 3 8× 10 9pfu mL ,cDNA插入片段大小在 0 5～ 3 0kb之间。文库经三轮血清学筛选共获得 2 3个阳性克隆 ,分别代表了 16个独立的cDNA插入片段 (抗原基因 )。其中 10个与已知基因高度同源 ,另外 6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鼻咽癌组织cDNA表达文库 ,有利于以cDNA文库为基础的进一步的实验研究。应用SEREX技术初步筛选鼻咽癌组织cDNA文库 ,共得到 16个鼻咽癌相关抗原基因 ,其中有 3个是新基因 ,可能为鼻咽癌的免疫学研究提供新的研究分子。

To obtain the NPC_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 performing serological analysis, a high quality cDNA library derived from human nasopharyngeal carcinoma (NPC) tissue was constructed. The primary library consisted of 3.64×10 6 recombinants and the recombinant rate was 94%. For better preserving the cDNA library, it was amplified. As a result, the titer of the amplified cDNA library was 3.8×10 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, 23 positive clones encoding antigenic genes were obtained after immunoscreening, and the nucleotide sequences of cDNA inserts were determined and analyzed with BLAST software in GenBank. Results showed that the 23 reactive clones were derived from 16 different genes. 10 of 16 genes had high homologous to the genes known in GenBank, such as RPL31、S100 A2、MT2A,etc. However, there were also 6 genes with low homology to the genes known in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be revealed by further research.