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目的 应用抑制性消减杂交技术构建人肾癌组织与正常肾组织差异表达的cDNA消减文库 ,并从中克隆鉴定出肾癌特异性表达的新基因。 方法 分别从肾癌及正常肾组织中提取mRNA并合成cDNA ,经酶切后将肾癌cDNA分为两组 ,分别与两种不同的接头衔接 ,再与正常肾组织 (cDNA)进行两次消减杂交及两次抑制性PCR ,将产物与T/A载体连接构建成功cDNA消减文库 ,并转染大肠杆菌进行文库扩增 ,随机挑取克隆进行酶切、测序分析。 结果 构建成功具有高消减效率的人肾癌cDNA消减文库 ,非特异性的cDNA片段被有效地消减 ,特异表达的cDNA得到富集。文库扩增后得到 35 0个白色克隆 ,随机挑取 2 0 0个制备质粒并酶切分析 ,其中 190个均得到 5 0～ 40 0bp插入片段。随机挑取 15个插入片段测序分析表明所获得的cDNA片段均为未知新基因片段。 结论 应用抑制性消减杂交技术所构建的人肾癌cDNA消减文库为大批量筛选、克隆肾癌特异性表达的未知新基因奠定了基础。初步筛选出的新基因片段为研究基因功能及其临床意义提供了依据。
Objective To construct a cDNA subtracted library differentially expressed between human kidney cancer tissues and normal kidney tissues by suppression subtractive hybridization and clone and identify new genes specifically expressed in kidney cancer. Methods mRNA was extracted from kidney cancer and normal kidney tissues, and cDNA was synthesized. After digestion, kidney cancer cDNA was divided into two groups, which were connected to two different types of adaptors, and then subtracted from normal kidney tissue (cDNA) twice. After hybridization and two inhibitory PCR, the product was ligated with the T/A vector to construct a cDNA subtracted library, which was then transfected into E. coli library for amplification. Clones were picked randomly for restriction analysis and sequencing analysis. RESULTS: A human subtractive cDNA library with high deletion efficiency was successfully constructed. Non-specific cDNA fragments were efficiently subtracted, and specifically expressed cDNA was enriched. After the library was amplified, 355 white clones were obtained and 200 plasmids were randomly picked and analyzed by enzyme digestion. Among them, 190 clones each received a 50-400 bp insert. Randomly picked 15 inserts and sequenced analysis showed that the obtained cDNA fragments were unknown new gene fragments. Conclusion The cDNA library of human kidney cancer cDNA subtracted by suppression subtractive hybridization has laid a foundation for large-scale screening and cloning of unknown genes that are specifically expressed in kidney cancer. The preliminary screening of new gene fragments provides a basis for studying gene function and its clinical significance.