抑制性消减杂交技术克隆肾癌差异表达基因

Suppression subtractive hybridization for cloning of renal cell carcinoma relative genes

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 subtractive library of human renal cell carcinoma (RCC) with suppression subtractive hybridization technique.The library contains only the differently expressin cDNAs between RCC and normal kidney. Methods Poly (A)+RNA were isolated from RCC and normal kidney tissues.Single strand cDNAs and double strand cDNAs were synthesized in turn.After enzyme restriction,cDNAs < 600bp were obtained.RCC cDNAs were then divided into two groups and li gated to the specific adaptor l and adaptor 2 respectively.After RCC cDNAs were hybridized with normal kidney cDNA twice and underwent two times of nested PCR and was then connected with arms of T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain Top 10F'. Results Human RCC subtractive library with high subtractive efficiency was set up successfully.The amplified library contains 350 positive clones.Random analysis of 200 clones with enzyme restriction shows that 190 plasmids in the clones contain 50～400bp inserts.Sequence analysis were performed in 15 clones.All of the sequences are unknown before. Conclusions The cDNA subtractive library of human RCC is a highly efficient one and lays solid foundation for screening and cloning new and specific oncogenes or tumor suppressor genes of RCC.The novel genes cloned proved to be an important clue for researching the mechanism of occurrence and development of RCC.