论文部分内容阅读
应用抑制性消减杂交技术(suppression subtractive hybridization, SSH), 成功地构建了高消减效率的人肾癌组织与正常肾组织差异表达的cDNA消减文库, 从中随机挑取200个克隆进行酶切分析, 结果显示其中190个均得到50~400 bp插入片段, 10个无插入片段. 对其中10个插入的cDNA片段进行测序后经GenBank检索表明10个片段均为未知新序列, 其中RCC18为5个拷贝, 这提示以上10个cDNA片段可能来自6个新基因. Northern杂交差异表达分析显示RCC18在肾癌组织中有明显表达, 而在正常肾组织中无表达, 这证明RCC18是肾癌特异表达的新基因, 依杂交信号估算其转录基因全长约3.5 kb. 人肾癌cDNA消减文库的建立为进一步大批量筛选、克隆肾癌特异性表达的未知新基因奠定了基础. 初步筛选出的特异性表达的新基因片段为进一步研究基因功能及其临床意义提供了依据.
Using subtractive subtractive hybridization (SSH), cDNA subtractive libraries differentially expressed in human renal cancer tissues and normal renal tissues with high efficiency were successfully constructed, and 200 clones were randomly selected for digestion analysis. Results The results showed that 190 of them all had 50-400 bp inserts and 10 non-insertions.All 10 inserts were sequenced and identified by GenBank, all 10 fragments were unknown new sequences, of which 5 were RCC18, This suggests that the above ten cDNA fragments may be derived from six new genes.Notably, Northern blot analysis of differentially expressed RCC18 in renal cell carcinoma, but no expression in normal renal tissue, which proves that RCC18 is a new gene specific for renal cell carcinoma , And the total length of the cDNA was estimated to be about 3.5 kb according to the hybridization signal.The establishment of cDNA subtractive library of human renal carcinoma lays the foundation for further screening and cloning of unknown new genes specifically expressed in renal carcinoma.The primary screening of specific expression The new gene fragment provides a basis for further study of gene function and clinical significance.