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学科主题: 泌尿外科学
题名:
肾癌差异表达基因的克隆及意义
其他题名: Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma
作者: 张强1; 辛殿旗1; 那彦群1; 郭应禄1; 张志文1
关键词: 肾肿瘤 ; ; 抑制性消减杂交 ; 文库 ; 基因 ; 克隆 ; kidney neoplasms ; carcinoma ; suppression subtractive hybridization ; library ; gene ; clone
刊名: 中华医学杂志(英文版)
发表日期: 2001
DOI: 10.3760/j.issn:0366-6999.2001.08.006
卷: 114, 期:8, 页:807-812
文章类型: Journal Article
摘要: 目的克隆并鉴定肾癌与正常肾之间差异表达的基因,为研究肾癌发生发展的分子生物学机制奠定基础。 方法应用抑制性消减杂交技术(suppression subtractive hybridization, SSH),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差异表达的基因。 结果构建成功高消减效率的人肾癌组织cDNA消减文库,对其中10个克隆插入的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 blotting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。应用SMART RACE技术获得RCC18基因的全长。 结论人肾癌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。初步筛选出的新基因为研究肾癌发生发展中的分子生物学机制提供了重要线索。 Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization. Methods Polyadenylated RNA [Poly (A)+ RNA] was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes. Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18. Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.
语种: 中文
原文出处: 查看原文
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内容类型: 期刊论文
版本: 出版稿
URI标识: http://ir.bjmu.edu.cn/handle/400002259/45631
Appears in Collections:北京大学第一临床医学院_泌尿外科_期刊论文

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作者单位: 1.北京大学第一医院泌尿外科研究所北京 100034
2.北京大学生理系北京 100083

Recommended Citation:
张强,辛殿旗,那彦群,等. 肾癌差异表达基因的克隆及意义[J]. 中华医学杂志(英文版),2001,114(8):807-812.
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