论文部分内容阅读
背景与目的:认识肾癌差异表达基因有助于阐明肾癌发生、发展的分子机制,但至今有关肾癌差异基因尤其肾癌特异相关基因的研究仍不令人满意。本实验应用抑制消减杂交技术筛选人肾癌组织与正常肾组织间差异表达的新基因,以期克隆出新的肾癌特异相关基因。方法:以肾癌组织mRNA为检测对象(Tester),正常肾组织mRNA为驱赶者(Driver),构建cDNA消减文库。随机挑取文库克隆进行酶切及测序,所得结果在GenBank中做同源性比对分析,对感兴趣的片段进行电子定位确定其在染色体的位置。用Northernblot、半定量RT-PCR方法检测新基因在肾癌组织与正常肾组织中的差异表达。结果:文库包含414个阳性克隆;随机挑取280个克隆提取质粒并酶切分析,其中265个有插入片段;将其中80个克隆进行测序,初步显示28、158、170、249号4个克隆为新基因片段,电子定位表明上述4个基因分别位于染色体21q22、4p15.3、9q34、22q11.2,已在GenBank中登录(BM181083,BI784487,BI863835,BI863386)。对其中28、170号克隆用Northern杂交、半定量RT-PCR检测,证实新基因在肾癌组织中表达较正常肾组织显著增高。结论:抑制消减杂交技术是筛选、克隆肾癌差异表达新基因的有效手段;筛选到的新基因片段为进一步克隆其全长、研究基因功能提供了实验基础。
BACKGROUND & OBJECTIVE: To understand differentially expressed genes in renal cell carcinoma is helpful to elucidate the molecular mechanism of the occurrence and development of renal cell carcinoma. However, the research on differentially expressed genes of renal cell carcinoma, especially those related to kidney cancer, remains unsatisfactory. In this study, we used suppression subtractive hybridization screening of new genes differentially expressed between human renal cell carcinoma and normal kidney tissue in order to clone a new kidney-specific gene. Methods: mRNA of renal cell carcinoma (Tester) and mRNA of normal renal tissue were used as driver to construct cDNA subtractive library. Randomly picked the library cloning for enzyme digestion and sequencing, the results obtained in GenBank homology comparison analysis, the fragment of interest for electron localization to determine its position in the chromosome. The differential expression of the new gene in renal cell carcinoma and normal renal tissue was detected by Northern blot and semi-quantitative RT-PCR. Results: The library contained 414 positive clones. 280 clones were randomly selected for plasmid extraction and digestion analysis. Among them, 265 were inserted and 80 clones were sequenced. The preliminary results showed that there were 4 clones 28,158,170,249 As a new gene fragment, the electronic localization indicated that the above four genes were located on chromosomes 21q22, 4p15.3, 9q34 and 22q11.2, respectively, and were registered in GenBank (BM181083, BI784487, BI863835, BI863386). Among them, 28,170 clones were detected by Northern hybridization and semi-quantitative RT-PCR. The results showed that the expression of new gene in renal cell carcinoma was significantly higher than that in normal kidney. Conclusion: Suppression subtractive hybridization is an effective method for screening and cloning new genes differentially expressed in renal carcinoma. The selected new gene fragments provide experimental basis for further cloning its full length and studying gene function.