【摘要】 目的利用抑制消减杂交技术(SuppressionSubtractiveHybridization,SSH)构建人大肠癌差异表达cDNA文库,分离并克隆出大肠癌发病的相关新基因。方法运用抑制消减杂交技术分离大肠癌组织及癌旁正常黏膜差异表达基因的cDNA片断,将其与T载体连接构建文库,从库中随机挑取200个白色菌落,使用PCR方法鉴定阳性克隆并对其中50个克隆进行测序,将测序结果登录GenBank进行同源性对比分析,并对部分有意义的差异表达片断进行VirtualNorthernBlot分析。结果成功构建人源性大肠癌消减cDNA文库,通过筛选获得20个差异表达的基因,其中有2个片断未检测到同源性序列,根据杂交结果考虑可能是在大肠肿瘤中差异表达的新基因。结论运用SSH技术成功构建了人大肠癌的差异表达的cDNA消减杂交文库,并筛选出2个在大肠肿瘤中差异表达的新基因。更多还原

【Abstract】 Aims To construct cDNA libraries of colorectal carcinoma, and screen and clone the new genes related to the colorectal carcinoma. Methods cDNA fragments of differently expressed genes in colorectal carcinoma and normal intestinal tissues were isolated with suppression subtractive hybridization (SSH). Then these cDNA fragments were directly inserted into T vector to set up the subtractive library. Two hundreds of positive bacteria clones were randomly picked and identified by colony PCR method. Then 50 differently expressed genes were sequenced and analyzed with BLAST program in GenBank wired NCBI website. Some genes were identified by Virtual Northern Blot. Results cDNA library of colorectal carcinoma and normal intestine tissue was successfully constructed, and 20 differently expressed genes were obtained after filtration. Two new genes did not match the identical sequence, and they should be new genes related to the colorectal cancer. Conclusions cDNA library of colorectal carcinoma and normal intestine tissue was constructed by SSH, and 2 new genes related to the colorectal cancer were found.更多还原