食管癌消减cDNA文库的构建

目的 :采用消减抑制杂交技术 ,研究正常食管粘膜和食管癌组织之间的基因表达差异状况 ,分离食管癌相关基因片段 ,构建食管癌的消减cDNA文库。方法 :以食管癌癌组织为测试子 (tester) ,以正常食管粘膜组织为驱赶子 (driver) ,用消减抑制杂交方法分离食管癌差异表达片段。将该差异表达片段克隆至T载体 ,经蓝白斑筛选后 ,再用PCR方法插入片段筛选出阳性重组质粒 ,构建食管癌消减cDNA文库。结果 :用消减抑制杂交技术分离食管癌差异表达片段 ,经蓝白斑筛选 ,得到 2 2 0个白色克隆 ;再用PCR方法快速筛选出 10 0个两端分别含连接子Adaptor1及Adaptor2R的阳性重组质粒 10 0个 ,从而成功地构建了中国人食管癌特异的消减cDNA文库。结论 :构建了中国人食管癌特异的消减cDNA文库。消减抑制杂交技术能快速有效地分离差异表达基因 ,方法简便、灵敏度高。使用PCR法快速筛选阳性克隆 ,对于消减文库的构建具有重要意义

Construction of subtractive cDNA library of esophageal cancer by using suppression subtractive hybridization

Aim: To construct subtractive cDNA library of esophageal cancer by using suppression subtractive hybridization(SSH). Method: SSH was used to obtain differentially expressed genes associated with esophageal cancer, comparing esophageal cancer tissues (as tester) with normal esophagus tissues(as driver).Those genes were ligated with T easy vector,transformed into E.coli JM109, screened through the blue white screening system,and then positive recombinant clones were confirmed by PCR method. Results: Through the blue white screening system, 220 white clones were picked out and 100 positive recombinants from 220 white clones were screened rapidly by PCR for successful construction of its subtracted cDNA library specific for Chinese esophageal cancer. Conclusion :In this study, a subtractive cDNA library specific for Chinese esophageal cancer has been constructed. Suppression subtractive hybridization is a simple and efficient method for isolating differentially expressed genes associated with esophageal cancer. Rapid screening positive recombinant by using PCR is of importance in the screening of subtractive cDNA library.