论文部分内容阅读
目的 构建肝癌及癌旁肝组织差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交方法 ,以肝癌组织及癌旁肝组织作为对比材料 ,分离肝癌组织中差异表达基因的cDNA片段 ,将其与T载体进行T/A连接构建文库 ,将连接产物用电穿孔法转化大肠杆菌进行文库扩增后 ,随机挑取 1 0 0个白色克隆用菌落PCR进行鉴定。结果 扩增消减cDNA文库获得 30 0 0余个白色阳性克隆 ,随机挑取 1 0 0个白色克隆用PCR进行扩增 ,95%的克隆中均有 1 0 0～ 6 0 0bp的插入片段 ,这些片段可能是肝癌差异表达基因的cDNA片段。结论 用SSH法及T/A克隆技术成功构建了肝癌与癌旁肝组织差异表达基因消减cDNA文库 ,该消减cDNA文库的建立为进一步筛选、克隆肝癌差异表达的新基因奠定了基础。
Objective To construct a subtracted cDNA library for differentially expressed genes in hepatocellular carcinoma and adjacent liver tissues. Methods The newly established suppression subtractive hybridization method was used to compare the cDNA fragments of differentially expressed genes in hepatocellular carcinoma with hepatocellular carcinoma and paraneoplastic liver tissues. The cDNA fragments were then T/A-ligated with T-vector to construct a library and used as a linker. Electroporation was used to transform E. coli into library and randomly selected 100 white clones were identified by colony PCR. Results Amplified cDNA library was amplified and more than 300 white positive clones were obtained. 100 white clones were randomly picked and amplified by PCR. There were 100 to 600 bp inserts in 95% of the clones. The fragment may be a cDNA fragment of a differentially expressed gene in liver cancer. Conclusion The differentially expressed cDNA library of hepatocellular carcinoma and paracancerous liver tissue was successfully constructed using SSH and T/A cloning technology. The establishment of the subtracted cDNA library laid the foundation for the further screening and cloning of new differentially expressed genes in liver cancer.