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目的构建正常人支气管上皮细胞(16HBE)与氯化镉诱导转化16HBE细胞间差异表达基因的消减cDNA文库。方法以16HBE为驱动子,氯化镉诱导转化16HBE细胞为检测子,应用抑制性消减杂交(SSH)方法构建cDNA文库,经2次消减杂交和2次PCR后,将巢式PCR产物插入载体,随机挑选克隆进行鉴定。结果得到纯度高及完整性好的总RNA和mRNA,并扩增出良好的双链cDNA,cDNA与接头的连接效率>25%,最终使差异表达基因得到富集;经蓝白菌落筛选,获得1 200余个白色阳性克隆;随机挑选50个白色克隆进行PCR扩增,显示96%克隆均有100～600 bp的插入片段,这些片段可能是差异表达基因cDNA片段,提示用SSH法及T/A克隆技术有效构建了两细胞株间差异表达基因的消减cDNA文库。结论成功创建正常16HBE细胞与镉转化16HBE细胞差异表达基因消减cDNA文库。
Objective To construct a subtractive cDNA library of normal human bronchial epithelial cells (16HBE) and cadmium chloride (CdCl2) -induced 16HBE differentially expressed genes. Methods 16HBE was used as a driver and cadmium chloride was used to induce 16HBE cells as the detector. The cDNA library was constructed by suppression subtractive hybridization (SSH). After two subtractive hybridizations and two PCRs, the nested PCR product was inserted into the vector, Random selection of clones for identification. As a result, the total RNA and mRNA with high purity and good integrity were obtained, and a good double-stranded cDNA was amplified. The efficiency of connection between cDNA and linker was> 25%, finally the differentially expressed genes were enriched. More than 1 200 positive clones were randomly selected. Fifty white clones were randomly selected for PCR amplification, which showed that 96% of the clones had 100-600 bp inserts. These fragments may be cDNA fragments of differentially expressed genes, suggesting that SSH and T / A cloning technology effectively constructed a subtractive cDNA library of differentially expressed genes between two cell lines. Conclusion The subtracted cDNA library of differentially expressed genes in 16HBE cells and normal 16HBE cells was successfully established.