正常细胞与镉转化细胞抑制消减cDNA文库构建

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。

Construction of suppression subtractive cDNA libraries of normal and cadmium-transformed cells

Objective To construct a subtractive cDNA library of differentially expressed genes in normal and cadmium-transformed cells.Methods The human bronchial epithelial cells(16HBE cells) transformed by cadmium chloride was used as a tester,and the normal 16HBE cells as a driver,to construct a cDNA library using suppression subtractive hybridization(SSH).The products were inserted into TA vector after two substractive hybridization and nested PCR.The clones were picked up randomly and analyzed with PCR.Results The total RNA and mRNA of purity and good integrity were extracted,and the duble-stranded cDNA was well produced.The link efficiency of cDNA and connector was greater than 25%.And finally the differentially expressed genes were enriched with substractive hybridization and nested PCR.After blue-white screening,the amplified library contained more than 1 200 white positive clones,and fifty of them were selected randomly and analyzed with PCR.Totally 96% of the clones had the inserted 100-600 bp segment and the segments might be the gene cDNA segments with differential gene expressions.Conclusion The subtractive cDNA library of differential genes in transformed 16HBE cells induced by cadmium was successfully established.