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目的 构建氯化血红素诱导性K5 62细胞抑制消减cDNA文库 ,以期鉴定出K5 62细胞中氯化血红素诱导性表达的珠蛋白合成调节因子cDNA基因。方法 采用不同浓度的氯化血红素诱导培养K5 62细胞 ,经联苯胺阳性细胞百分率及血红蛋白浓度分析 ,选择其最佳诱导浓度。分别提取氯化血红素诱导培养的K5 62细胞 (tester ,检测子 )和未加诱导剂培养的K5 62细胞 (driver ,驱赶子 )mRNA ,逆转录合成双链cDNA。经两轮消减杂交 ,两轮PCR扩增后 ,正向消减的PCR产物与T载体连接 ,构建K5 62细胞抑制消减cDNA文库 ,文库经蓝 -白筛选后 ,纯化阳性克隆质粒 ,EcoRⅠ酶切及PCR扩增插入片段。结果 以 5 0 μmol/L氯化血红素诱导K5 62细胞 ,其联苯胺阳性细胞百分率及血红蛋白浓度均达到最大值 ,故选择 5 0 μmol/L氯化血红素诱导培养K5 62细胞并构建K5 62细胞抑制消减cDNA文库。文库鉴定证实其阳性克隆中分别含有不同长度的插入片段。结论 成功构建了氯化血红素诱导性K5 62细胞抑制消减cDNA文库 ,该消减cDNA文库结合生物信息学 (Bioinformatics)分析可用于深入研究K5 62细胞内氯化血红素诱导性表达基因的结构和功能
OBJECTIVE: To construct the hemin-induced inhibitory subtractive cDNA library of K562 cells in order to identify the heme-inducible cDNA of globin synthesis regulator in K562 cells. Methods K562 cells were induced by using different concentrations of hemin. The optimal concentrations of benzidine-positive cells and hemoglobin were determined. The mRNA of K562 cells (tester, detector) induced by hemin and K562 cells cultured without inducing agent were extracted and double-stranded cDNA was reverse transcribed. After two rounds of subtractive hybridization, two PCR amplification products were ligated with T vector to construct K562 cell subtractive cDNA library. After blue-white screening, the positive clones were purified and digested with EcoRⅠ PCR amplification of insert. Results K562 cells were induced with 50 μmol / L hemin to maximize the benzidine-positive cells and hemoglobin concentration. K562 cells were induced by 50 μmol / L hemin and K562 cells were constructed Cell Suppression subtractive cDNA library. Library identification confirmed that the positive clones contained inserts of different lengths respectively. Conclusion The hemin-inducible K562 cell subtractive cDNA library was successfully constructed. The subtracted cDNA library combined with bioinformatics analysis can be used to further study the structure and function of hemin-induced genes in K562 cells