K562细胞消减cDNA文库的构建  

Construction of Subtractive cDNA Library of K562 Cells

作　　者：陈汉春[1] 孟巧[1] 王吉伟[1] 

机构地区：[1]中南大学湘雅医学院分子生物学研究中心,湖南长沙410078

出　　处：《中国医师杂志》2003年第1期3-6,共4页Journal of Chinese Physician

基　　金：中华医学基金资助项目 (CMBNo.99- 698)

摘　　要：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K5Objective To construct the suppression subtractive library of hemin-inducing K562 cells for identifying the cDNA genes of globin synthesis regulatory factors expressed in K562 cells induced by hemin. Methods K562 cells were cultured under different concentration of hemin, both the percentage of positive benzidine staining cells and hemoglobin content were measured, the most reasonable concentration of hemin was chosen for inducing incubation of K562 cells. The poly (A) positive RNA (mRNA) was isolated from the hemin-induced K562 cells (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNA molecules were synthesized by reverse transcription. After two times subtractive hybridization followed by two times polymerase chain reaction (PCR) amplification, the forward subtracted PCR products were ligated with pGEM T-Easy vector and the subtracted cDNA library was constructed. The library clones were selected by blue-white screening. The plasmid DNAs of the single positive colony were purified and digested by EcoRⅠ, and the inserts in plasmid were amplified by PCR. Results The maximum of positive benzidine staining cells percentage and hemoglobin content of K562 cells were obtained in 50μmol/L of hemin inducing condition. The suppression subtractive library of hemin-inducing K562 cells was constructed after K562 cells were induced by 50μmol/L of hemin. The library identification showed that the positive clones contain inserts in different length respectively. Conclusions The suppression subtracted cDNA library of hemin-inducing K562 cells were successfully constructed. This subtracted cDNA library combined with bioinformatics analysis can be used to explore structures and functions of hemin-inducing expression genes in K562 cells.

关 键 词：构建 K562细胞 抑制性消减杂交 CDNA文库 氧化血红素 

分 类 号：R346[医药卫生—基础医学]