急性白血病多药耐药相关消减cDNA文库的构建与鉴定

Construction and Analysis of Subtractive cDNA Library Associated with Multidrug Resistance of Acute Leukemia

研究目的是构建急性白血病多药耐药相关消减cDNA文库 ,以用于白血病多药耐药 (MDR)相关基因的筛选。采用改良的消减杂交方法建立急性白血病耐药细胞差异表达基因cDNA文库 ,提取HL 6 0 /VCR与HL 6 0细胞mRNA ,将HL 6 0 /VCR细胞mRNA用Cap Finder法反转录成cDNA ,与以常规反转录的HL 6 0细胞cDNA为模板 ,PCR合成生物素标记的扣除探针进行杂交。杂交产物用SephacrylS 4 0 0柱和具有链亲和素作用的磁珠纯化后 ,得到差异表达cDNA。PCR法特异扩增 ,进行T A克隆建成消减cDNA文库 ,并通过反向点杂交鉴定其质量。结果表明 :构建急性白血病多药耐药相关消减cDNA文库所需样本量少且时间短 ,全长或近全长的cDNA分子较多 (约达总数的 2 / 3) ,质量较高。结论 :成功构建急性白血病多药耐药相关消减cDNA文库 ,为筛选白血病耐药相关基因奠定了基础。

The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method,and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing,filtrating through the sephacryl S-400 column,absorbing by the magnetic beads,and amplifying by PCR method,the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction,the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.