人髓性白血病细胞系U937定向克隆cDNA表达文库的构建及其意义  

Construction and Significance of Directional Expression cDNA Library from Myeloid Leukemia Cell Line U937

作　　者：陈刚[1] 张王刚[1] 付杰[2] 曹星梅[1] 赵万红[1] 赵爱志[3] 韩月恒[3] 李福洋[3] 刘新平[3] 药立波[3] 

机构地区：[1]西安交通大学第二医院血液内科,西安710004 [2]西安交通大学第二医院感染科,西安710004 [3]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国实验血液学杂志》2003年第4期355-358,共4页Journal of Experimental Hematology

基　　金：卫生部部属(管)医疗机构临床学科重点项目基金资助项目编号 2 0 0 12 13 1

摘　　要：本研究的目的是构建髓性白血病U937细胞系表达型cDNA文库 ,为肿瘤抗原的筛选奠定工作基础。提取U937细胞总RNA ,分离mRNA ,反转录合成双链cDNA ,消平cDNA末端 ,连接EcoRⅠ适配子 ,磷酸化EcoRⅠ适配子 5′端 ;XhoⅠ酶切后 ,丙烯葡聚糖凝胶 S4 0 0柱除去小于 4 0 0bpcDNA片段 ,与λZAP表达载体连接 ,包装蛋白包装后建成cDNA文库 ;取出 1μl倍比稀释感染大肠杆菌XL1 Blue MRF′ ,测定文库大小、重组率 ;随机挑取 10个噬斑 ,在ExAssist辅助性噬菌体的作用下 ,释放出PBK CMV噬菌粒 ;感染大肠杆菌XLOLR ,铺于卡那霉素抗性的LB平板 ;挑取克隆 ,37℃震摇过夜 ;抽提质粒 ,经EcoRI和XhoI酶切后 ,初步确定片段的大小及多样性。结果 :构建成含 2 .87× 10 6重组子的U937细胞cDNA文库 ,重组子平均插入外源片段长约 1.7kb。结论 :所建文库合格 ,适合用于筛选目的cDNA克隆。To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl S400 spin column, the fragments longer than 400 bp were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1 Blue MRF′ for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87×10 6 recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.

关 键 词：U937细胞系 髓性白血病细胞系 CDNA表达文库 

分 类 号：Q78[生物学—分子生物学] R733.7[医药卫生—肿瘤]