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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。
The aim of this study was to construct an expression cDNA library of myeloid leukemia U937 cell line, which laid the foundation for the screening of tumor antigens. Total RNA was extracted from U937 cells, mRNA was isolated, double-stranded cDNA was reverse transcribed, and the end of Hyphanapha cDNA was ligated with EcoRI adapter to phosphorylate the 5 ’end of EcoRI adapter. After XhoⅠ digestion, 0 0 column was removed less than 400bpcDNA fragment, and λZAP expression vector was connected, packaging protein packaging to build a cDNA library; remove 1μl multiples of dilutions of infected E. coli XL1 Blue MRF ’, determination of library size, recombination rate; Plaque release of PBK CMV phagemid by ExAssist helper phage; infecting E.coli XLOLR on kanamycin resistant LB plates; picking clones and shaking at 37 ° C overnight; plasmid extraction, After digestion with EcoRI and XhoI, the size and diversity of the fragments were initially determined. Results: The U937 cell cDNA library containing 2.87 × 10 6 recombinants was constructed. The average insert size of the recombinants was about 1.7 kb. Conclusion: The constructed library is qualified, suitable for screening the purpose of cDNA cloning.