K562细胞cDNA表达文库的构建和鉴定

Construction and characterization of K562 cell cDNA bank

目的 构建K5 6 2细胞表达型cDNA文库。方法 提取K5 6 2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 ,Sephacryl S4 0 0柱除去小于 4 0 0bpcDNA片段 ,与去磷酸化的λgt11噬菌体连接 ,体外包装后建成cDNA文库。取出 1μL倍比稀释感染E .coliY10 90 ,测定文库大小、重组率 ,并以PCR鉴定cDNA插入片段的大小。结果 构建成含 1.0 2× 10 6重组子的K5 6 2细胞cDNA文库 ,重组子平均插入外源片段长约 1.3kb。结论 所建文库合格 ,适合用于筛选目的cDNA克隆。

Objective To construct a human K562 cell cDNA expression bank. Methods Total RNA and purified mRNA were extracted from leukemia cell line K562. First and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with Eco R I adapters, and the end of Eco R I adapters was phosphorylated. The cDNA smaller than 400 bp were removed by Sephacryl S400 spin column, and the remaining were ligated with the dephosphorylated arms of λgt11. The recombinants were packaged in vitro , and a small portion of packaged phage was used to infect E.coli Y1090 for titration. Results The cDNA bank consisting of 1.02×10 6 recombinant bacteriophages was constructed. The average exogenous insertion of the recombinants was about 1.3?kb. Conclusion The constructed cDNA bank can be used to screen target clones.