HMy2细胞cDNA表达文库的构建和鉴定

Construction and characterization of human myeloma cell cDNA library

目的 :构建人骨髓瘤细胞株HMy2cDNA表达文库 .方法 :提取HMy2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 ,与去磷酸化的噬菌体λgt11连接 ,体外包装后建成cDNA文库 .取出一部分倍比稀释感染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 1.5× 10 6重组子的人骨髓瘤细胞cDNA文库 ,重组子平均插入外源片段长约 1.4kb .结论 :所建文库合格 ,适合用于筛选目的cDNA克隆 .

AIM: To construct human myeloma cell cDNA expression library. METHODS: Total RNA were extracted from human myeloma cell line HMy2 and mRNA were purified with Oligotex mRNA kit. 1 st and 2 nd strands of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with Eco RI adapters, and the end of Eco RI adapters was phosphorylated. The cDNA smaller than 400 bp were removed by Sephacryl S400 spin column,the remaining fragments were ligated with the dephophrylated arms of λ gt11. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli Y1090 for titration. RESULTS: The HMy2 cell line cDNA library consisting of 1.5 ×10 6 recombinant bacteriophages was constructed for the first time. The average exogenous insert of the recombinants was about 1.4 kb. CONCLUSION: The constructed cDNA library can be used to screen target clones.