人膀胱癌BLZ211细胞cDNA文库的构建分析

Analysis of Construction of cDNA Library from Human Bladder Cancer BLZ211 Cells

目的:构建人膀胱癌BLZ211细胞表达型cDNA文库,为肿瘤抗原的筛选奠定工作基础。方法:提取BLZ211细胞总RNA,分离mRNA,反转录合成双链cDNA,削平cDNA末端,连接EcoRI适配子,磷酸化EcoRI适配子5′端,XhoI酶切,Sephacryl-S400柱除去小于400bpcDNA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7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1.39×106重组子的BLZ211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 cDNA expression library from human bladder cancer BLZ211 cells for the screening of tumor antigen. METHODS： Total RNA in bladder cancer cell line BLZ211 was extracted and the mRNA was isolated, which were synthesized to double strands through reverse transcription. After blunting the cDNA termini, the EcoRI adapters were conjoined, with 5＇ end of EcoRI adapters phosphorylated and then digested by XhoI. cDNA fragments smaller than 400bp were removed by Sephacryl - S400 spin column, the fragments longer than 400bp were conjugated with XZAP expression vector. The cDNA expression library was established after the recombinants had been packaged. Then 1μl multiple proportion dilution of XL1 - Blue - MRF infected with E coli was taken out for the determination of the size of the library and the recombination rates. 7 bacteriophage plaques were randomly selected to release BK - CMV phasmids under the action of ExAssist helper phage. The phasmid infected with E coli XLOLR were spread out on the kanamycin resistant LB flat plate. The clones were picked out and shaken overnight at 37 ℃. Plasmids were excised for the determination of size and diversity of cDNA fragments after being digested by XhoI and EcoRI. RESULTS： The BLZ211 cell line cDNA library consisting of 1.39 × 10^6 recombinant bacteriophages was constructed, with the average length of exogenous insert in the recombinants at about 1.3kb. CONCLUSION： The constructed cDNA library is up to the standard and suitable to be used to screen target cDNA clones.