摘要
目的构建云南个旧人肺鳞癌YTMLC-90细胞cDNA文库.方法从培养的人肺鳞癌YTMLC-90细胞株中提取总RNA,利用经修饰的oligo(dT)引物(含SfiⅠB酶切位点)合成cDNA第一链,同时根据真核生物mRNA5'端帽子结构特点,利用SMART寡核苷酸(含SfiⅠ A酶切位点)作为cDNA第一链在mRNA 5'端延伸出去的模板,进而以此序列为引物,利用LD-PCR(long-distance PCR)合成双链cDNA,双链cDNA经Sfi Ⅰ(Ⅰ A和Ⅰ B)酶切和过柱分级分离后,克隆入经Sfi Ⅰ酶切的载体后经体外包装而成cDNA文库.结果人肺癌细胞YTMLC-90的cDNA文库获得1.01×106 pfu/ml个重组子,重组率为93.2%,文库扩增后,滴度达5.24×109pfu/ml,插入cDNA的长度为7.50～3000bp.结论所构建的cDNA文库具有较好的质量,该文库为进一步筛选、克隆肺癌特异表达基因莫定了基础.
Objective To construct Yunnan Gejiu human lung cancer cell YTMLC -90 cDNA library. Methods The total RNA was separated from cultured human lung cancer cell and the frist - strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained Sfi ⅠB site) while the SMART oligonucleotide (contained Sfi ⅠA site) was utilized as a template so that the frist - strand cDNA could be extended over the 5' end of mRNA.'nle double - strand cDNA was amplified by LD - PCR (long- distance PCR) with the above two primers and then digested by Sfi Ⅰ(ⅠA& ⅠB) restriction enzyme. After cDNA size fractionation through CHROMA SPIN column, the double - strand cDNA was ligated into the SfiⅠdigedted λTripTE×2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human lung cancer cell cDNA library consists of 1.01×106 pfu/ml independent clones in which the percentage of recombinant clones is about 93.2%.The ther of the amplified cDNA Ubrary is 5.24×109 pfu/ml and the exogenous inserts of the recombinants is 750-3000bp. Conclusions The human lung cancer cell cDNA Ubrary has an excellent quality and lays solid foundation for screening and cloning new tissue-specific genes of human lung cancer.
出处
《医学研究通讯》
2004年第1期14-16,共3页
Bulletin of Medical Research
基金
国家十五科技攻关项目资助(No.2001BA703B11)
