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目的 :构建云南个旧肺鳞癌YTMLC 90细胞株的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1链。同时 ,根据真核生物mRNA 5′端帽子结构的特点 ,利用SMART核苷酸 (含SfiⅠA酶切位点 )作为cDNA第 1链在mR NA 5′端延伸出去的模板。以此序列为引物 ,利用LD PCR(long distancePCR)合成双链cDNA。双链cDNA经SfiⅠ (ⅠA和ⅠB)酶切和过柱分级分离后 ,克隆入经SfiⅠ酶切的载体λTripIE× 2 ,经体外包装即为cDNA文库。结果 :人肺癌细胞株YTMLC 90的cDNA文库中 ,含有 1.0 1× 10 9pfu/L个重组子 ,重组率为 93.2 %。将该文库扩增后 ,克隆数可达 5 .2 4× 10 12 pfu/L ,插入cDNA的长度为 75 0～ 30 0 0bp。结论 :所构建的cDNA文库具有较好的质量 ,为进一步筛选、克隆肺癌特异性表达基因奠定了基础。
Objective: To construct a cDNA library of YTMLC 90 cell line from Gejiu, Yunnan. Methods: Total RNA was extracted from human lung squamous cell carcinoma cell line YTMLC90 and the first strand of cDNA was synthesized by using modified oligo (dT) primer (containing SfiIB restriction enzyme site). At the same time, SMART nucleotides (including SfiⅠA restriction sites) were used as templates for the extension of the first strand of cDNA at the 5 ’end of mR NA according to the structure of the 5’ end cap of the eukaryotic mRNA. Using this sequence as a primer, double-stranded cDNA was synthesized by using LD PCR (long distance PCR). The double-stranded cDNA was digested with SfiⅠ (ⅠA and ⅠB) and separated by column. The double-stranded cDNA was cloned into vector λTripIE × 2 digested with SfiI and packaged in vitro. RESULTS: The cDNA library of human lung cancer cell line YTMLC 90 contained 1.01 × 10 9 pfu / L recombinants with a recombination rate of 93.2%. After the library was amplified, the number of clones reached 52.4 × 10 12 pfu / L, and the length of inserted cDNA was 75 0 ~ 30 0 0 bp. Conclusion: The constructed cDNA library has good quality and lays the foundation for further screening and cloning of lung cancer-specific genes.