人肺癌细胞NHE-1基因部分正调控序列的克隆

Cloning and identification of partial positive regulatory sequence of Na^+/H^+ exchanger-1 gene from human lung cancer cells

目的 克隆人肺癌细胞Na+ /H+ 交换泵 1(NHE 1)基因上游正调控序列部分 ,为进一步将该片段导入肺癌细胞株 ,竞争性结合转录因子 ,达到抑制细胞内NHE 1基因的表达奠定基础。方法 采用PCR技术从人肺癌A5 49细胞基因组中扩增长约170bp的NHE 1基因调控序列中起正调控作用的片段。在上、下游引物的 5’ 端带上BamHⅠ和EcoRⅠ酶切位点。然后将该片段连接到pUC18载体上。最后对产生的重组子进行酶切、PCR和测序鉴定。结果 经酶切及PCR鉴定 ,所克隆的目的片段大约为180bp ,而DNA测序证实克隆并插入到载体中的片段为目的片段 ,长度为 168bp ,与报道的序列比较缺失 2个t。结论 本实验已成功地克隆了人肺癌细胞NHE 1基因调控序列中正调控序列片段。

Objective To clone the partial positive regulatory fragment of Na +/H + exchanger 1 (NHE 1) gene from human lung cancer cells. Methods After BamH Ⅰ and EcoR Ⅰ cut sites were added to the 5' ends of the upstream and downstream primers respectively, the partial positive regulatory sequence of NHE 1 gene was cloned with the length of 170 bp from genomic DNA of lung cancer cell line A549 cells with PCR method. The cloned fragment was ligated to plasmid pUC18. Finally, the constructed recombinant was identified with enzyme cut, PCR and DNA sequencing. Results The cloned fragment was about 170 bp in size and successfully ligated to pUC18 with identifiation of double enzyme cut and PCR. DNA sequencing approved that the fragment cloned was objective one with 168 bp in length. Compared with the reported sequence, two t were lost. Conclusion The positive regulatory fragment of NHE 1 gene from human lung cancer cells was successfully cloned.