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目的 构建人肺癌细胞Na+/H+交换泵 1(Na+/H+exchanger 1,NHE 1)基因的反义表达载体 ,为将来能在体内应用抑制NHE 1基因表达的肿瘤治疗奠定基础。方法 采用PCR技术从人肺癌A5 49细胞基因组中克隆出长为 45 4bp的NHE 1基因外显子部分序列 ,在上、下游引物的 5’ 端带上BamHⅠ和EcoRⅠ酶切位点。然后将该片段反向插入逆转录病毒载体pLXSN的多克隆位点。最后对产生的重组子进行琼脂糖凝胶电泳和测序鉴定。结果 经琼脂糖凝胶电泳鉴定 ,所克隆的目的片段大约为 46 7bp ,并成功地连接到pLXSN载体上 ,而DNA测序证实克隆并插入到载体中的片段为目的片段。结论 本实验已成功地克隆了NHE 1的目的片段 ,并将其反向插入到pLXSN中 ,构建了NHE 1反义表达载体pNHE 1
Objective To construct an antisense expression vector of Na + / H + exchanger 1 (NHE1) gene in human lung cancer cells and lay a foundation for the future treatment of tumors that inhibit the expression of NHE1 gene in vivo. Methods The 45 bp sequence of NHE 1 gene was cloned from human lung adenocarcinoma A549 cells by PCR. The upstream and downstream primers were cloned into BamH Ⅰ and EcoR Ⅰ restriction sites. The fragment was then reverse inserted into the multiple cloning site of the retroviral vector pLXSN. Finally, the resulting recombinants were subjected to agarose gel electrophoresis and sequencing. The result was identified by agarose gel electrophoresis. The cloned target fragment was about 46 7 bp, and was successfully ligated into the pLXSN vector. DNA sequencing confirmed that the fragment cloned and inserted into the vector was the target fragment. Conclusions The target fragment of NHE 1 has been successfully cloned and inserted into pLXSN in the reverse direction. The constructed expression vector NHE 1 antisense pNHE 1