人肺癌细胞NHE-1基因片段的克隆及其反义表达载体的构建

Cloning of Na^+/H ^+ exchanger-1 gene fragment from human lung cancer cells and construction of its antisense expression vector

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

Objective To construct an antisense expression vector of Na +/H + exchanger 1 (NHE 1) of human lung cancer cell in order to found a basis for tumor gene therapy by inhibiting the expression of NHE 1 gene in vivo. Methods Partial sequence of the exon of NHE 1 gene was cloned with the length of 454bp from genomic DNA of A549 cells with PCR method. Bam HI and Eco RI cleaving sites were added to 5' ends of the upstream and downstream primers respectively. The cloned fragment was reversely inserted into the multiclone site of plasmid reverse transcription virus vector pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophresis and DNA sequencing. Results The cloned fragment was in the length of 461bp and successfully bound to pLXSN. It also proved to be the objective one by DNA sequencing. Conclusion The objective fragment was cloned and reversely inserted into pLXSN in our study. The antisense vector of NHE 1, namely, pNHE 1 was constructed successfully.