hPOT1基因正反义真核表达载体的构建及鉴定

目的 构建人端粒保护蛋白（human protection of telomeres，hPOT1）基因的正、反义真核表达载体，以进一步研究hPOT1蛋白的功能及作用机制。方法用限制性内切酶将hPOT1 cDNA从pLPCNMyc POT1质粒上切下来，经适当改造后以正、反方向插入到质粒pcDNA3．1（-）中，构建hPOT1基因的正、反义真核表达载体；用PCR、酶切及DNA测序的方法鉴定hPOTI基因正、反义表达载体序列和方向的正确性。结果PCR、酶谱分析及DNA测序结果表明hPOT1正，反义真核表达载体序列和方向正确。结论成功地构建了hPOT1正、反义真核表达载体，为进一步研究hPOT1在端粒保护，细胞衰老和凋亡中的作用奠定了基础。

Construction and identification of hPOT1 sense and antisense eukaryotic expression vector

Objective To construct eukaryotic expression vector of sense and antisense hPOT1(human protection of telomeres) gene in order to further study on the roles and mechanism of hPOT1.Methods hPOT1 cDNA gene was cleaved from the pLPCNMyc plasmid using proper restriction enzymes, and inserted into plasmid pcDNA3.1(-) in both normal and reverse sequence after proper reformed. PCR,restriction enzyme analysis and sequencing method were used to confirm the orientation and quality of the hPOT1 cDNA in the pcDNA3.1(-) vector respectively.Results PCR,restriction enzyme mapping and sequencing assay performed on DNA from transformed clones demonstrated the presence of the hPOT1 cDNA insert cloned in the sense and antisense orientation in the pCDNA3.1(-) vector.Conclusion Eukaryotic expression vectors of sense and antisense hPOT1 were successfully constructed. This lays good foundation for further study on the roles of hPOT1 in telomere protection, human cell aging and apoptosis.