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目的探讨LRP15基因在K562细胞中对紫外线诱导的DNA损伤的修复作用,以进一步研究该基因的功能。方法利用真核表达质粒pcDNA3.1构建LRP15基因开放阅读框架(ORF)序列的正义及反义重组质粒;两种质粒经阳性脂质体介导法分别转染K562细胞,G418筛选阳性克隆;采用单细胞凝胶电泳(SCGE)技术观察两组细胞对紫外线诱导的DNA损伤的修复作用的差异。结果PCR鉴定证实,正义及反义重组质粒中LRP15基因ORF序列均按预定方向正确插入;SCGE实验表明,经紫外线照射后两组细胞的DNA基础损伤无明显差异(P=0.156)。但经45及90min修复后实验组细胞的DNA迁移长度明显小于对照细胞(P<0.001)。结论成功构建LRP15基因的正义及反义真核表达质粒;LRP15基因对紫外线诱导的DNA损伤具有修复作用,可能是一个DNA修复基因。
Objective To investigate the repair effect of LRP15 gene on UV-induced DNA damage in K562 cells to further investigate its function. Methods Eukaryotic expression plasmid pcDNA3.1 was used to construct sense and antisense recombinant plasmids of LRP15 open reading frame (ORF). Two plasmids were transfected into K562 cells by positive liposome-mediated method and positive clones were screened by G418. Single cell gel electrophoresis (SCGE) technique was used to observe the difference between the two groups of cells in UV-induced DNA damage repair. Results PCR identification confirmed that the ORF sequences of LRP15 gene in sense and antisense plasmids were correctly inserted in a predetermined direction. SCGE showed that there was no significant difference in DNA damage between the two groups (P = 0.156) after UV irradiation. However, after 45 and 90 min of repair, the DNA migration length of the experimental group was significantly smaller than that of the control cells (P <0.001). Conclusion The sense and antisense eukaryotic expression plasmids of LRP15 gene were constructed successfully. The LRP15 gene could repair DNA damage induced by ultraviolet light and may be a DNA repair gene.