稳定表达人Fhit突变体细胞系的建立

目的:构建人脆性组氨酸三联体(Fhit)突变体真核表达载体并建立稳定表达人Fhit突变体的细胞株,以便进一步研究Fhit与复制蛋白A(RPA)在体内的相互作用。方法:将3种人Fhit突变体cDNA克隆至带有HA标签的真核表达载体pREP10上,构建人Fhit突变体真核表达载体,转染HeLa细胞,经潮霉素B加压筛选阳性克隆,用Western印迹鉴定稳定表达Fhit突变体蛋白FhitA、FhitD和FhitF的阳性细胞株。结果:经PCR鉴定及序列分析,Fhit突变体基因真核表达载体pREP10/FhitA/D/F-HA构建正确,转染人HeLa细胞,筛选出Fhit突变体表达较高的细胞株。结论:建立了3株稳定表达Fhit突变体的细胞株HeLa-FhitA/D/F,为研究Fhit与RPA的相互作用在DNA损伤应答中发挥的作用奠定了基础。

Establishment of Human Fhit Mutant Stable Expressed Cell Lines

Objective： To construct the eukaryotic expression vectors of human fragile histidine triad（Fhit） muta tion genes and to establish stable expressed cell lines of human Fhit mutants in order to confirm Fhit and replica tion protein A（RPA） interaction in mammalian cells. Methods： Three Fhit mutant eDNA were inserted into the eukaryotic expression vector pREP10 containing HAtag, HeLa cells were transfected with pREP10/HA vector alone or the plasmids pREPIO/FhitA/D/FHA, stable cell lines were selected from hygromycin B resistant colonies and the expressed Fhit mutants were verified by Western blot with HA antibody. Results The eukaryotic expres sion vector pREPIO/FhitA/D/FHA was verified by PCR and sequencing, human Fhit mutants were highly ex pressed in the transfected HeLa cells. Conclusion： Three stable cell lines HeLaFhitA/D/F expressing Fhit mutant were established, it is the basis to study the role of the interaction between Fhit and RPA in DNA damage re sponse.