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目的:构建人脆性组氨酸三联体(fragile histidine triad gene,fhit)基因与报告基因egfp的双顺反子表达载体及融合表达载体,研究fhit基因过表达对HeLa细胞生长及凋亡的影响。方法:根据已知fhit基因的mRNA序列,RT-PCR得到493bp的cDNA序列,将其构建到pMD18-T中,筛选目的片段插入正确的重组质粒,经限制性内切酶切割,回收目的片段分别插入双顺反子表达载体pIRES2-EGFP和融合表达载体pEGFP-C1的多克隆位点中。再以脂质体介导转染HeLa细胞,G418筛选稳定转染的克隆,分别通过基因组PCR、RT-PCR、免疫细胞化学及TUNEL法在DNA、RNA和蛋白质水平检测其表达情况。结果:转基因细胞基因组PCR获得493bp片段,证明目的基因已整合到细胞基因组中;RT-PCR检测其在RNA水平得到表达;免疫细胞化学及TUNEL法分析表明fhit基因在HeLa细胞内实现了稳定遗传与表达。结论:实验证明fhit基因过表达可有效促进HeLa细胞的凋亡。
OBJECTIVE: To construct the bicistronic expression vector and the fusion expression vector of fragile histidine triad gene (fhit) and egfp reporter gene to study the effect of fhit gene overexpression on HeLa cell growth and apoptosis. Methods: According to the mRNA sequence of known fhit gene, a 493bp cDNA sequence was obtained by RT-PCR and was cloned into pMD18-T. The target fragment was screened and inserted into the correct recombinant plasmid. After restriction endonuclease digestion, Was inserted into the bicistronic expression vector pIRES2-EGFP and the multi-cloning site of the fusion expression vector pEGFP-C1. The recombinant plasmid was transfected into HeLa cells by lipofectamine. The stable transfected clones were screened by G418, and the expression of them was detected by DNA microarray, RNA and protein level respectively by genomic PCR, RT-PCR, immunocytochemistry and TUNEL. Results: The 493bp fragment was obtained from genomic DNA of transgenic mice and the target gene was integrated into the cell genome. The expression of the target gene was detected at the RNA level by RT-PCR. Immunocytochemistry and TUNEL analysis showed that fhit gene was stable in HeLa cells expression. Conclusion: The experiment proved that fhit gene overexpression can effectively promote HeLa cell apoptosis.