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目的构建人多梳家族环指蛋白1(PCGF1)基因的慢病毒载体,建立稳定表达PCGF1基因的细胞系。方法根据人PCGF1序列设计并合成引物,以A549细胞c DNA为模板,PCR扩增目的基因。双酶切目的基因并插入p LVXIRES-puro质粒,对重组质粒进行慢病毒包装,用包装好的病毒感染A549细胞系,通过嘌呤霉素筛选阳性表达细胞,Western blotting验证PCGF1表达情况。结果重组质粒经测序分析正确,瞬时转染293T细胞后,Western blotting验证PCGF1表达明显升高。包装慢病毒颗粒后感染A549细胞,经嘌呤霉素筛选后,Western blotting验证得到PCGF1稳定过表达的A549细胞系。结论成功构建了p LVX-IRES-PCGF1-puro慢病毒过表达载体和PCGF1稳定过表达的A549细胞系,为进一步研究PCGF1的功能奠定了基础。
Objective To construct a lentiviral vector containing human PCDNA1 gene and establish a cell line stably expressing PCGF1 gene. Methods According to the sequence of human PCGF1, primers were designed and synthesized. The target gene was amplified by PCR using A549 cell c DNA as a template. The target gene was double-digested and inserted into p LVXIRES-puro plasmid. The recombinant plasmids were packaged in lentivirus. A549 cells were infected with the packaged virus. The positive cells were screened by puromycin. The expression of PCGF1 was confirmed by Western blotting. Results The recombinant plasmid was correctly sequenced. After transient transfection of 293T cells, the expression of PCGF1 was significantly increased by Western blotting. A549 cells were infected after the lentivirus particles were packaged. After puromycin selection, the A549 cell line stably overexpressed by PCGF1 was confirmed by Western blotting. Conclusion The overexpression vector p LVX-IRES-PCGF1-puro and the A549 cell line stably overexpressed by PCGF1 were successfully constructed, which laid the foundation for the further study on the function of PCGF1.