人NIRF基因真核表达质粒的构建及其在HepG2.2.15细胞中的表达

Construction of Eukaryotic Expression Vector for Human NIRF Gene and Its Expression in HepG2.2.15 Cells

目的构建人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达NIRF蛋白。方法从HeLa细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增NIRF基因编码区全长序列,插入到pIRES2-EGFP真核表达载体中,构建重组真核表达质粒pIRES2-EGFP-NIRF,转染HepG2.2.15细胞,检测细胞中NIRF基因mRNA的转录水平及蛋白的表达水平。结果重组真核表达质粒pIRES2-EGFP-NIRF经双酶切及测序鉴定证明构建正确,转染HepG2.2.15细胞后,可检测到细胞中NIRF基因mRNA的转录及蛋白的表达。结论已成功构建了人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达了NIRF蛋白,为下一步研究其在肿瘤组织中的功能奠定了基础。

Objective To construct a eukaryotic expression vector for human NIRF gene and express NIRF protein in HepG2.2.15 cells. Methods Total RNA was extracted from HeLa cells, from which the full-length sequence at encoding region of NIRF gene was amplified by RT-PCR using designed specific primers, and inserted into eukaryotic expression vector plRES2-EGFP. The constructed recombinant plasmid pIRES2-EGFP-NIRF was transfected to HepG2.2.15 cells, and the transcription of NIRF mRNA and expression of NIRF protein were determined. Results Both restriction analysis and sequencing proved that recombinant plasmid pIRES2-EGFP-NIRF was constructed correctly, and both transcription of NIRF mRNA and expression of NIRF protein were proved in transfected HepG2.2.15 cells. Conclusion The eukaryotic expression vector for human NIRF gene was successfully constructed, and NIRF protein was expressed in HepG2.2.15 cells, which laid a foundation of study on function of NIRF protein in tumor tissue.