IL28RA真核表达载体的构建和对HaCaT细胞迁移能力的影响

Construction of eukaryotic expression vector of IL28RA gene and its effects on the migration ability of HaCaT cells

目的构建白介素28受体ɑ(IL28RA)基因的真核表达载体,并转染HaCaT细胞,观察该基因对HaCaT细胞迁移能力的影响。方法从HepG2细胞中提取总RNA,逆转录后以其cDNA为模板PCR扩增IL28RA编码片段。将其插入质粒pCMV6构建pCMV6-IL28RA重组表达体;重组体经酶切分析及测序鉴定,将其转染至HaCaT细胞中,经G418筛选出稳定过表达IL28RA细胞株。为检测转染效率,反转录实时荧光定量多聚核苷酸链式反应(RT-qPCR)分析转染细胞中IL28RA mRNA相对表达水平。伤口愈合实验检测过表达IL28RA对HaCaT细胞迁移能力的影响。结果IL28RA基因成功构建并在HaCaT中表达,RT-qPCR检测转染效率显示与对照细胞相比,过表达细胞中IL28RA mRNA相对表达水平显著升高。细胞伤口愈合实验检测显示与对照细胞相比,IL28RA过表达抑制HaCaT细胞迁移能力。结论IL28RA基因过表达降低HaCaT细胞迁移能力。

Objective To construct an eukaryotic expression vector of interleukin 28 receptorα(IL28RA)and then transfect it into HaCaT cells to examine its effect on the migration ability of HaCaT cells.Methods Total RNA was extracted from HepG2 cells and CDS of human IL28RA gene was amplified by PCR with HepG2′s cDNA as the template.The recombinant expression vector of pCMV6-IL28RA was constructed by inserting the CDS of IL28RA into pCMV6 plasmid.After identification by restriction enzyme digestion and sequencing,the recombinant was transfected into HaCaT cells and screened by G418 to get overexpressed cells.To testify the efficacy of transfection,the relative expression mRNA level of IL28RA gene was detected by RT-qPCR.The effect of IL28RA on the migration ability of HaCaT cells was detected by wound healing.Results The eukaryotic expression vector of pCMV6-IL28RA was constructed successfully and expressed in HaCaT cells.The relative expression level of IL28RA in transfected HaCaT cells was significantly higher than the control cells examining by RT-qPCR.Meanwhile,compared with the control cells,overexpression of IL28RA inhibiting the migration ability of HaCaT cells was detected by wound healing assay.Conclusion Overexpression of IL28RA can decrease the migration ability of HaCaT cells.