MIR-122慢病毒表达载体构建及稳定转染HepG2细胞系  

Construction of Lentiviral Vector of MIR-122 and Establishment of its Stable Transfected HepG2 Cell Line

作　　者：张旭[1] 于芳[1] 聂勇战[1] 唐红卫[1] 梁琳琳[1] 陈蕊蕊[1] 

机构地区：[1]第四军医大学西京消化病医院肿瘤生物学国家重点实验室,陕西西安710032

出　　处：《现代生物医学进展》2012年第16期3008-3011,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30770959)

摘　　要：目的:构建人mir-122慢病毒表达载体,感染肝癌细胞HepG2,建立稳定表达mir-122的HepG2细胞系。方法:以人has-mir-122成熟序列,设计并合成引物,采用PCR的方法扩增目的基因,并连接到慢病毒表达质粒pGCSIL-GFP(含绿色荧光蛋白GFP基因)中。对重组质粒进行双酶切鉴定后,进行mir-122基因慢病毒(pGCSIL-GFP-miR-122)的包装及病毒滴度测定,用构建好的慢病毒表达载体感染HepG2细胞,qPCR检测感染后细胞中MIR-122的变化。通过流式细胞仪检测荧光蛋白GFP,westernblot检测mir-122靶分子CAT-1,验证pGCSIL-GFP-miR-122在HepG2细胞中的表达效果。结果:pGCSIL-GFP-miR-122经双酶切分析及测序,插入序列正确。qPCR检测显示转入病毒后mir-122在细胞中的表达显著提高。表明mir-122慢病毒表达载体构建成功。流式细胞仪根据GFP荧光筛选纯化感染后细胞,感染率达90%以上。Western blot显示mir-122明显抑制其靶分子表达。进一步验证pGCSIL-GFP-miR-122在细胞中的稳定表达。结论:成功构建mir-122慢病毒表达载体,并建立稳定表达的细胞系,为研究mir-122在人体所起的作用及功能机制打下基础。Objective： To construct human lentiviral vector of mir-122,and establish a new hepatoma sub cell line of HepG2 stabi-lized infected with mir-122 virus.Methods： Primers were designed and synthesized,according to the human has-mir-122 precursor se-quence.MiR-122 was amplified using PCR,and connected to the lentiviral expression plasmid pGCSIL-GFP.Lentiviral vector of mir-122（pGCSIL-GFP-miR-122） was constructed.After infection of HepG2 cells with the miR-122 lentiviral vector,miR-112 expression was confirmed by qPCR and fluorescent protein GFP expression using FACS.Expression of one of mir-122 targeted molecules CAT-1 was validated by Western blot in pGCSIL-GFP-miR-122 HepG2 cells.Results： PGCSIL-GFP-of miR-122 was confirmed by sequencing analysis,indicating that Mir-122 lentiviral expression vector was successfully constructed.miR-122 expression in the lentiviral mir-122 infected cell line was significantly increased comparing to the parent cell line HepG2.Conclusion： Flow cytometry based on GFP fluores-cence filter purification,also shows the infection rate of more than 90%.Inhibited the expression of its target molecules was confirmed by Western blot.

关 键 词：MicroRNA MIR-122 慢病毒表达载体 稳定转染 HEPG2细胞 

分 类 号：R351[医药卫生—基础医学]