张旭于芳聂勇战唐红卫△ 梁琳琳陈蕊蕊.MIR-122 慢病毒表达载体构建及稳定转染HepG2 细胞系[J].现代生物医学进展英文版,2012,12(16):3008-3011. |
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 StableTransfected HepG2 Cell Line |
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DOI: |
中文关键词: MicroRNA Mir-122 慢病毒表达载体 稳定转染 HepG2 细胞 |
英文关键词: MicroRNA Mir-122 Lentiviral vector Stable transfection HepG2 cells |
基金项目:国家自然科学基金项目(30770959) |
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中文摘要: |
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,western
blot 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。 |
英文摘要: |
Objective: To construct human lentiviral vector of mir-122, and establish a new hepatoma sub cell line of HepG2 stabilized
infected with mir-122 virus. Methods: Primers were designed and synthesized, according to the human has-mir-122 precursor sequence.
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 fluorescence
filter purification, also shows the infection rate of more than 90%. Inhibited the expression of its target molecules was confirmed by
Western blot. |
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