| 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].,2012,12(10):1847-1852 |
| 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 Establishmentof Its Stable Transfected 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 by using PCR, and then it was 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 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, which indicated that mir-122 lentiviral expression vector was successfully constructed. miR-122 expression in the
lentiviral mir-122 infected cell line increased significantly compared with that in the parent cell line HepG2. Flow cytometry based on
GFP fluorescence filter purification, also showed the infection rate of more than 90%. Inhibited the expression of its target molecules was
confirmed by Western blot. Conclusion: mir-122 lentiviral expression vector was successfully constructed anda stable expression cell
lines was established, laying the foundation for the further study of the role and mechanism of mir-122 in humans. |
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