| 李婷 王安平 汪保安 哈斯 母义明.shRNA慢病毒载体稳定抑制LRP16 表达的HepG2 细胞的构建及鉴定[J].,2016,16(8):1406-1410 |
| shRNA慢病毒载体稳定抑制LRP16 表达的HepG2 细胞的构建及鉴定 |
| Construction and Identification of Recombinant HepG2 Cells with shRNALentiviral Vector Targeting LRP16 |
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| DOI： |
| 中文关键词: LRP16 短发卡RNA 慢病毒载体 HepG2 细胞 |
| 英文关键词: LRP16 RNA interference Lentiviral vector HepG2 cell |
| 基金项目:国家自然科学基金项目（面上项目）（81471026） |
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| 中文摘要: |
| 目的：构建LRP16基因抑制表达的HepG2稳定细胞系，鉴定其LRP16 基因抑制表达的效果。方法：构建LPP-HSH008357-
LVRH1GP-200短发卡RNA (shRNA) 慢病毒表达载体，用该抑制表达慢病毒感染HepG2 细胞系，通过荧光筛选及药筛，获得
LRP16 基因稳定抑制表达细胞系；最终运用Q-PCR和Western blot 鉴定该稳定细胞系中LRP16 的表达。结果：构建了LRP16 基
因抑制表达及抑制表达对照的HepG2 稳定细胞系，并通过Q-PCR和Western blot 进行了鉴定。Q-PCR 结果显示相对于对照稳转
株（LPP-CSHCTR001-LVRH1GP-100），抑制表达稳转株（LPP-HSH008357-7-LVRH1GP-200）LRP16 基因的抑制效率是4 组抑制表
达细胞中效果最理想的一组，其抑制效率高达98%；Western blot 结果也显示该抑制表达稳转株（LPP-HSH008357-7-
LVRH1GP-200）的LRP16 蛋白表达量明显低于野生型HepG2 细胞及对照稳转株（LPP- CSHCTR001-LVRH1GP-100），其结果与
Q-PCR 一致。结论：构建并鉴定了人LRP16 基因抑制表达HepG2 稳定细胞系及其相应的对照稳转株。 |
| 英文摘要: |
| Objective:To construct a recombinant lentiviral vector of short hairpin RNA (shRNA) targeting LRP16 gene, and to
identify its inhibitory effect in HepG2 cells.Methods:The LRP16 shRNA sequence was designed and inserted into the lentiviral vector
HSH008357-LVRH1GP. The lentiviral vector was further packaged with accessory plasmids into lentivirus in HEK293T cells. After
verification, virus infecting, cell screening, real time PCR and Western blotting, we got recombinant HepG2 cells with shRNA lentiviral
vector targeting LRP16.Results:LRP16 stable knockdown HepG2 cell lines were established. The efficiency of inhibitory effect were
confirmed by Q-PCR and Western blot. Q-PCR results showed that the inhibition efficiency was as high as 98 %which is the most ideal
of 4 groups. Western blot results showed that the LRP16 stable knockout HepG2 cell lines was significantly lower than its control cell
and the wild HepG2 cell, and the results were consistent with Q-PCR.Conclusion:HepG2 cell lines stable expressing LRP 16 shRNA
were successfully established with lentiviral systemand its control cell. |
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