GPC3过表达载体的构建和表达检测

Construction and detection of GPC3 overexpression vector

目的构建并检测磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)真核表达重组质粒。方法提取肾组织RNA进行逆转录,扩增GPC3基因的编码区,将其克隆到真核表达载体pcDNA3.1中,酶切鉴定并测序;将克隆成功的质粒转染至Huh7细胞,用荧光定量PCR和Western blot检测GPC3的表达水平。结果成功扩增GPC3编码区,并克隆至载体pcDNA3.1中;GPC3过表达载体转染至Huh7细胞后,荧光定量PCR和Western Blotting检测结果显示GPC3mRNA和蛋白表达水平均明显增加。结论成功构建GPC3过表达载体可用于后续研究。

Objective To construct and detect the glypican-3 （GPC3） recombinant eukaryotic expression plasmid. Methods The extracted renal RNA underwent reverse transcription. The coding region of GPC3 gene was amplified and then cloned into the eukaryotic expression vector pcDNA3.1, followed by endonuclease digestion and sequencing. The successful cloned plasmid was transfected into Huh7 cells, and GPC3 expression was determined by fluorescence quantitative PCR and Western blot. Results The coding region of GPC3 gene was successfully amplified, and cloned into the pcDNA3.1. After transfected with GPC3 overexpression vectors, expression of GPC3 mRNA and protein was upregulated in Huh7 cells. Conclusion GPC3 overexpression vector is successfully constructed and can be used in the post-study.