人HNF4α基因真核表达载体的构建与鉴定*

doi:10.3969/j.issn.1672-5069.2016.02.008

摘要/Abstract

摘要： 目的体外构建人肝细胞核因子4α（HNF4α）基因真核表达载体,并初步鉴定其在HepG2细胞的表达。方法从肝癌手术患者获得肝组织,分离得到肝组织总RNA,将其逆转录合成cDNA,经特异性引物扩增HNF4α基因片段,将其定向连接到pcDNA3.1(+）真核表达载体,经抗生素筛选后,酶切及测序鉴定其序列正确。提取质粒,转染HepG2细胞,48 h后裂解细胞,应用抗HNF4α行Western blot检测目的蛋白。结果从临床肝癌患者肝组织中成功分离得到总RNA,扩增出HNF4α基因,经筛选、酶切鉴定和测序,确认pcDNA3.1-4α真核表达载体构建成功。在转染HepG2细胞48 h后,检测显示53 kDa位置有明显融合蛋白条带。结论我们成功构建了HNF4α基因真核表达载体,体外转染HepG2细胞成功表达HNF4α蛋白,为后续研究奠定了基础。

关键词: HepG2细胞, 肝细胞核因子4α, 真核表达, 细胞因子

Abstract: Objective To construct eukaryotic expression vector of human hepatocyte nuclear factor (HNF）-4α gene and identify its expression in vitro. MethodsThe total RNA was isolated from the liver tissues of two patients with hepatocellular carcinoma (HCC） who had underwent surgical operation, and HNF4α cDNA was synthesized by reverse transcription and amplified with the existence of specific primers. Then, the HNF4α fragment was directionally linked to pcDNA3.1 positive-eukaryotic expression vector. After antibiotic screening, the sequence analysis was conducted. The vector was transfected into HepG2 cells, and the expression of target protein after 48-hour incubation was detected by Western blot. Results Enzyme digestion and sequencing illustrated that pcDNA3.1-4α positive-eukaryotic expression vector was successfully constructed, and the results of Western blot revealed that obvious band of fusion protein was present at 53 kDa position. Conclusion The eukaryotic expression vector of HNF4α gene is successfully constructed, and it works well in HepG2 cells in vitro, which is good for further study of HNF4α protein.

Key words: HepG2 cells, Hepatocyte nuclear factor-4α, Eukaryotic expression, Cytokines

丁宁, 张明香. 人HNF4α基因真核表达载体的构建与鉴定^*[J]. 实用肝脏病杂志, 2016, 19(2): 156-159.

Ding Ning, Zhang Mingxiang. Construction and identification of eukaryotic expression vector of human hepatocyte nuclear factor 4α gene in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(2): 156-159.

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人HNF4α基因真核表达载体的构建与鉴定^*

Construction and identification of eukaryotic expression vector of human hepatocyte nuclear factor 4α gene in vitro

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