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

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Hep G2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c DNA,经特异性引物扩增HNF4α基因片段,将其定向连接到pc DNA3.1(+)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Hep G2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pc DNA3.1-4α真核表达载体构建成功。在转染Hep G2细胞48 h后,检测显示53 k Da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Hep G2细胞成功表达HNF4α蛋白,为后续研究奠定了基础。

Objective To construct eukaryotic expression vector of human hepatocyte nuclear factor（HNF）-4α gene and identify its expression in vitro. Methods The total RNA was isolated from the liver tissues of two patients with hepatocellular carcinoma（HCC） who had underwent surgical operation, and HNF4α c DNA was synthesized by reverse transcription and amplified with the existence of specific primers. Then, the HNF4αfragment was directionally linked to pc DNA3.1 positive-eukaryotic expression vector. After antibiotic screening, the sequence analysis was conducted. The vector was transfected into Hep G2 cells, and the expression of target protein after 48-hour incubation was detected by Western blot. Results Enzyme digestion and sequencing illustrated that pc DNA3.1-4α positive-eukaryotic expression vector was successfully constructed, and the results of Western blot revealed that obvious band of fusion protein was present at 53 k Da position. Conclusion The eukaryotic expression vector of HNF4α gene is successfully constructed, and it works well in Hep G2 cells in vitro, which is good for further study of HNF4α protein.