戊型肝炎病毒与ING5相互作用的研究

Interaction of hepatitis E virus and ING5

目的构建ING5基因真核表达载体和ING5荧光素酶载体,将pMIR-Report-ING5荧光素酶质粒与PUCHEV质粒共转染293T细胞后,验证HEV与ING5之间相互关系。方法通过提取293T细胞总RNA,RT-PCR法扩增ING5基因序列,并克隆到真核表达载体PGC3.1(+)上。利用脂质体转染法将PGC-ING5真核表达质粒转染HepG2细胞后,通过Western Blot法检测ING5表达情况;将pMIR-Report-ING5荧光素酶质粒和PUC-HEV质粒共转染293T细胞进一步探究ING5与HEV之间相互作用关系。结果经双酶切和测序鉴定真核表达质粒PGC-ING5构建成功,ING5基因在HepG2细胞中成功表达;将pMIR-Report-ING5荧光素酶质粒与PUC-HEV质粒共转染293T细胞后,发现HEV明显抑制239T细胞中pMIR-Report-ING5荧光素酶的活性。结论成功构建了PGC-ING5真核表达载体和pMIR-Report-ING5荧光素酶载体,并且证明HEV与ING5之间具有相互作用。

The ING5 gene eukaryotic expression vector and the ING5 luciferase vector were constructed. The relationship between ING5 luciferin plume and PUC-HEV co-transfected 293T cells was verified in our studying. The ING5 gene fragment was amplified by RT-PCR by extracting total RNA from 293T cells and cloned into the eukaryotic expression vector PGC （＋）. After the PGC-ING5 plasmid was transfected into HepG2 cells by liposome transfection,the expression of ING5 was detected by Western Blot. The ING5 luciferase vector and PUC-HEV were constructed to co-transfect 293T cells to further explore the relationship between ING5 and HEV. Interaction relationship. The recombinant eukaryotic expression plasmid PGC-ING5 was successfully constructed by double restriction enzyme digestion and sequencing. The ING5 gene has successful expressed in HepG2 cells. ING5 luciferase vector and PUC-HEV were co-transfected into 293T cells,the HEV significantly inhibited the activity luciferase from pMIR-Report-ING5 in 293T cell. It indicates that the PGC-ING5 and pMIR-Report-ING5 are successfully constructed, and it proves that there is interaction between HEV and ING5.