关键词: HNF, HNF4A, 慢病毒, 载体构建

Abstract: 【Abstract】〓Objective〓To construct, package and identify the recombinant lentivirus overexpressing hepatocyte nuclear factor 4 alpha, HNF4A, and to lay the foundation for further study the functions and mechanism of action of HNF4A. Methods〓The human HNF4A cDNA was amplified from the human genomic DNA by polymerase chain reaction (PCR), which was constructed to the lentiviral expression vector PCDH-CMV-MCS-EF1a-GFP-puro. After enzymatic digestion and sequencing,the positive recombinant PCDH-CMV-HNF4A-EF1a-GFP- puro expression vector, pCMV-VSV-G and pCMV-dR8.91, three plasmids were cotransfected into HEK-293T cells, next the supernatants were harvested. After getting the virus suspension concentrated, we make the gradient dilution to infect HEK-293T cells and determine the virus titer by the green fluorescence protein. Finally the expression of HNF4A in primary cultured human skin fibroblasts HFFs infected with recombinant lentivirus was analyzed by real-time quantitative PCR. Results〓Enzymatic digestion and DNA sequencing demonstrated that the recombinant lentiviral expression vector PCDH-CMV- HNF4A-EF1a-GFP-puro was successfully constructed. The lentivirus, with the concentration of 1×108 TU/ml, can significantly increased the level of HNF4A expression in HFFs which was infected. Conclusion〓The recombinant lentivirus with PCDH-CMV- HNF4A-EF1a-GFP-puro was successfully constructed and highly overexpresses HNF4A in HFFs, what make a contribution to the better carrier for HNF4A with high efficiency and stability.

Key words: HNF, HNF4A, Lentivirus, Vector construction