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

摘要： 【摘要】〓目的〓构建肝细胞核因子4α(hepatocyte nuclear factor 4 alpha, HNF4A)的过表达慢病毒载体,并对其进行病毒包装、鉴定与滴度测定,为进一步研究HNF4A的功能和作用机制奠定基础。方法〓利用PCR法扩增人类基因组DNA中HNF4A的cDNA序列,并将其克隆至PCDH-CMV-MCS-EF1a-GFP-puro慢病毒表达载体上,经双酶切及测序鉴定,将阳性重组PCDH-CMV-HNF4A-EF1a-GFP-puro表达载体、pCMV-VSV-G 和pCMV-dR8.91三质粒共转染到HEK-293T细胞,收集上清,将获得的病毒悬液浓缩后梯度稀释后感染HEK-293T细胞,并利用绿色荧光蛋白检测病毒滴度。将重组慢病毒感染原代培养人皮肤成纤维细胞HFFs,并利用荧光定量PCR检测HNF-4A的表达。结果〓重组慢病毒表达载体PCDH-CMV-HNF4A-EF1a-GFP-puro酶切及测序鉴定证明载体构建成功,并得到滴度为1×108 TU/mL的病毒液。病毒感染人皮肤成纤维细胞后HNF4A表达显著增强。结论〓通过优化载体构建方法成功构建人HNF4A的重组慢病毒载体并可在HFFs内显著过表达HNF4A,为HNF4A的研究提供更高效稳定的基因载体。

关键词: HNF, HNF4A, 慢病毒, 载体构建