Abstract: 【Abstract】〓Objective〓To construct the restructuring lentiviral expression vector FoxA1 gene and lay a foundation for us to explore the functions and mechanisms of FoxA1. Methods〓The primers were designed for the amplification of FoxA1 by polymerase chain reaction(PCR),and cDNA was constructed to the lentiviral expression vector PCDH-CMV-MCS-EF1a-GFP-puro. After PCR,double digests and DNA sequencing, the positive recombinant FoxA1 expression vector, pCMV-VSV-G and pCMV-dR8.91 were cotransfected into HEK-293T cells to produce lentiviral particles, and then the latter was transfected into 293T cells to calculate the virus titer by observing the GFP expression. Finally the mRNA expression of transfected FoxA1 in primary human skin fibroblasts HFFs was identified by real-time quantitative PCR. Results〓PCR, double digests and DNA sequencing assays demonstrated that the inserted sequences was correct. The recombinant lentiviral expression vector FoxA1 was successfully constructed with a concentration of 1×108 TU/mL of viral titer.The level of FoxA1 expression was significantly increased after infection with PCDH-CMV-FoxA1-EF1a-GFP-puro. Conclusion The lentiviral vector over-expressing FoxA1 gene was successfully constructed and highly overexpresses in HFFs. It lays an experiment foundation to explore the functions and mechanisms of FoxA1 for further study.

Key words: lentivirus, Vector construction, Over-expression, FoxA1

摘要： 【摘要】 目的 构建叉头盒蛋白A1(Forkhead-boxA1,FoxA1)的重组慢病毒表达载体,为探究其功能和作用机制奠定基础。方法〓设计基因引物,采用聚合酶链反应(PCR)扩增出FoxA1的cDNA序列,然后将其克隆至PCDH-CMV-MCS-EF1a-GFP-puro慢病毒表达载体上,经PCR、双酶切反应及DNA测序鉴定,将阳性重组FoxA1表达载体、pCMV-VSV-G 和pCMV-dR8.91包装质粒共转染到HEK-293T细胞进行病毒包装收集病毒浓缩液,然后再感染293T细胞,通过观察绿色荧光蛋白来计算病毒滴度,最后用病毒转染原代培养人皮肤成纤维细胞(HFFs),通过实时荧光定量PCR检测FoxA1 mRNA的表达水平。结果〓重组慢病毒表达载体FoxA1经PCR扩增、酶切及测序鉴定正确,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表达显著增强。结论〓成功构建了人FoxA1重组慢病毒载体,并可在HFFs内高效表达,为后续FoxA1的功能和机理研究奠定了实验基础。

关键词: FoxA1, 慢病毒, 载体构建, 过表达