人FoxA1基因重组慢病毒载体的构建和鉴定

Construction and identification of the human FoxA1 gene recombinant lentivirus

目的构建叉头盒蛋白A1（Forkhead-box A1,Fox A1）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Fox A1的c DNA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Fox A1表达载体、p CMV-VSV-G和p CMV-d R8.91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Fox A1 m RNA的表达水平。结果重组慢病毒表达载体Fox A1经PCR扩增、酶切及测序鉴定正确,并得到滴度为1×10~8 TU/m L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Fox A1重组慢病毒载体,并可在HFFs内高效表达,为后续Fox A1的功能和机理研究奠定了实验基础。

Objective To construct the restructuring lentiviral expression vector Fox A1 gene and lay a foundation for us to explore the functions and mechanisms of Fox A1. Methods The primers were designed for the amplification of Fox A1 by polymerase chain reaction（PCR）,and c DNA was constructed to the lentiviral expression vector PCDH-CMV-MCS-EF1a-GFP-puro. After PCR,double digests and DNA sequencing, the positive recombinant Fox A1 expression vector, p CMV-VSV-G and p CMVd R8.91 were cotransfected into HEK-293 T cells to produce lentiviral particles, and then the latter was transfected into 293 T cells to calculate the virus titer by observing the GFP expression. Finally the m RNA expression of transfected Fox A1 in primary human skin fibroblasts HFFs was identified by realtime quantitative PCR. Results PCR, double digests and DNA sequencing assays demonstrated that the inserted sequences was correct. The recombinant lentiviral expression vector Fox A1 was successfully constructed with a concentration of 1 ×10~8TU / m L of viral titer.The level of Fox A1 expression was significantly increased after infection with PCDH-CMV-Fox A1-EF1a-GFP-puro. Conclusion The lentiviral vector over-expressing Fox A1 gene was successfully constructed and highly overexpresses in HFFs. It lays an experiment foundation to explore the functions and mechanisms of Fox A1 for further study.