人Runx3基因重组慢病毒载体的构建与鉴定

目的构建人Runx3基因重组慢病毒载体。方法 PCR扩增Runx3基因,应用In-Fusion技术将Runx3基因PCR扩增产物交换进入线性化慢病毒载体pGC-FU以构建重组慢病毒质粒pGC-FU-Runx3,并转化大肠杆菌DH5α感受态细胞。对长出的阳性克隆进行PCR鉴定,并进行测序和比对分析。将构建成功的重组慢病毒质粒pGC-FU-Runx3与包装质粒pHelper 1.0、包膜质粒pHelper 2.0共转染293T细胞进行病毒包装。荧光显微镜下观察重组慢病毒质粒pGC-FU-Runx3转染293T细胞后细胞内绿色荧光表达情况。Western blot检测Runx3与EGFP融合蛋白表达情况。实时定量PCR测定病毒滴度。结果重组慢病毒质粒pGC-FU-Runx3经测序和比对分析证实目的基因序列正确。三质粒共转染293T细胞后荧光显微镜下观察细胞内可见明显的绿色荧光。Western blot证实Runx3与EGFP融合蛋白在293T细胞内稳定表达。浓缩病毒后测定滴度为2.0×108TU/ml。结论成功构建携带人Runx3基因的重组慢病毒载体pGC-FU-Runx3,为进一步研究Runx3过表达对慢性乙型肝炎患者外周血Th细胞分化的影响奠定基础。

Construction and identification of the recombinant lentiviral vector containing human Runx3 gene

Objective To construct a recombinant lentiviral vector carrying human Runx3 gene.Methods Runx3 gene was amplified by PCR.Next,the Runx3 gene fragment and the lentiviral vector（pGC-FU）were ligated with In-Fusion technique to generate the recombinant lentiviral plasmid（pGC-FU-Runx3）.Then pGC-FU-Runx3 was transformed into Escherichia coli DH5α competent cells.The positive clones were screened by PCR and were confirmed by sequencing and comparative analysis.The recombinant lentiviral plasmid（pGC-FU-Runx3）was mixed with the packaging plasmid（pHelper 1.0）and the envelope plasmid（pHelper 2.0）and then they were co-transfected into 293T cells.The expression of Runx3-EGFP was detected by both fluorescence microscopy and Western blot.The viral titer was measured by real-time quantitative PCR.Results The recombinant lentiviral plasmid（pGC-FU-Runx3）was confirmed that the target gene sequence was correct by sequencing and comparative analysis.Strong green fluorescence was observed in 293T cells under fluorescent microscope following co-transfection with three plasmids（pGC-FU-Runx3,pHelper 1.0 and pHelper 2.0）into the cells.Western blot identified the stable expression of Runx3-EGFP fusion protein in the transfected 293T cells.The virus in the supernatant reached a titer of 2.0×108TU/ml.Conclusions The recombinant lentiviral vector（pGC-FU-Runx3）carrying human Runx3 gene was successfully constructed,which would lay the foundation of investigating the effect of Runx3 overexpression on Th cell differentiation in peripheral blood from the patients with chronic hepatitis B.