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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。
Objective To construct human lentiviral vector of Runx3 gene. Methods Runx3 gene was amplified by PCR. In-fusion technique was used to exchange the amplified product of Runx3 gene into linearized lentiviral vector pGC-FU to construct recombinant lentiviral plasmid pGC-FU-Runx3 and transformed into E. coli DH5α competent cells. Positive clones were identified by PCR and sequenced and aligned. The constructed recombinant lentiviral plasmid pGC-FU-Runx3 was co-transfected into 293T cells with packaging plasmid pHelper 1.0 and envelope plasmid pHelper 2.0 for viral packaging. Fluorescence microscopy was used to observe the green fluorescent protein expression in 293T cells transfected with recombinant lentiviral plasmid pGC-FU-Runx3. Western blot assay Runx3 and EGFP fusion protein expression. Real time quantitative PCR was used to determine the virus titer. Results The recombinant lentiviral plasmid pGC-FU-Runx3 was confirmed by sequencing and alignment analysis. Three plasmids co-transfected 293T cells under fluorescence microscope showed obvious green fluorescence. Western blot confirmed that Runx3 and EGFP fusion proteins were stably expressed in 293T cells. After the virus was concentrated, the titer was determined to be 2.0 × 10 8 TU / ml. Conclusion The recombinant lentiviral vector pGC-FU-Runx3 carrying human Runx3 gene was successfully constructed, which lays the foundation for the further study on the effect of Runx3 overexpression on the differentiation of Th cells in peripheral blood of patients with chronic hepatitis B.