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目的:将已构建的SP-TAT-Apoptin融合基因真核表达载体包装成感染性慢病毒颗粒,并用病毒颗粒感染肝癌HepG2细胞,测定其诱导凋亡的效率。方法:通过脂质体Li-pofectamine TM2000将SP-TAT-Apoptin融合基因真核表达载体与其他包装质粒共同转导入293FT细胞,包装并收集感染性病毒颗粒,用实时定量PCR法测定病毒滴度,免疫荧光法检测重组慢病毒感染的293FT细胞中SP-TAT-Apoptin融合基因的表达,同时通过流式细胞术(FCM)测定慢病毒感染后HepG2细胞的凋亡率。结果:SP-TAT-Apoptin重组慢病毒感染293FT细胞后,用V5抗原单克隆抗体(mAb)进行免疫荧光化学检测,示SP-TAT-Apoptin融合基因可成功表达于293FT细胞;通过Anexin-VPI法检测示SP-TAT-Apoptin融合基因慢病毒感染肝癌HepG2细胞后可引起细胞凋亡,其凋亡效率明显高于单纯脂质体转染组。结论:成功包装出可表达SP-TAT-Apoptin融合基因且具感染性的慢病毒颗粒,感染HepG2肝癌细胞后可引起其凋亡,为进一步研究融合基因SP-TAT-Apoptin体内治疗效果极其在临床中的应用奠定了基础。
OBJECTIVE: To construct the eukaryotic expression vector of SP-TAT-Apoptin fused gene and construct the infective lentivirus particles. HepG2 cells were infected with virus particles to determine the efficiency of apoptosis induction. METHODS: The eukaryotic expression vector of SP-TAT-Apoptin fusion gene and other packaging plasmids were co-transducted into 293FT cells by Lipofectamine TM2000, and the infectious virus particles were packaged and collected. The virus titers were determined by real-time quantitative PCR. The expression of SP-TAT-Apoptin fusion gene in 293FT cells infected with recombinant lentivirus was detected by immunofluorescence method. The apoptosis rate of HepG2 cells after lentivirus infection was determined by flow cytometry (FCM). RESULTS: The 293T cells were infected with SP-TAT-Apoptin recombinant lentivirus. The immunofluorescence assay with monoclonal antibody against V5 antigen (mAb) showed that the fusion gene SP-TAT-Apoptin was successfully expressed in 293FT cells. Anexin-VPI The results showed that the apoptosis of HepG2 cells induced by SP-TAT-Apoptin fusion gene lentivirus was significantly higher than that of lipofectamine alone. CONCLUSION: Lentiviral particles capable of expressing SP-TAT-Apoptin fusion gene are successfully packaged and cause apoptosis after infection with HepG2 hepatoma cells. In order to further study the in vivo therapeutic effect of fusion gene SP-TAT-Apoptin in clinic In the application laid the foundation.