【摘要】 目的用Tet-on慢病毒载体系统构建稳定表达弓形虫棒状体蛋白18(ROP18)的293T细胞株。方法PCR扩增ROP18基因序列插入慢病毒载体p LVCT-t TR-KRAB中,构建带有ROP18活性片段的慢病毒载体p LVCT-t TRKRAB-ROP18。经鉴定后将该重组载体(6μg)与辅助质粒ps PAX2(4μg)和p MD2.G(2μg)共同转染293T细胞,荧光显微镜下观察细胞荧光。于转染后48和72 h收集含有慢病毒颗粒上清液,感染293T细胞。以1μg/ml强力霉素(DOX)诱导293T细胞表达ROP18,荧光显微镜下观察细胞荧光,RT-PCR法检测293T细胞中的ROP18表达情况。结果 PCR扩增rop18片段为960 bp,与预期大小一致。经PCR和酶切鉴定重组慢病素载体构建成功。转染48 h后,293T细胞于荧光显微镜下可见绿色荧光。经DOX诱导24 h即可观察到293T细胞中明亮绿色荧光。RT-PCR检测结果显示,293T细胞ROP18可产生960 bp特异条带,与预期结果一致。结论采用Tet-on慢病毒载体系统构建了稳定表达弓形虫ROP18的293T细胞株。更多还原

【Abstract】 Objective To construct a 293 T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Teton lentivirus expression system. Methods Rop18 gene of T. gondii was amplified by PCR, and inserted into a lentiviral vector p LVCT-t TR-KRAB. The recombinant plasmid p LVCT-t TR-KRAB-ROP18(6 μg) and 293 T human embryonic kidney cells were co-transfected with ps PAX2(4 μg) and p MD2.G(2 μg) for the packaging. The result of cotransfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection, the supernatant of the packaging lentivirus was collected for the 293 T cell infection. The doxycycline(DOX) was added into the medium to induce the ROP18 expression in 293 T cells. The ROP18 fusion expression was observed under fluorescence microscope and detected by RT-PCR after induction. Results PCR product of the gene fragment encoding ROP18 was 960 bp. The recombinant plasmid p LVCT-t TR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293 T cells at 48 h post-transfection. Bright green fluorescence was observed in 293 T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band(ROP18 gene) was detectable in 293 T cells. Conclusion 293 T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.更多还原