Abstract:

Objective  To construct a 293T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Tet-on lentivirus expression system.  Methods  Rop18 gene of T. gondii was amplified by PCR， and inserted into a lentiviral vector pLVCT-tTR-KRAB. The recombinant plasmid pLVCT-tTR-KRAB-ROP18（6 μg） and 293T human embryonic kidney cells were co-transfected with psPAX2 （4 μg） and pMD2.G （2 μg） for the packaging. The result of co-transfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection， the supernatant of the packaging lentivirus was collected for the 293T cell infection. The doxycycline（DOX） was added into the medium to induce the ROP18 expression in 293T 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 pLVCT-tTR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293T cells at 48 h post-transfection. Bright green fluorescence was observed in 293T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band（ROP18 gene） was detectable in 293T cells.  Conclusion  293T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.

Key words: Toxoplasma gondii, ROP18, Lentivirus, Tetracycline inducible expression system