关键词: 弓形虫, 突变株, 绿色荧光蛋白, 荧光显微镜, 流式细胞仪

Abstract: Objective   To construct a Toxoplasma gondii mutant for stably expressing green fluorescent protein （GFP），and establish method to determine the rate of mutant-infected HeLa cells.  Methods   Freshly lysed-out tachyzoites of T. gondii RH strain were transfected with plasmid ptubP30-GFP/sag-CAT. Stable transformants were selected with chloramphenicol and limited dilution. The expression of GFP in mutant tachyzoite was determined by RT-PCR and fluorescence microscopy. When infected with 1×10⁴-1×10⁷ mutant tachyzoites respectively for 24 h，the total number of HeLa cells with green fluorescence was determined by fluorescent microscope in 10 high-power fields，and the rate of HeLa cells with parasitophorous vacuole was determined by flow cytometry.  Results   Untransfected tachyzoites were killed by chloramphenicol，while the stable transformants showed resistance to chloramphenicol. The expression of GFP gene was detected by RT-PCR. The P30-GFP transfectants displayed fluorescence outside the parasite. The rate of mutant-infected HeLa cells increased with the rise of the number of mutant for infection. When infected with 1×104-1×107 tachyzoites，the numbers of HeLa cells with fluorescence were （14±6），（133±45），（332±93）  and （443±90），and the rates of infected cells were （0.49±0.09）%，（8.76±0.50）%，（21.02±1.49）% ，and （39.00±3.47）% by flow cytometry，respectively.   Conclusion   T. gondii mutant with GFP tag is constructed，which provides a new method to determine the proliferation when cultured in host cells.

Key words: Toxoplasma gondii, Mutant, Green fluorescent protein, Fluorescence microscopy, Flow cytometry