关键词: 刚地弓形虫, 棒状体蛋白18, 膜表面抗原30, 核酸疫苗, 免疫保护

Abstract:

Objective To study the immunoprotective effect of a nucleic acid vaccine dual-targeting rhoptry protein 18 (ROP18) and surface antigen 30 (P30) of Toxoplasma gondii. Methods The p30 and rop18 genes of T. gondii were amplified by PCR to construct recombinant plasmids pVAX1-p30 and pVAX1-rop18-p30, which were verified by PCR, enzyme digestion, and sequencing. HeLa cells were transfected with pVAX1 plasmids (2 μg) and pVAX1-rop18-p30 plasmids using Lipofectamine^TM 3000 transfection reagents, and the expression of target protein was detected by indirect immunofluorescence assay after 24 hours. Seventy-five female BALB/c mice were randomly divided into five groups (pVAX1-rop18-p30 group, pVAX1-rop18 group, pVAX1-p30 group, pVAX1 group, and PBS group), with each group containing 15 mice. The plasmids [100 μg (100 μl)] and PBS (100 μl) were injected into the quadriceps femoris muscle, with a total of three injections at two-week intervals, and the last injection had a plasmid dosage of 200 μg. The levels of serum IgG were quantified by ELISA before each injection and two weeks after the final dose. The spleen cells were collected aseptically from three mice in each group two weeks after the last dose, and the levels of IFN-γ, IL-2, IL-4, IL-10, and IL-12 in the culture supernatant were measured by ELISA. Two weeks after the last immunization, 12 mice were selected from each group, and 1 000 tachyzoites of T. gondii RH strain were injected intraperitoneally into each mouse to induce acute infections. The survival time of the mice was recorded, and their immune protection was evaluated. Single-factor analysis of variance was used for inter-group comparison. Results PCR amplification fragments of p30 and rop18 genes with lengths of 789 bp and 1 665 bp. The pVAX1-rop18-p30 recombinant plasmids were successfully identified through PCR, enzyme digestion, and sequencing. HeLa cells transfected with pVAX1-rop18-p30 exhibited yellow-green fluorescence in the cytoplasm, while no such fluorescence was observed in cells transfected with pVAX1. ELISA results indicated that two weeks after the last immunization, the A₄₅₀ value in the pVAX1-rop18-p30 group (0.788 ± 0.025) was significantly higher than that in the pVAX1-rop18 group (0.512 ± 0.027), pVAX1-p30 group (0.498 ± 0.027), pVAX1 group (0.122 ± 0.014), and PBS group (0.109 ± 0.011) (F = 77.6, P < 0.05). There was no statistically significant difference in A₄₅₀ values between the pVAX1-rop18 group and the pVAX1-p30 group (F = 67.80, P > 0.05). The serum IgG levels increased with the frequency and duration of immunization. Two weeks after the final immunization, the levels of IFN-γ, IL-2, IL-4, IL-10, and IL-12 in the supernatant of splenocyte cultures were the highest in the pVAX1-rop18-p30 group [(678.77 ± 3.69), (375.28 ± 2.65), (130.82 ± 4.72), (279.68 ± 3.67), (579.68 ± 3.67) pg/ml], followed by the pVAX1-rop18 group [(448.59 ± 6.52), (256.34 ± 5.58), (126.28 ± 7.48), (156.58 ± 4.59), (231.50 ± 3.21) pg/ml] and pVAX1-p30 group [(423.67 ± 4.82), (277.92 ± 4.23), (115.17 ± 4.37), (137.18 ± 5.62), (190.47 ± 4.18) pg/ml], while the pVAX1 group [(48.97 ± 2.65), (47.65 ± 2.76), (47.14 ± 2.04), (45.29 ± 2.31), (46.21 ± 2.17) pg/ml] and PBS group [(47.69 ± 3.42), (46.77 ± 3.35), (48.59 ± 4.75), (44.92 ± 4.91), (46.88 ± 3.56) pg/ml] showed significantly lower levels (F = 1 582.0, 531.5, 268.7, 215.0, 170.5; all P < 0.05). The levels of IFN-γ, IL-10, IL-12 were elevated in the pVAX1-rop18-p30 group compared with the pVAX1-rop18 group and the pVAX1-p30 group (F = 1 620.8, 1 208.0, 728.0; all P < 0.05), the levels of IL-2 and IL-4 didn’t show significant changes between the pVAX1-rop18-p30 group, the pVAX1-rop18 group and the pVAX1-p30 group (F = 53.21, P > 0.05). After acute infection, the mice mean survival time in the pVAX1-rop18-p30 group, the pVAX1-rop18 group, the pVAX1-p30 group, the pVAX1 group and the PBS group were (288 ± 2), (196 ± 3), (230 ± 6), (144 ± 2), (146 ± 11) h, the mean survival time of mice in the pVAX1-rop18-p30 group was significantly longer than that in the pVAX1 group and PBS group (F = 100.1, P < 0.05), as well as the pVAX1-rop18 and pVAX1-p30 groups (F = 38.7, P < 0.05). Conclusion The pVAX1-rop18-p30 dual-targeting nucleic acid vaccine could induce cellular and humoral immunity in mice; its immunprotectiv effect against T. gondii infection is better than that of the vaccine mono-targeting either pVAX1-rop18 or pVAX1-p30.

Key words: Toxoplasma gondii, Rhoptry protein 18, Surface antigen 30, Nuclei acid vaccine, Immunoprotection