Abstract:

Objective To investigate the effect of autophagy related 5 (ATG5) protein on the maturation of dendritic cells (DCs) stimulated by Toxoplasma gondii. Methods Total RNA was extracted from Kunming mice using TRIzol, reverse transcribed into cDNA, and amplified by PCR to receive ATG5 gene. The recombinant plasmid pET-32a-ATG5 was constructed and transformed into Escherichia coli Rosetta strain. The expressed recombinant ATG5 protein was purified by urea and identified by Western blotting with anti-His tag mAb. Mouse DCs were isolated, and then infected with T. gondii, which were treated by recombinant ATG5 protein in vitro. There were three groups: control group, T. gondii group (T. gondii + DCs), and ATG5 protein group (ATG5 + T. gondii + DCs), each with 3 replicates. The T. gondii tachyzoites (1 × 10⁴) were added to the DCs and incubated at 37 ℃ under 5% CO₂ for 2 h. The T. gondii in the ATG5 protein group were pre-treated with ATG5 protein (50 μg) in vitro for 2 h. The percentage of CD11cCD83 mature DCs among all DCs was assessed by flow cytometry. The effect of ATG5 protein on the maturation of DCs stimulated by T. gondii was evaluated. Result The amplified product of ATG5 was 729 bp, and the relative molecular mass (M_r) of the recombinant ATG5 protein was 46 000. Western blotting showed that the expressed recombinant ATG5 protein had good reactivity. Results of flow cytometry showed that the percentages of CD11cCD83 mature DCs in the control group, T. gondii group, and ATG5 protein group were (13.3 ± 0.5)%, (52.5 ± 0.3) % and (47.8 ± 0.6) %, respectively, with a significant difference between the latter two groups (P < 0.01), and between the control group and the other two (P < 0.01). Conclusion The ATG5 protein can reduce the ability of T. gondii to stimulate the maturation of DCs in vitro.

Key words: Toxoplasma gondii, Autophagy-related 5 protein, Dendritic cells