[Preparation and application of the polyclonal antibody of Toxoplasma gondii autophagy protein 8 (TgAtg8)].

ABSTRACT

OBJECTIVE: To prepare and evaluate specific-TgAtg8 polyclonal antibody. METHODS: The known Saccharomyces cerevisiae Atg protein sequences were used to identify Toxoplasma gondii homologous protein through bioinformatics analysis. TgAtg8 cDNA was amplified and cloned into prokaryotic expression vector pGEX-6p-1. The constructed pGEX-6p-1-TgAtg8 was transformed into E. coli BL21 cells and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE and Western blotting. The recombinant TgAtg8 protein with an N-terminal glutathione-S transferase tag was used to immunize rabbits and raise specific polyclonal antibody against TgAtg8. Subsequently, the antibody was applied for Western blotting and IFA assay. RESULTS: Recombinant expression plasmid of pGEX-6p-1-TgAtg8 was confirmed correct by restriction enzyme digestion and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg8 protein with the predicted molecular weight (M(r)40000) was expressed highly in E. coli BL21. After immunization, the specific antibodies against TgAtg8 protein were produced. The anti-TgAtg8 polyclonal antibody reacted specifically with TgAtg8 fusion protein or endogenous TgAtg8. Importantly, IFA assay determined that the TgAtg8 signal was generally distributed throughout the cytoplasm of the tachyzoites. However, the green fluorescence signal gathered into one or more green spots after induction of autophagy. CONCLUSION: The specific polyclonal antibody against TgAtg8 could be used to observe the dynamics of autophagosome formation in T. gondii, which is useful tool to investigate the autophagic machinery in this parasite.