Plasmid Constructs for Toxoplasma Vaccination

The pVAX1 plasmids encoding T. gondii profilin (pVAX1-PF), rhoptry protein 16 (pVAX1-ROP16), rhoptry protein ROP18 (pVAX1-ROP18), microneme protein 6 (pVAX1-MIC6), and calcium-dependent protein kinase 3 (pVAX1-CDPK3) were constructed as previously reported (31 (link)–35 (link)), with the fidelity of all plasmids confirmed by sequencing (Sangon, China). The five eukaryotic plasmids were each transformed into E. coli DH5α for propagation and were isolated by anion exchange chromatography (EndoFree Plasmid Giga Kit, Qiagen Sciences, MD, USA) following the manufacturer’s instruction. Plasmid concentration and purity was determined by measuring the optical density ratio A₂₆₀/A₂₈₀. The purified plasmids were stored at −20°C until used in the mouse immunization protocols.