Inject freund s complete adjuvant

To create the corresponding expression plasmids (Supplementary Data 1), cDNA fragments encoding full or partial polypeptides of BIP, Tubulin, Cen1, and GRASP from T. gondii were cloned into pET28a using primers. The proteins were expressed in Escherichia coli BL21DE3 (Transgene Biotech, CD601-02) and purified by HisPur Ni-NTA rotary columns (Thermofisher Scientific, 88225). Following the manufacturer’ protocols, the recombinant proteins prepared by Inject Freund’s complete adjuvant (Sigma-Aldrich, F5881) was used to produce antiserum in mice or rabbits. BALB/C mice and New Zealand white rabbits were used in this study, following the guidelines and regulations provided by the Veterinary Office of China Agricultural University (Issue No. AW11402202-2-1). The antisera were subjected to a series of dilutions for evaluation via IFA and western blots. Further assessment was conducted based on the intraparasitic localization and the molecular weights of the corresponding proteins in T. gondii.

T. gondii cDNA fragments encoding full or partial polypeptides of known proteins, i.e., Hsp60, GAP45, actin, ACP and IMC1, were cloned into pET28a (Thermo Fisher Scientific) using primers to create corresponding expression plasmids (Supplementary Data 2 and 3), and transformed into E. coli BL21DE3 (CD601-02, Transgene Biotech). The recombinant proteins were purified using HisPur Ni-NTA spin columns (88225, Thermo Fisher Scientific), and prepared for antiserum generation in mice (5 mice per antibody generation) or rabbits (2 rabbits per antibody generation) using Inject Freund’s complete adjuvant (F5881, Sigma-Aldrich), as recommended by the manufacturer’ protocols. The anti-sera were tested using serial dilutions on IFA and western blots, followed by an assessment of protein localization and molecular weights of the corresponding T. gondii proteins.

T. gondii cDNA fragments encoding full or partial polypeptides of IPPS, STX6, and MLC1, respectively, were cloned into pET28a (Thermo Fisher Scientific) using primers to create corresponding expression plasmids (Table S2 and S3). The proteins were then expressed in and purified from Escherichia coli BL21DE3 (Transgene Biotech) by HisPur Ni-NTA spin columns (Thermo Fisher Scientific). The recombinant proteins were prepared for anti-serum generation in mice or rabbits using Inject Freund’s complete adjuvant (Sigma-Aldrich), as recommended by the manufacturer’s protocols. The anti-sera were tested by serial dilutions on IFA and western blots, further assessed by the localization in parasites, and by molecular weights of the corresponding T. gondii proteins.