Bamhi and sali restriction enzymes

Codon-optimized A01 and A05 T cell TCR sequences were assembled into an expression cassette. In this construct, the TCR α and TCR β chain constant regions are replaced with modified murine TCR ɑ chain and TCR β chain constant regions (mTCRBC) to facilitate measurement of TCR expression by flow cytometry. It also contains an extra cysteine residue to encourage pairing between exogenous TCR chains. The TCR cassettes were synthesized through Thermo Fisher GeneArt Synthesis service and were then cloned into pRRL.PPT.MP.GFPpre^{59 (link),60 (link)} using BamHI and SalI restriction enzymes (New England BioLabs, Ipswich, MA). All plasmids were purified using Maxi Prep kits (Qiagen, Hilden, Germany).

The coding sequence (CDS) of ROP18 of T. gondii RH strain (GenBank No. JX045330) was amplified from total RNA extracted from tachyzoite of T. gondii RH strain using the primers: ROP18-F (5’-GGGGGATCCATGACACTTGGTCCTTCAAAACTCG-3’) and ROP18-R (5’-GGGGTCGACTTCTGTGTGGAGATGTTCCTGCTGTTC-3’). The PCR conditions were set as follows: pre-denaturation for 5 min at 98°C followed by 35 cycles of 98°C for 20 s, 56°C for 18 s, and 72°C for 30 s; 72°C for 5 min and hold at 4°C. The PCR product was purified using Gel Extraction kit (OMEGA, China). The purified ROP18 CDS was cloned into PCMV-N-HA vector using BamHI and SalI restriction enzymes (NEB, USA), according to the manufacturer’s instructions. The constructed plasmid (PCMV-N-HA-ROP18) was transformed into E. coli DH5α competent cells (TIANGEN, China). Single bacterial colony was randomly selected and identified using PCR primers ROP18-F and ROP18-R. Positive colonies were sequenced by Genscript Corporation (Nanjing, China). The plasmid of PCMV-N-HA-ROP18 bacterial colony was extracted using Endofree Plasmid Kit (TIANGEN, China) following the manufacturer’s instructions, and the extracted plasmid was stored at −20°C until use.