Table 1

The primers used to construct pSAG1::CAS9-U6::sgROP5 plasmid"

Table 2

Primers for the construction of pROP5::CAT-D plasmid"

Table 3

Primers for identification of TR-ROP5-KO strain"

Table 4

Primers of RT-PCR used to detect the mRNA transcription levels of cells infected by different strains of T. gondii"

Fig. 1

Construction and identification of pSAG1::CAS9-U6::sgROP5 plasmid A, C. Amplification of pSAG1::CAS9-U6::sgROP5-1 plasmid and pSAG1::CAS9-U6::sgROP5-2 plasmid fragments by PCR [M. DL 15000 marker; 1. Fragment 1 of plasmid (3 000 bp); 2. Fragment 2 of plasmid (4 021 bp); 3. Fragment 3 of plasmid (2675 bp)]; B, D. Identification of pSAG1::CAS9-U6::sgROP5-1 plasmid and Identification of pSAG1::CAS9-U6::sgROP5-2 plasmid by PCR (M. DL 2000 marker; 1-2. Identify 220 bp of gRNA1/gRNA2); E. Amplification of gRNA2 (including Kpn Ⅰ and Xho Ⅰ restriction sites) fragment of ROP5 by PCR (M. DL 2000 marker; 1-2. 676 bp of gRNA2 fragment); F. Identification of pSAG1::CAS9-U6::sgROP5 plasmid by PCR (M. DL 2000 marker; 1-3. Identify 220 bp of gRNA1); G. Identification of pSAG1::CAS9-U6::sgROP5 plasmid by PCR (M. DL 2000 marker; 1-6. Identify 778 bp of gRNA2)"

Fig. 2

Construction and identification of pROP5::CAT-D plasmid A. Amplification of pROP5::CAT-D plasmid fragments by PCR (M. DL 15000 marker; 1. Fragment 1 of plasmid; 2. Fragment 2 of plasmid; 3. Fragment 3 of plasmid; 4. Fragment 4 of plasmid); B. Identification of pROP5::CAT-D plasmid by PCR (M. DL 15000 marker; 1. Blank control; 2-4. Positive plasmid, the two fragments are 3 146 bp at the junction of 5′UTR and CAT and 2 875 bp at the junction of CAT and 3′UTR)"

Fig. 3

Identification of TR-ROP5-KO strain by PCR and Western blot A. Identification of TR-ROP5-KO strain by PCR (M. DL 5000 marker; 1. Amplification of 820 bp fragment in TR gene; 2. Amplification of 901 bp fragment in ROP5 gene; 3. Amplify 1546 bp of CAT resistant fragment; 4. Amplify 3 163 bp of DHFR resistant fragment); B. Identification of TR-ROP5-KO strain by Western blot (M. Protein marker; 1. RH strain of T. gondii; 2. TR-ROP5-KO strain of T. gondii)"

Fig. 4

Study on biological activity of TR and ROP5 double-gene deletion strains of T. gondii A. Proliferation assay of TR-ROP5-KO strain in Vero cells; B. Invasion assay of TR-ROP5-KO strain in Vero cells; C. Plaque assay of TR-ROP5-KO strain in Vero cells; D. Virulence assay of TR-ROP5-KO strain in mice"

Fig. 5

Oxidative stress levels in TR and ROP5 gene deletion strains of T. gondii A. MDA levels in TR and ROP5 gene deletion strains of T. gondii; B. T-AOC levels in TR and ROP5 gene deletion strains of T. gondii; C.ROS levels in TR and ROP5 gene deletion strains of T. gondii. *. P < 0.05, **. P < 0.01"

Fig. 6

Effects of TR and ROP5 gene deletion of T. gondii on ROS levels in RAW264.7 cells **. P < 0.01"

Fig. 7

Effects of TR and ROP5 gene deletion strains of T. gondii on the expression of NF-κB, IL-12, IFN-γ mRNA in RAW264.7 cells A. Effects of TR and ROP5 gene deletion strains of T. gondii on the expression of NF-κB mRNA in RAW264.7 cells; B. Effects of TR and ROP5 gene deletion strains of T. gondii on the expression of IL-12 mRNA in RAW264.7 cells; C. Effects of TR and ROP5 gene deletion strains of T. gondii on the expression of IFN-γ mRNA in RAW264.7 cells. *. P < 0.05, **. P < 0.01, ns. P > 0.05"

Fig. 8

Effects of TR and ROP5 gene deletion strains of T. gondii on IL-12 in mice **. P < 0.01"