Fig. 2
Verification of the interference effect of TgeIF-5A knockdown tachyzoites. Three specific siRNAs were transfected into tachyzoites in 1 μM, 2 μM and 4 μM, respectively. A nonspecific siRNA group was established as the control group, and a phosphate-buffered saline (PBS) group was established as a blank group. After culture for 24 h, the tachyzoites were collected. a Real-time PCR assessment of the knockdown of TgeIF-5A. The total RNA of the tachyzoites was extracted for reverse transcription. The relative abundance was estimated using the 2−∆∆Ct method, following normalization to β-tubulin. *** P < 0.001. Two-way ANOVA with Bonferroni post hoc test. b Western blot of the total soluble protein of T. gondii tachyzoites. (Lane M) protein marker; (Lane 1) The total soluble protein of T. gondii tachyzoites probed by sera from rats immunized by rTgeIF-5A, a band at about 17 kDa was identified that was consistent with the molecular weight of native TgeIF-5A protein; (Lane 2) The total soluble protein of T. gondii tachyzoites probed by sera of normal rats, no specific band was identified. c Western blot of rTgeIF-5A. (Lane M) Protein marker; (Lane 1) Recombinant protein TgeIF-5A probed by sera from rats experimentally infected with T. gondii as primary antibody, a band about 36 kDa was identified that was consistent with the molecular weight of rTgeIF-5A (fused with the polyhis-tag protein of pET-32a (+) vector); (Lane 2) Recombinant protein TgeIF-5A probed by sera of normal rats as the primary antibody, no specific band was identified. d Western blot analysis of TgeIF-5A knockdown parasites. The total soluble proteins of TgeIF-5A knockdown and RH tachyzoites were generated. (Line eIF-5A-I) The total soluble proteins of TgeIF-5A knockdown tachyzoites were probed by antibody against β-actin and rTgeIF-5A, respectively. (Line Control) The total soluble proteins of RH tachyzoites were probed by antibody against β-actin and rTgeIF-5A, respectively. e Analysis of TgeIF-5A expression level in TgeIF-5A knockdown and control tachyzoites by Western blotting. The protein level of TgeIF-5A is shown as a percentage of actin in each sample. The data are indicative of three individual experiments, ** P < 0.01, Student’s t test