2.1 Cells and parasites
Human foreskin fibroblast (HFF) cells were purchased from ATCC (SCRC-1041) and routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, BI, Israel), 100 µg/mL penicillin and 100 µg/mL streptomycin (Sigma, USA), and maintained in an incubator at 37°C and 5% CO2. Toxoplasma gondii RH strain, green fluorescent protein-RH strain (GFP-RH), TgCtwh3 and TgCtwh6 were propagated in HFF cells.
2.2 RNA extraction and quantitative real-time PCR assays
Total RNA was extracted from the parasites, using Trizol Reagent (Invitrogen, USA). Total RNA was used for reverse transcription with the cDNA synthesis kit (TaKaRa, Japan), according to the manufacture’s protocol. cDNA synthesis was performed using SYBR-Green Master Mix (TaKaRa, Japan). The primer sequences used were as follows:
TgMIC1 forward, TCGGTTTATGCTGAGTGTGC;
TgMIC1 reverse, GGCGAATTCCTTCCTCTTCT;
TgMIC4 forward, GACATGACGGGATCCAGAAC;
TgMIC4 reverse, CATGCAACTTGGCAGTCTGT;
TgMIC6 forward, CATATCACCTGCAAGCGTGT;
TgMIC6 reverse, GGCTCACGACTTTCACCTTC;
β-tublin forward, GTCTCCACTTCTTCCTCATTG;
β-tublin reverse, GTTCTTTGCGTCGAACATC.
β-tublin was used as an internal control. Relative expression levels were calculated according to the standard 2−ΔΔCt method. All experiments were performed in triplicate and repeated at least three times.
2.3 Protein isolation and western blot assays
Parasites was lysed with RIPA lysis buffer (Beyotime, China) with 1% phenyl methyl sulfonyl fluoride and total proteins were subjected to electrophoresis in 8-10% polyacrylamide gel. The proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, USA) by a standard western blot procedure. The membrane was blocked with 5% non-fat milk and incubated with specific primary antibodies diluted in the blocking buffer overnight. Following incubation with the corresponding secondary antibodies conjugated to horseradish peroxidase for 1 h. The membranes were visualized using ECL Western blotting substrate (Bio-RAD, USA).
2.4 Expression and purification of recombinant TgMIC1 proteins
The Coding sequence (CDS) of TgMIC1 was optimized by using the OptigeneTM codon optimized analysis platform (Shanghai Jierui Bioengineering Co., Ltd.). The optimized TgMIC1 sequence was synthesized and cloned into pET30a. The recombinant pET30a-MIC1 plasmid was transformed into Escherichia coli BL21 (DE3), cultivated in Luria-Bertani (LB) at 37 °C. The recombinant protein expression was induced by adding 0.5mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma, USA) at 16°C for overnight with constant shaking at 200 rpm. Then, the bacterial cells were harvested by centrifugation at 8000 rpm for 6 min, and resuspended in 20ml 10mM Tris-HCL buffer. The bacteria suspension was sonicated on ice (500 W, 180 times, 5 s each time, 5 s interval). The lysate was centrifuged at 12,000 rpm for 10 min to separate the supernatant and bacteria debris. The level of rTgMIC1 expression was analyzed by 12% SDS-PAGE. Furthermore, the separated supernatants were collected and purified by Ni column (Ni Sepharose 6 Fast Flow, GE Healthcare). The purification efficiency was analyzed via 12% SDS–PAGE.
2.5 TgMIC1 polyclonal antibody preparation and specific identification
For polyclonal antibody production, about 2 kg New Zealand white rabbits were immunized with 400 µg of purified rTgMIC1 diluted in 200 µL PBS was mixed with equal volume of complete Freund’s adjuvant (Sigma, USA) and multiple injected intradermally to the back of each rabbits. Two weeks later, the immunization was boosted with 200 µg rTgMIC1 protein in Freund's incomplete adjuvant, followed by a booster immunization once every 2 weeks. The ear vein blood was collected 7 days after the fourth immunization to measure the titer of antibodies by enzyme-linked immunosorbent assay (ELISA). The rabbit heart was then bled, and the antibodies were purified by Protein A. The serum mixed with equal volume of the binding buffer to equilibrate the column. After serum sample was loaded, rinse the column with binding buffer until the binding solution contains no protein. The presence of TgMIC1 polyclonal antibody was further identified by western blot and immunofluorescence assays.
2.6 ELISAanalysis
The titer in the serum from immunized rabbits was performed with ELISA. The purified rMIC1 protein was used to coat plate at 4°C overnight, followed by blocked with 5% BSA at 37°C for 60 min. The serum sample was serially diluted from 1:200 to 1:204800, and then 100 µL of diluted sample was added to each well incubating 60 min at 37°C. Following incubation with the secondary anti-rabbit antibodies conjugated to horseradish peroxidase to each well for 1 h. Thereafter, each well was incubated with 100 µL of tetramethylbenzidine (TMB, Beyotime, China) at 37°C for 10 min. Finally, 2 M H2SO4 (50 µL) was used to stop the reaction in the well, and the absorbance was read at 450 nm.
2.7 Immunofluorescence assays (IFA)
HFF monolayers were grown on coverslips placed in wells of a 6-well plate at a density of 105 cells per well and were incubated for 24 h. Then, cells were challenged with T. gondii GFP-RH strains for 24 h. After washing with phosphate buffered saline (PBS) three times, the coverslips were fixed with 4% paraformaldehyde (PFA) for 20 min. The coverslips were permeabilized with 0.1% Triton X-100 (Sigma, USA) for 30 min, and blocked for 30 min at 37°C with 5% BSA in PBS. After washing again, the coverslips were then incubated with primary antibodies overnight at 4°C, followed by goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, USA). Images were recorded using a Zeiss LSM880 confocal microscope.
2.8 Invasion assay
HFF monolayers were grown on coverslips placed in wells of a 6-well plate at a density of 105 cells per well and were incubated for 24 h. Then, cells were challenged with T. gondii TgCtwh3 and TgCtwh6 strains for 1 h. Non adherent parasites were washed away with PBS before fixation with 4% PFA for 20min. Adherent external parasites were detected using rabbit anti-T. gondii glides associated protein 45 (TgGAP45) antibodies, followed by secondary anti-rabbit antibodies coupled to Alexa488. After cell permeabilization with 0.1% Triton X-100 for 30 min, invaded intracellular and adherent external parasites were labeled with anti-GAP45 antibodies, followed by secondary anti-rabbit antibodies coupled to Alexa594. Fields were randomly selected in the same pattern for all samples and the number of external and internal parasites was counted.
2.9 Intracellular growth assay
The TgCtwh3 or TgCtwh6 parasites were allowed to invade HFF cells for 24 h. After washing with PBS three times, the coverslips were fixed with methyl alcohol for 5 min, followed by Wright Giemsa method and observed under microscope. About 50 PVs were randomly selected and the number of T. gondii tachyzoites in each PV was counted.
2.10 Statistical analysis
Typical results are shown with values expressed as means ± standard deviation of at least three independent experiments. The statistical significance of differences was performed using one-way ANOVA and t-test. The value of p < 0.05 was considered statistically significant.