Reagents
In this study we use many reagents including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), which bought from Wisent (Montreal, QC, Canada). Puromycin (PM), penicillin and streptomycin were purchased from Sigma (St. Louis, Missouri, USA).
Parasites, Plasmid construction, and lentvirus infection
The acquisition of Toxoplasma gondii tachyzoites (type II) from the PRU strain was initially passaged through homogenate of the brain containing the encapsulated mouse. The open reading frame encoding TgGRA15II (missing signal peptide 15bp, http://ToxoDB.org) was amplified by real-time (RT)-PCR in the total RNA of the fast-growing spores, and the recombinant plasmid pEGFP-gra15II was obtained to obtain a recombinant lentivirus (LV) vector ( LV-pEGFP-gra15II)[16].
Cell culture
The murine HCC cells, Hepa1-6, were purchased from the Chinese Academy of Sciences Cell Bank in Shanghai. Mouse cell line RAW264.7 is kept by laboratory; Mouse peritoneal macrophage was injected into the mouse peritoneal cavity with 200 PRU tachyzoites, and the mice were anesthetically killed at 48, 72, 96 and 120h respectively. Peritoneal lavage solution (PEC) was collected under aseptic conditions, and then cultured in a 9cm petri dish with complete medium RPMI1640. After 3h, the non-adherent cells were washed away, and the adherent cells were counted as peritoneal macrophages, and the complete medium RPMI1640 was added. The medium used for all cells was 10%-15% serum and penicillin-streptomycin mixture in DMEM, and the culture conditions were 37 ° C and 5% CO 2.
Flow cytometry assay
Flow cytometry was used to detect GRA15II-transfected macrophages and M1 and surface markers. Specifically, four groups of cells (M1 LV-vector, M1 LV-gra15, M2 LV-vector, M2 LV-gra15) were separately prepared as single cell suspensions washed with PBS containing 1% fetal bovine serum and adjusted in concentration for to 1 × 106 cells per 100 µl PBS with 1% FBS, respectively. All cells were incubated at 4 ° C for 20 min in the dark, washed twice with PBS, and detected by flow cytometry, then use Flow Jo to analysis results.
Tumor Models
As for the subcutaneous tumor model, saline containing 3×106 hepa1-6 cells were injected subcutaneously into the right lower groin of mice. After three days, a lump was palpable and the tumor volume reached 50mm3. Tumors were observed in all infected mice. Thirty mice were randomly divided into 10 mice per group, namely, LV-gra15- Mφ, LV - Mφ and saline (NS) control. On the third and fifth days after inoculation, mice in the first and second groups were injected with saline containing 2.5 × 106 corresponding macrophages per 100ul through the tail vein, while mice in the control group were injected with saline of equal volume. On days 3, 5, 7, and 10 of inoculation, tumor size was measured using vernier calipers and 0.52* long tumor diameter * short tumor diameter. The animals were euthanized 10 days later before the results were analyzed.
RNA Extraction and qRT-PCR
Total RNA of the LV-vector and LV-gra15 groups was extracted with Trizol reagent, and RNA was reverse-transcripted into cDNA according to the manual using the Prime Script first Stand cDNA Synthesis Kit. Relative expressions of TNF-α, IL-12, IL-10 and Arg1 were detected by qRT-PCR using SYBR Premix Ex Taq Kit. The instrument used in the experiment was ABI Prism 7500 sequence detection system. The internal reference gene was GAPDH, and each sample was repeated three times to increase the accuracy of the data. Finally, the relative expression quantity is calculated by 2 - Δ Δ Ct to calculate.
Western Blotting Analyse
Total protein was extracted from the five groups of LV-gra15II, TNF-α positive, TNF-α negative, LV-vector, and TRAF6-KO co-cultured with Mφ, respectively. Separated with 12% SDS-polyacrylamide gel. Then, the membrane was transferred, 5% skim milk was blocked, the primary antibody was incubated overnight at 4 ° C, the secondary antibody was incubated for two hours, developed, and the band intensity was observed.
RNA Chip
Mouse ascites were taken and washed with PBS two to three times to collect toxoplasma tachyzoites. RNA was extracted with Trizol and sent to the company for related experiments.
ELISA
LV-gra15II-M1, LV-vector-M1, LV-gra15II-M2 and LV-vector-M2 were separately seeded in 6-well plates (2 × 106 cells per well), suspended in 2 ml common culture medium, and cultured at 37°C with 5% CO2 for 48 h. Afterwards, we collected four cell supernatants, LV-gra15II-M1, LV-vector-M1, LV-gra15II-M2 and LV-vector-M2, and determined IL-10, IL-12, respectively, using an ELISA kit. The concentration of these three cytokines was statistically different.
Immunoprecipitation
We collected LV-gra15II- Mφ and cell IP lysis buffer (containing protease inhibitor) added in, then lysed at 4 ° C for 30 minutes, and centrifuged at 12000 rpm for 30 minutes, last took the supernatant. A small amount of lysate was taken for WB analysis. For the remaining lysate, we added the corresponding 1 ul antibody and 10-50 ul protein A/G-beats to the corresponding lysate and incubate overnight at 4 °C. After co-immunoprecipitation, centrifuge at 3000r for 5 min at 4 °C, centrifuge protein A/G-beats to the bottom of the tube, discard the supernatant, rinse 3 times with 1 ml of lysis buffer, and finally add 15 ul of 2×SDS. Add sample buffer and boil for 10 minutes. Ultimately, the SDS-PACE, Western Blotting was to understand comprehend if GRA15 is connected with TRAF6, MyD88, RIP1I, and RAK1.
Animal care and ethical statements
C57BL/6 female mice (6 weeks old) were purchased from changzhou gavens laboratory animal company, China (production license no. Scxk2013-003).The relevant animal care and experimental programs are implemented strictly in accordance with the guidelines for the care and use of experimental animals of the national institutes of health of China (1998) and approved by the institutional review committee of the institute of biomedical sciences of anhui medical university (license no. Amu26-080610).Try to minimize animal suffering during the study.
Immunofluorescence technique
The five groups of cells (LV-gra15- Mφ, LV- gra15II -M1, LV-vector-M1, LV- gra15II -M2 and lv-vector-M2) were counted respectively. After culture for 24h, the culture solution was discarded and washed with PBS twice. Formaldehyde fixation, membrane penetration, sealing, overnight incubation with primary antibody, incubation with secondary antibody for one hour, and finally sealing tablet observation