Animals and Parasites Preservation
BALB/c mice (body-weight 18-20g) and SD (Sprague-Dawley) rats (body-weight 220-250g) were bought from Qinglongshan, Animal breeding farm, Nanjing, Jiangsu P.R. China (certified: SCXK 2008-0004); maintained under supervised condition by Animal House of Nanjing Agricultural University. T. spiralis (ISS534) utilized in this experiment was isolated from a pig in Nanyang, Henan Province, China, and were preserved by serial passage in BALB/c mice after every 6–8 months. T. spiralis muscle larvae (ML) were restored from BALB/c mice by 40 dpi (days post-infection) with standardized HCl-pepsin digestion technique (27). Adult worms (AL) were retrieved from intestine at 6 dpi, and new borne larvae (NBL) were recovered from a female adult at 6 dpi from the RPMI-1640 culture media at 37°C for 24 h as previously described by (28, 29). The parasite collected at different development phases were homogenized and chilled in liquid nitrogen.
Sequence Analysis of Ts-MAPRC2
The whole-genome coding sequence of the Trichinella spiralis (Ts) membrane-associated progesterone receptor component-2 (Ts-MAPRC2) gene (GeneBank Accession No. XM_003375886.1) was primarily obtained from the online GeneBank, The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Sequence properties of Ts-MAPRC2 were studied from the Expasy website (http:// www.expasy.org/). The Phylogenetic analysis of the MAPRC2 (Trichinella spiralis) protein sequence was carried out with identical genes from the other strains using the Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Prediction of transmembrane in protein and N-terminal Signal peptide prediction were confirmed by online tools TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/).
Cloning of Ts-MAPRC2
The full-length sequence of Ts-MAPRC2 comprises 234 amino acids (aa) (705 bp). Fragment size of 97-234aa (225-705bp) from the Ts-MAPRC2 having conserved domain (104-173aa) was expressed in the current study. The Ts-MAPRC2 gene of T. spiralis was amplified using RT-PCR analysis. Specific sense (5’-GAATTC AATAGATTTCGTATAAAATGGACATCT-3’) and anti-sense (5’- AAGCTT TCACTGATCATCAACATCACAATCAGAG − 3) primers were used with restriction enzymes EcoR Ⅰ and Hind III. The amplified PCR materials were electrophoresed and cleansed through Gel-Extraction Kit (Omega, USA) and eventually ligated into cloning vector pMD19-T (TaKaRa, China). The recombinant (pMD19-T/ Ts-MAPRC2) plasmid was further processed into E. coli (DH5α) strain (Invitrogen, China) and cultivated in LB (Luria Bertani) medium with ampicillin (100 µg/mL). Positive bacterial (pMD19-T/ Ts-MAPRC2) clones were assured by digestion of restriction enzymes and confirmed through sequencing (Invitrogen, China).
Development of recombinant Ts-MAPRC2 (rTs-MAPRC2)
The restriction digestion of the plasmid (pMD19-T/ Ts-MAPRC2) was carried out by using the enzymes viz., EcoR Ⅰ, and Hind III were cloned into the prokaryotic expression vector pET-32a (+) (Novagen, USA). Recombinant plasmid (pET32a (+)/Ts-MAPRC2) was then processed into BL21 (DE3) and induced protein expression by 1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich). Further, cells were harvested and lysed by lysozyme (10 µg/mL) (Sigma-Aldrich, USA), pursued by sonication. The sonicated outputs were subsequently confirmed on the SDS-PAGE (12% w/v). Recombinant Ts-MAPRC2 (rTs-MAPRC2) protein was purified by the His-TrapTM FF column following the manufacturer's instructions (GE Healthcare, USA), and protein concentration was calculated by Pierce-TM BCA-Protein Assay Kit (Thermo Scientific, USA). Also, empty pET32a (histidine-tagged) protein was purified and expressed using the same procedure mentioned above for Ts-MAPRC2, and utilized vector-protein as a negative control. Pictures of the SDS-PAGE carrying the rTs-MAPRC2 purified protein were taken. The stock of rTs-MAPRC2 protein was prepared and preserved at − 80°C until the next experiments.
Generation of the rat-polyclonal antibody of rTs-MAPRC2
For anti-sera preparation, SD-rats (n = 3) were immunized subcutaneously with rTs-MAPRC2 protein (300 µg) combined with Freund's complete adjuvant (Sigma–Aldrich, Darmstadt, Germany) equally. After two weeks, the second dose of 200 µg rTs-MAPRC2 protein was inoculated with Freund's Incomplete Adjuvant (Sigma–Aldrich). Again, we repeated the same two booster doses at an interval of one week. After one week of the last dose, serum samples were collected and stored at − 80°C for further experiments. Serum collected from non-treated rats (n = 3) was used as a negative control.
Immuno-blot assay of rTs-MAPRC2
Separation of rTs-MAPRC2 protein carried by SDS-PAGE (12%), and were transferred to nitrocellulose membrane (Millipore, USA). Afterward, the membrane was blocked with skim milk (5% w/v) powder in TBST (0.5% Tween-20 in TBS) at 37°C\(\)for 2h. Then washed three times with TBST, and membrane again incubated with primary antibody (rat- anti-sera- against rTs-MAPRC2) at 37°C for 1h (1:300 dilution). For negative control, we used the normal rat serum. Following this, the strips were washed three times and again incubated with secondary antibody conjugated-Horseradish peroxidase (HRP) goat-anti-rat IgG (1:3000 dilutions) (Sigma, St. Louis, MO, USA) for 2h at 37°C. In the end, the immune-reaction were appeared within 3–5 min, as per the manufacturer's directions of the DAB-HRP color development kit (Beyotime, China).
Determine binding ability of rTs-MAPRC2 protein with progesterone
In order to determine the binding ability of rTs-MAPRC2 protein with progesterone, we used the progesterone antibody (PROG-Ab) kit (Sandwich ELISA kit, FY95030-B, Feiya-Biotechnology, China), the pET-32a and PBS (phosphate buffer saline) were used as a control for the relative comparison. Initially, a standard curve was prepared using different concentrations (ng/mL) of the standard sample. According to kit instructions, 50 µl of the standard sample was added into three wells. Sample diluent (40 µl) was combined with rTs-MAPRC2 protein (10 µl) and pET-32a (10 µl) protein and incubated at 37°C for 30 min. After this, it was washed with the diluted washing solution (20x) 5 times for 30 sec each. Then 50 µl HRP-Conjugate solution was poured in each well then incubated again at 37°C for 30 min. Again, it was washed with the diluted washing solution (20x) 5 times for 30 sec. Afterward, the 50 µl Chromogen-A and Chromogen-B were added and incubated for 10 min at 37°C. Finally, 50 µl of a stop solution was added, and the color change from blue to yellow was observed. The optimum density (OD) values were measured at 450 nm with a plate reader and compared (Thermo Fisher, Life Technologies).
Immunofluorescent assay of rTS-MAPRC2 at the different developmental stage
The cross-section of T. spiralis samples at different developmental stages, i.e., ML, F-AL, M-AL, & NBL were fixed in 4% formaldehyde-0.2% glutaraldehyde with PBS for 45 min, and liquid nitrogen was used as a frozen snap. With the use of a cryotome (CM1950-Frankfurt, Germany), worms were cut into cross-section pieces (10 µm thick) and cleansed by PBS. First, the 5% BSA (Bovine serum albumin) was treated with slides to block non-specific binding and continued by incubation with primary antibody (rat- anti-sera- against rTs-MAPRC2) as well as normal rat serum (control group) at 37°C for 2 h (1:300 dilution). Then both group slides were washed with PBS then incubated with Cy3-goat labeled anti-rat as a secondary antibody (Beyotime-Shanghai, China) for 1h at 37°C. Thereafter, the DAPI (diamidino-2-phenylindole) (Sigma, USA) stain was used for staining the nuclei of the worm cells for 5 min under darkness. Lastly, a laser confocal microscope (PerkinElmer, USA) was used to observed worm cross-section cells.
Relative mRNA expression of Ts-MAPRC2 gene at different stages incubated with progesterone (P4) and mifepristone (RU486) by in vitro
The powder form of progesterone (P4) and mifepristone (RU486) were purchased from Sigma (Sigma-Aldrich, USA), and dissolved in absolute ethanol to prepare the desired stock solution following company instruction. Furthermore, the stock solution was sterilized by using the filtration process with a 0.2 mm Millipore filter. The high levels of progesterone range were 100–200 ng/mL in rats, 81.9 ng/mL in mice, 25–30 ng/mL in pigs, and 25 ng/mL in pregnant women were determined (30–33). So, five different concentration of agonist progesterone (P300 ng/mL, P100 ng/mL, P30 ng/mL, P10 ng/mL, and P3 ng/mL) and also three concentrations of antagonist mifepristone (M300 ng/mL, M100 ng/mL, and M30 ng/mL) were prepared. Also, control (only RPMI), and control vehicle ethanol (EtOH-RPMI)(0.125%) were prepared described by (34). All the stages of the parasites viz., F-AL, M-AL, ML, and NBL were collected as described previously by (28, 29). The 2000 worms/well at all above parasite stages (ML, AL, NBL) were cultured in 6-well plates, and a 6-well precondition was used. The cultured medium consists of RPMI-1640, 10% heat-inactivated FBS (Fetal bovine serum), and 2% antibiotics (100 U/mL penicillin; 100 mg/mL streptomycin) (Gibco, Paisley, UK), and incubated at the 37°C and 5% of CO2 for 48 h, and the medium was changed after 24 h for all treatments. The female (F-AL) and male (M-AL) adult worms were separated under light microscopy using Axiovert Zeiss Microscope (25x Neo Plan objectives). Gene (Ts-MAPRC2) expression of all treated groups at various developmental stages was measured by relative quantitative PCR (qRT-PCR) as previously described by (35). Briefly, the Trizol technique was used for RNA extraction from treated groups at all stages of worms using a prime script RT reagent kit (Takara, CA, USA). The isolated RNA from each group at all parasite stages were reverse transcribed by using HiScript II Q RT SuperMix (Vazyme, Nanjing, China) kit using manufacturer instructions. The Ts-MAPRC2 gene-specific primers were used as follows: forward (5′-ACGATGTGACCCGAAAGAGA-3′) and reverse (5′-CATGCATAGCCCATTCACGT-3′). Quantitative amplifications were performed by BI 7500 Fast Real-time PCR System (Applied Biosystem, USA) with the use of Cham-QTM SYBR qRT-PCR master mix-Kit (Vazyme, Nanjing, China). GenBank Accession No. AF452239 GADPH (Glyceraldehyde-3-phosphate dehydrogenase) of Trichinella was used as an internal control. The primer designed for GADPH were followed as forward (5′-GTCGTGGCTGTGAATGATC-3′) and reverse (5′-GCTGCCCCACTTAATTGCTT-3′), and data were computed using the comparative Ct (2 − ΔΔCt) technique (35).
In-Vitro phenotypic effect of P4, RU486, and rTs-MAPRC2-Ab on F-AL and ML stages
In this experiment, the selected concentration of progesterone (P30 ng/mL), mifepristone (M100 ng/mL), and rTs-MAPRC2-Ab (rat- anti-sera- against rTs-MAPRC2 ) ratio (1:300 dilution) were used at F-AL and ML stages with both controls (only RPMI and EtOH-RPMI) collected as described above, and the previously described procedures were followed according to (28, 29). The 2000 worms/well of both stages (F-AL, ML) were cultured in a 6-well plate, and a 6-well precondition was used. The cultured medium consists of RPMI-1640, 10% heat-inactivated FBS (Fetal bovine serum), and 2% antibiotics (100 U/mL penicillin; 100 mg/mL streptomycin) (Gibco, Paisley, UK), and incubated at 37°C and 5% CO2 for 48 h with medium changed after 24 h for all treatments. The female (F-AL) and male (M-AL) adult worms were separated under light microscopy using Axiovert Zeiss Microscope (25x Neo Plan objectives) and observed the phenotypic appearance at both stages (F-AL and ML) by using an inverted bright field microscope (Olympus, Shibuya, Japan). In the F-AL stage, the pregnancy maintenance or aborted to new borne larvae (NBL) were observed. While at the ML stage ecdysis (molting process) and motility of parasites were noted.
The phenotypic effect and relative mRNA expression of mifepristone on F-AL stage by In-Vivo
To determine the phenotypic effect of mifepristone on F-AL (female adult worm) of Trichinella spiralis, a total of 12 BALB/c mice were divided into three groups (i) mifepristone (M100 ng/mL) administration, (ii) adjuvant group EtOH (Olive oil-ethanol 3:1), and (iii) control group. Mifepristone (M100 ng/mL) powder from Sigma (Sigma-Aldrich, USA) was dissolved in a mixture of Olive oil-ethanol (EtOH) and injected subcutaneously (SC) into BALB/c mice every 24 h till retrieved AL (Adult worms) from the intestine at 6 dpi (days post-infection) with 0.1 mL of the suspension comprising boost dose of steroid according to the protocol of González et al. (1997). In the adjuvant group EtOH (Olive oil-ethanol), BALB/c mice were administrated only adjuvant EtOH with the same protocol, and the control group was kept without any drug therapy. On day six of mifepristone (M100 ng/mL) treatment, all groups of mice have orally injected 500 T.spiralis muscle larvae (ML) with the help of gastric cannula (27). Adult worms (AL) were collected from the intestine at 6 dpi (days post-infection) and adult worms (F-AL, M-AL) were separated using Axiovert Zeiss Microscope (25x Neo Plan objectives) and observed the phenotypic appearance, especially pregnancy, occurred or not using an inverted bright field microscope (Olympus, Shibuya, Japan). The gene (Ts-MAPRC2) expression of all the three groups at the F-AL ( female adult worm) developmental stage was also measured by relative quantitative PCR (qPCR) as described above and previously by (35).
The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's and LSD (Least significant difference) using analytical statistics software (Statistix 8.1, USA, 2003). The qRT-PCR data were normalized using 2^−ΔΔCt in Microsoft Excel 2010 (Redmond, Washington, USA). Origin software (Origin Pro 2021) was used to perform figures (Origin Lab Corporation, Northampton, MA, USA). Statistical data were presented as mean ± SD (n = 3). P ≤ 0.05, P ≤ 0.01 were considered significant.