All animal experiments were performed according to the guidelines of the Animal Welfare Law and Regulations of the Department of Health and Human Services, China and were reviewed and approved by the South China Agricultural University Animal Care and Use Committee (approval number: SYXK(Yue)2019-0136).

The Ace-CRT gene was amplified using the specific primer pair Ace-CRT-F (5′-ACGGCCAGTGAATTCATGGCTGT-3′) and Ace-CRT-R (5′-CTATAAAAGCTTGGCG-3′) designed according to the A. ceylanicum CRT gene sequence (GenBank accession no.: EYB83200.1). Each PCR reaction (25 μl) contained 0.5 μl cDNA, 1 μl each of the forward and reverse primers, 12.5 μl 2× PrimeStar HS PCR Master Mix (TaKaRa, Dalian, China), and 10 μl ddH₂O. The cycling conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 30 sec, 54.8°C for 40 sec, and 72°C for 90 sec, and 72°C for 8 min. The PCR products were visualized following 1% agarose gel electrophoresis and extracted and purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). Then, the purified PCR product was ligated to a pUC-57 vector (Tsingke, Beijing, China) and transformed into E. coli DH5α competent cells (TaKaRa). Bacterial PCR was used to screen for positive clones, which were then sequenced by Sangon Biotech (Shanghai, China). The amino acid sequences of Ace-CRT were predicted using DNAMAN software 6.0. The size and position of the signal peptide in the coding sequence region of the Ace-CRT amino acid sequence were analyzed using the online software SignalP5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/). The transmembrane domain of the Ace-CRT amino acid sequence was investigated using the online tool TMPred (https://bio.tools/TMPred). ProtScale software (https://www.expasy.org/search/protscale) was used to determine Ace-CRT hydrophobicity/hydrophilicity. The tertiary structure was predicted using the online tool SWISS-MODEL (http://swissmodel.expasy.org/).

The purified PCR products and expression vector pET28a were digested using NheI and HindIII restriction enzymes (New England Biolabs, Ipswich, UK), ligated using T4 DNA ligase, and transformed into E. coli BL21 competent cells (Sangon Biotech) for protein expression. After culture (200 rpm, 37°C) and induction for 16 h with 0.5 mM IPTG, the bacteria were collected by centrifugation and suspended in 10 ml PBS. The bacterial suspension was ultrasonicated on ice and the resulting cell lysates were centrifuged at 12,000 g for 5 min. The supernatant was transferred to a Ni-NTA affinity chromatography column (Beyotime, Shanghai, China), allowed to stand overnight at 4°C, and eluted in imidazole solution with a 0–50 mM gradient. The supernatants were precipitated. We eluted proteins after which were separately analyzed by SDS-PAGE with Coomassie brilliant blue staining (Solarbio, Beijing, China).

Three dogs were infected by gavage with third-stage A. ceylanicum larvae. At 8 weeks after infection, blood samples (2 ml) were collected from the cephalic vein of infected dogs (positive serum) and healthy dogs (n=3, negative serum) in centrifuge tubes and refrigerated at 4°C overnight. Then, the serum was collected from each sample by centrifuging at 4,000 g for 15 min. The purified Ace-CRT protein was electrotransferred to a nitrocellulose membrane (Sangon Biotech). Western blot was performed with mouse anti-His tag antibody (1:3,000; Sangon), positive canine serum, and negative canine serum as primary antibodies. Horseradish peroxidase-conjugated goat anti-mouse and rabbit anti-dog antibodies (1:3,000; Sangon) were used as secondary antibodies. The specificity and antigenicity of the recombinant protein were tested using a DAB western blot detection kit (Solarbio).

We sacrificed 10 healthy 6-week-old BALB/c mice and soaked them in alcohol for disinfection before removing the spleen from the abdominal cavity, crushing it into pieces, and filtering it through a 200-mesh sieve to collect the primary splenocytes. Then, RPMI-1640 medium supplemented with 10% fetal bovine serum was added to the isolated splenocytes and centrifuged at 2,400 g for 5 min. After decanting the supernatant, splenocyte washing and centrifugation were repeated. Then, the cell pellets were harvested and resuspended in RPMI-1640 medium, and the number of viable cells was counted following trypan blue staining (Sangon Biotech). Samples with >95% viability were selected for subsequent analysis.

The mouse splenocytes (2×10³) were added to each well of a 96-well plate and cultured for 24 h in a 5% CO₂ cell culture incubator at 37°C. After stabilization, 100 μl of rAce-CRT at concentrations of 15, 30, 60, and 90 μg/ml and BSA (negative control) were added to each well and placed in a 5% CO₂ cell culture incubator (37°C, 24 h). Finally, the absorbance values at 570 nm were measured in each group following the addition of MTT.

We used a Lymphocyte Isolation Kit (TBD, Tianjin, China) to isolate canine PBMCs from a 2-ml sample of anticoagulated blood collected from a healthy dog, as described previously [12]. After lymphocyte growth was stabilized, the experimental groups (with purified protein), blank control, and negative control (BSA) were prepared. Eight working concentrations of purified protein (5, 10, 15, 30, 45, 60, 75, and 90 μg/ml) were used in the experimental groups, with 3 repetitions per group. The prepared plate was incubated in a 5% CO₂ cell culture incubator (37°C, 24 h). Then, the cell suspension was centrifuged, and the precipitates were collected for extraction of PBMC total RNA using an E.Z.N.A. Total RNA Kit (Omega Bio-Tek) according to the manufacturer’s instructions. A microplate reader was used to assess the RNA quality (optical density [OD]_{260 nm}/OD_{280 nm}≈2.0), and agarose gel electrophoresis was performed to determine RNA integrity. The recovered PBMC total RNA was used to synthesize first-strand cDNA by reverse transcription using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Fluorescence quantitative reverse-transcription PCR was performed using a LightCycler 96 Instrument (Roche, Basel, Switzerland) to detect cytokine expression in PBMCs stimulated with different concentrations of Ace-CRT. Supplementary Table S1 shows the sequences and lengths of the real-time PCR primers used to target canine 18S rRNA and the genes for cytokines IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-23, and IFN-γ. Each reaction (10 μl) comprised 5 μl ChamQ Universal SYBR qPCR Master Mix (Vazyme), 0.4 μl of each forward and reverse primer, 1 μl cDNA, and 3.2 μl ddH₂O. The qPCR reaction conditions were 50 cycles of 95°C for 1 min, 95°C for 10 sec, and 60°C for 30 sec. 18S rRNA was used as the internal reference gene, and each sample was assayed in triplicate. The relative cytokine expression levels were calculated using the 2-ΔΔCT method [21].

We performed a hemolysis assay with a slight modification of the previous method [22]. Briefly, whole blood from a healthy dog was collected into a clean centrifuge tube and left to stand for 1 h at room temperature, followed by 2 h at 4°C. Then, the sample was centrifuged at 6,000 g for 10 min at 4°C to collect the serum. Next, the serum was diluted to 1:2, 1:5, 1:10, 1:20, 1:40, 1:80, and 1:160 with barbitone buffer solution (BBS-GM) to determine the optimal serum dilution. Each serum dilution was mixed with sensitized sheep erythrocytes and BBS-GM (200 μl each) for the hemolysis test, with 3 replicates per group. A mixture of sheep erythrocytes (2%, 100 μl) and ultrapure water (500 μl) was considered the complete hemolysis group, and its absorbance was used as the standard to calculate the hemolysis rate. Next, the optimal dilution of canine serum (200 μl) was mixed with 200 μl of rAce-CRT at different concentrations (5, 10, 15, and 25 μg/ml). The serum/rAce-CRT suspensions were then gently mixed with 200 μl of sensitized sheep erythrocytes, incubated at 37°C for 30 min in a water bath, and centrifuged at 2,400 g for 10 min. The supernatant (150 μl) was collected from each group to measure the absorbance at 405 nm (A1). BBS-GM, rAce-CRT, and sensitized sheep red blood cells were used as the control group to measure absorbance at 405 nm (A2). BBS-GM, canine serum, and sensitized sheep erythrocytes (200 μl each) were used as the complement group for measuring the absorbance at 405 nm (A0). The hemolysis inhibition rate was calculated as follows: (1− [A1−A2]/A0)×100 and expressed as a percentage.

Data were expressed as the mean±SD. All results were analyzed using SPSS 22.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 6.2 (GraphPad, San Diego, CA, USA). A P-value <0.05 was considered statistically significant.

Using A. ceylanicum cDNA as the template, PCR targeting Ace-CRT yielded a PCR product of approximately 1,200 bp (Supplementary Fig. S1A). Following cloning, PCR of the bacterial suspension resulted in amplified bands corresponding to the expected fragment size (Supplementary Fig. S1B). After double-enzyme digestion of the recombinant plasmid pET28a-Ace-CRT, a fragment of approximately 1,200 bp (Supplementary Fig. S1C) was obtained, consistent with the expected target size.

We used SDS-PAGE to analyze the bacterial culture (before and after expression induction), after which purified protein. The protein of interest was predominantly expressed in the supernatant, and optimal purification was achieved using 50 mm imidazole. The size of the recombinant protein was approximately 50 kDa (Supplementary Fig. S2), consistent with the expected size of the target protein. Western blot analysis revealed that the recombinant protein was recognized by anti-His tag antibodies, showing a specific band at 50 kDa (Supplementary Fig. S3A). The recombinant protein was also recognized by positive canine serum, showing a distinct band at 50 kDa. No reaction bands were observed with negative canine serum (Supplementary Fig. S3B), indicating reliable antigenicity of the recombinant protein.

We measured the OD_{570 nm} of mouse primary splenocytes after coculturing with different rAce-CRT concentrations to assess the effect of rAce-CRT on splenocyte proliferation. rAce-CRT significantly stimulated mouse primary splenocyte proliferation (P<0.05) (Fig. 1). The rate of splenocyte proliferation following coculture with 15, 30, 60, and 90 μg/ml rAce-CRT was significantly increased (P<0.05) and was highest in the coculture with 15 and 30 μg/mL rAce-CRT (Fig. 1). These findings suggest that Ace-CRT promotes immune cell proliferation and plays an immunomodulatory role.

After stimulating canine PBMCs with different concentrations of rAce-CRT, the expression levels of IL-4, IL-10, IL-12, and IL-13 were increasecl compared with the expression levels in the control group (P<0.05), but the difference between the rAce-CRT-stimulated group and the control group was only significant for IL-13 (P<0.05). In contrast, the expression levels of IL-5, IL-6, and IFN-γ decreased in the rAce-CRT-stimulated group compared with the expression levels in the control group (Fig. 2).

Increasing the canine serum dilution ratio resulted in a gradual reduction in the rate of hemolysis of sensitized sheep erythrocytes. The hemolysis rate was 90.7% when the serum dilution ratio was 1:50 (Fig. 3A). Compared with the control group, the different concentrations of rAce-CRT inhibited hemolysis of sensitized sheep erythrocytes. The hemolysis inhibition rate increased with an increasing protein concentration (Fig. 3B).