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The protein containing the histidine acid phosphatase domain of Haemonchus contortus is an antigen stimulating the Th1 immune response of goat PBMC | Parasites and vectors

Cloning, expression and western blot analysis of Hc-HAP

No signal peptide and transmembrane structure were found in the Hc-HAP protein sequence (Supplementary File 2: Figures S1, S2). Adult cDNA H. contortion worms was used as a template to amplify the Hc-HAP gene using HAP-F and HAP-R primers, and the size of the amplified target gene was 990 bp (Fig. 1a). The Hc-HAP gene was successfully inserted into the pET32a vector and verified by enzymatic digestion and online BLAST analysis (Fig. 1b). The recombinant plasmid (pET32a/Hc-HAP) was induced by IPTG to be expressed in E.coli BL21 (DE3). After separation by SDS-PAGE, Coomassie Brilliant Blue staining revealed that the size of the rHc-HAP fusion protein was approximately 52 kDa (Fig. 1c). The rHc-HAP protein was expressed as inclusion bodies (Fig. 1d) and purified by passage through the HisTrap TM FF column. SDS-PAGE showed a single band for purified rHc-HAP, indicating good purification (Fig. 1e).

Fig. 1

Cloning, expression and Western blot analysis of Hc-HAP. a Amplification of Hc-HAP. Routes: M DL 2000 DNA marker, 1 amplification products of the Hc-HAP gene. b Routes: M DL10000 DNA marker, 1 digestion of the plasmid pET-32a/Hc-HAP by enzymes. vsF Expression and purification of rHc-HAP and pET-32a. Way: M Standard protein molecular weight marker. vs Routes: 1, 2 pET-32a induced by IPTG for 0 and 5 h, 3–8 pET-32a/Hc-HAP induced by IPTG for 0 to ca. 5h. D Routes: 1 Supernatant of expression products, 2 inclusion bodies of expression products. e Way: 1 purification of rHc-HAP. F Way: 1 purification of pET-32a. g Western blot analysis of rHc-HAP protein. Routes: M Standard protein molecular weight marker, 1 rHc-HAP detected by serum incubated with H. contortion goat, 2 no protein was detected with normal goat sera. Abbreviations: Hc-HAP, histidine acid phosphatase gene H. contortion; IPTG, isopropyl-β-D-thiogalactopyranoside; rHC-HAP, purified recombinant Hc-HAP

As shown in Fig. 1g, rHc-HAP was recognized by sera from goats infected with H. contortion, while normal goat sera did not recognize rHc-HAP. This result suggests that Hc-HAP is exposed to the host immune system during H. contortion infection and has the potential to be a candidate vaccine antigen.

Sequence alignment analysis and molecular modeling of Hc-HAP

The sequence of the cloned Hc-HAP gene was confirmed by BLAST analysis and the nucleotide sequence was translated into 329 amino acid residues by the ExPASy Translate tool (https://web.expasy.org/translate/). Multiple sequence alignment of Hc-HAP with homologous sequences available on the NCBI database revealed that Hc-HAP was closely related to the HAP of Teladorsagia circumcincta (72.46%), Ancylostoma ceylanicum (57.67%), American Necator (48.48%), Caenorhabditis briggsae (48.77%), Caenorhabditis remanei (49.24%), Caenorhabditis elegans (47.26%), Oesophagostomum dentatum (42.81%), Teladorsagia circumcincta (55.65%), Loa loa (35.0%) and brugia malayi (32.41%) (Fig. 2a). The SWISS-MODEL server provided the best model to predict the three-dimensional Hc-HAP model and rat acid phosphatase (1rpa.1.A) with 31.36% identity (Fig. 2b).

Figure 2
Figure 2

Sequence alignment analysis and molecular modeling of Hc-HAP. a Multiple sequence alignment analysis of amino acid sequences of Hc-HAP with HAP homologs from other species [Teladorsagia circumcincta (72.46%), Ancylostoma ceylanicum (57.67%), Necator americanus (48.48%), Caenorhabditis briggsae (48.77%), Caenorhabditis remanei (49.24%), Caenorhabditis elegans (47.26%), Oesophagostomum dentatum (42.81%), Teladorsagia circumcincta (55.65%), Loa loa (35.0%), Brugia malayi (32.41%)]. b The SWISSMODEL server provided the best model to predict the three-dimensional Hc-HAP model and rat acid phosphatase (1rpa.1.A) with 31.36% identity

Relative abundance of the Hc-HAP gene transcript at different life stages of H. contortion

The relative levels of Hc-HAP gene transcription at different developmental stages of the H. contortion were examined by qPCR. As shown in Figure 3, Hc-HAP transcript levels were significantly upregulated at the L3 level [t-test, t(8) = 22.29, P < 0.0001] and xL3 [t-test, t(8) = 7.916, P = 0.0014] stages relative to the egg stage. However, transcription was significantly downregulated in adults. [female (t-test, t(8) = 9.737, P = 0.0006] and male [t-test, t(8) = 60.91, P < 0.0001) stages and was higher in female adults than in male adults [t-test, t(8) = 6.466, P = 0.0029].

Figure 3
picture 3

Transcriptional analysis of the Hc-HAP gene at different developmental stages of H. contortion. The relative quantities (with respect to the state of the egg: Egg = 1) are represented by mean values ​​± SEM. The results presented here are representative of three independent experiments. Asterisks indicate significant differences to *PPPP

Immunolocalization of native Hc-HAP protein in adults H. contortion

The localization of the Hc-HAP protein in adult nematodes was examined by IFA. Figure 4 shows the longitudinal sections of female and male worms, respectively, where the red fluorescent signal represents the Hc-HAP protein. Expression of rHc-HAP was observed in intestinal microvilli (the main sites of expression of the Hc-HAP protein), gonads and body wall of H. contortion. Control sections showed no red fluorescent signal.

Figure 4
number 4

Immunolocalization of Hc-HAP in adults H. contortion. Red color indicates location of target protein stained with Cy3 dye in adult worms, and blue color indicates location of nuclei stained with DAPI fluorescent stain. Merge the image of DAPI and Cy3. Organs annotated in the Merge image are intestinal microvilli (white arrow), gut cavity (asterisks). Scale bars: 100 μm

Binding of rHc-HAP protein to goat PBMCs

Goat PBMCs were incubated with rHc-HAP protein, and the ability of PBMCs to bind rHc-HAP protein was determined by IFA. IFA results showed that red fluorescence was observed on the surface of rHc-HAP protein-treated PBMCs, while no red fluorescence was detected in the control group (Fig. 5). These results suggest that the rHc-HAP protein can bind to goat PBMCs.

Figure 5
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Binding of rHc-HAP protein to goat PBMCs. The red color indicates the localization of the target protein stained with Cy3 on the PBMCs and the blue color indicates the localization of the nuclei stained with DAPI. Merge the image of DAPI and Cy3. No red fluorescence was observed in the control. Scale bars: 20 μm. Abbreviation: PBMC, Peripheral Blood Mononuclear Cells

Effect of rHc-HAP protein on the proliferation of goat PBMCs

The effect of rHc-HAP protein on PBMC proliferation was examined using a CCK-8 kit. The results showed that the rHc-HAP protein did not affect the proliferation of goat PBMCs [pET-32a: ANOVA, F(4, 10) = 1.591, P = 0.7556; 10 μg/ml: ANOVA, F(4, 10)  = 1.591, P = 0.2507; 20 μg/ml: ANOVA, F(4, 10) = 1.591, P = 0.6182; 40 μg/ml: ANOVA, F(4, 10) = 1.591, P = 0.2736] (Fig. 6).

Figure 6
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Influence of rHc-HAP on PBMC proliferation. PBMCs were treated with phosphate-buffered saline (negative control), purified pET-32a protein (positive control), and serial concentrations of rHc-HAP for 24 h. The cell proliferation index was determined by fixing the OD450 100% control group values. Data are presented as the mean ± SEM of three independent experiments

Effect of rHc-HAP protein on goat PBMC apoptosis

The effect of rHc-HAP protein on PBMC apoptosis was examined by Annexin V-FITC Apoptosis Detection Kit. Results showed that rHc-HAP protein did not affect apoptosis of goat PBMCs [pET-32a: ANOVA, F(4, 10) = 0.609, P = 0.2317; 10 μg/ml: ANOVA, F(4, 10) = 0.609, P = 0.4777; 20 μg/ml: ANOVA, F(4, 10) = 0.609, P = 0.1987; 40 μg/ml: ANOVA, F(4, 10) = 0.609, P = 0.5198] (Fig. 7).

Picture 7
number 7

Effect of rHc-HAP on apoptosis of goat PBMCs. Cells were incubated with serial concentrations of rHc-HAP and pET-32a for 24 h at 37°C and 5% CO2. Data are presented as the mean ± SEM of three independent experiments

Effect of rHc-HAP protein on NO secretion from goat PBMCs

As shown in Figure 8, rHc-HAP protein increased nitrate levels in cell supernatant in a dose-dependent manner, indicating that rHc-HAP protein upregulates NO production in PBMCs from goat. [pET-32a: ANOVA, F(4, 10) = 15.417, P = 0.9230; 10 μg/ml: ANOVA, F(4, 10) = 15.417, P = 0.0298; 20 μg/ml: ANOVA, F(4, 10) = 15.417, P = 0.0011; 40 μg/ml: ANOVA, F(4, 10) = 15.417, P < 0.0001] (Fig. 8a). As expected, rHc-HAP protein promoted iNOS expression in goat PBMCs in a dose-dependent manner [pET-32a: ANOVA, F(4, 10) = 27.770, P = 0.5905; 10 μg/ml: ANOVA, F(4, 10) = 27.770, P = 0.0083; 20 μg/ml: ANOVA, F(4, 10) = 27.770, P = 0.0002; 40 μg/ml: ANOVA, F(4, 10) = 27.770, P < 0.0001] (Fig. 8b).

Figure 8
figure 8

Effect of rHc-HAP on NO secretion from goat PBMCs. Cells were incubated with serial concentrations of rHc-HAP and pET-32a for 24 h at 37°C and 5% CO2. a The effect of rHc-HAP on NO release from PBMCs was examined using a commercial kit. b The effect of rHc-HAP on the expression level of iNOS in PBMCs was analyzed using Western blot assays. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate a significant difference to *PPPP

rHc-HAP protein promoted immune responses in helper T cells of goat PBMCs

The effect of rHc-HAP protein on IFN-γ transcript levels [t helper type 1 (Th1)], IL-4 (Th2), IL-9 (Th9) and IL-17 (Th17) in goat PBMCs were analyzed in qPCR assays. The results are shown in Figure 9a. The rHc-HAP protein dose-dependently upregulated the transcriptional abundance of IFN-γ [pET-32a: ANOVA, F(4, 10) = 3.492, P = 0.8125; 10 μg/ml: ANOVA, F(4, 10) = 3.492, P = 0.0448; 20 μg/ml: ANOVA, F(4, 10) = 3.492, P = 0.0274; 40 μg/ml: ANOVA, F(4, 10) = 3.492, P = 0.0234] in goat PBMCs and dose-dependently downregulated the transcriptional abundance of IL-4 [pET-32a: ANOVA, F(4, 10) = 85.052, P = 0.1759; 10 μg/ml: ANOVA, F(4, 10) = 85.052, P < 0.0001; 20 μg/ml: ANOVA, F(4, 10) = 85.052, P < 0.0001; 40 μg/ml: ANOVA, F(4, 10) = 85.052, P < 0.0001] in goat PBMCs. However, rHc-HAP protein did not affect IL-9 transcription [pET-32a: ANOVA, F(4, 10) = 0.447, P = 0.5977; 10 μg/ml: ANOVA, F(4, 10) = 0.447, P = 0.8713; 20 μg/ml: ANOVA, F(4, 10) = 0.447, P = 0.3283; 40 μg/ml: ANOVA, F(4, 10) = 0.447, P = 0.6646] and IL-17 [pET-32a: ANOVA, F(4, 10) = 0.436, P = 0.3884; 10 μg/ml: ANOVA, F(4, 10) = 0.436, P = 0.2796; 20 μg/ml: ANOVA, F(4, 10) = 0.436, P = 0.5787; 40 μg/ml: ANOVA, F(4, 10) = 0.436, P = 0.8156] in goat PBMCs. We also analyzed the effect of rHc-HAP protein on the expression levels of IFN-γ, IL-4, IL-9, IL-17, STAT1 and of p-STAT1 in goat PBMCs by Western blot assays. The results are shown in Figure 9b. rHc-HAP protein dose-dependently promoted IFN-γ expression [pET-32a: ANOVA, F(4, 10) = 24.013, P = 0.8274; 10 μg/ml: ANOVA, F(4, 10) = 24.013, P = 0.0022; 20 μg/ml: ANOVA, F(4, 10) = 24.013, P < 0.0001; 40 μg/ml: ANOVA, F(4, 10) = 24.013, P < 0.0001] and p-STAT1 [pET-32a: ANOVA, F(4, 10) = 65.669, P = 0.8063; 10 μg/ml: ANOVA, F(4, 10) = 65.669, P < 0.0001; 20 μg/ml: ANOVA, F(4, 10) = 65.669, P < 0.0001; 40 μg/ml: ANOVA, F(4, 10) = 65.669, P < 0.0001], which indicated that the rHc-HAP protein activated the IFN-γ/STAT1 signaling pathway in goat PBMCs. Consistent with qPCR results, rHc-HAP protein dose-dependently inhibited IL-4 [pET-32a: ANOVA, F(4, 10) = 10.425, P = 0.4348; 10 μg/ml: ANOVA, F(4, 10) = 10.425, P = 0.0023; 20 μg/ml: ANOVA, F(4, 10) = 10.425, P = 0.0024; 40 μg/ml: ANOVA, F(4, 10) = 10.425, P = 0.0004] expression in goat PBMCs. However, rHc-HAP protein had no significant effect on IL-9 expression. [pET-32a: ANOVA, F(4, 10) = 1.281, P = 0.3957; 10 μg/ml: ANOVA, F(4, 10) = 1.281, P = 0.1385; 20 μg/ml: ANOVA, F(4, 10) = 1.281, P = 0.3424; 40 μg/ml: ANOVA, F(4, 10) = 1.281, P = 0.0601]IL-17 [pET-32a: ANOVA, F(4, 10) = 0.198, P = 0.8814; 10 μg/ml: ANOVA, F(4, 10) = 0.198, P = 0.4789; 20 μg/ml: ANOVA, F(4, 10) = 0.198, P = 0.9921; 40 μg/ml: ANOVA, F(4, 10) = 0.198, P = 0.6766] and STAT1 in goat PBMCs. These results suggest that the rHc-HAP protein upregulates the Th1 immune response in PBMCs while downregulating the Th2 immune response in PBMCs.

Figure 9
number 9

To assess the effect of rHc-HAP on the transcriptional expression of IFN-γ, IL-4, IL-9, IL-17 and p-STAT1 in goat PBMCs . a Transcript levels of IFN-γ, IL-4, IL-9 and IL-17 were detected by qPCR assays. b Expression levels of IFN-γ, IL-4, IL-9, IL-17, p-STAT1 and STAT1 were detected by Western blot assays. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate significant differences to *PPPP