Phylogenetic analyses of amino acid sequence data
The amino acid sequences of Hc-TGH-2 (GenBank Accession no. ACQ84508.1) and the homologues from 17 species (Ancylostoma caninum, Ascaris suum, Brugia malayi, Caenorhabditis briggsae, Caenorhabditis elegans, Capra hircus, Danio rerio, Heligmosomoides polygyrus, Homo sapiens, Mus musculus, Nippostrongylus brasiliensis, Parastrongyloides trichosuri, Strongyloides stercoralis, Strongyloides ratti, Teladorsagia circumcincta, Toxocara canis and Trichinella spiralis; Table 1) were aligned and then phylogenetic analyses of aligned sequence data were conducted using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods employing the Jones-Taylor-Thornton (JTT) model [32]. Confidence limits were assessed using a bootstrap procedure employing 1000 pseudo-replicates for NJ, MP and ML in MEGA v.6.0 [32]. A 50% cut-off value was implemented for the consensus tree. A TGF-β type II receptor from C. elegans (GenBank Accession no. CCD63118.1) was used as an outgroup for phylogenetic analyses.
Table 1
Sequences used for phylogenetic analyses in the present study.
Species | GenBank accession number | References |
Ancylostoma caninum | AAY79430.1 | [21] |
Ascaris suum | ADY41407.1 | [33] |
Brugia malayi | CDQ03041.1 | [34] |
Caenorhabditis briggsae | CAP21409.1 | [35] |
Caenorhabditis elegans | CCD61866.1 | [14] |
Caenorhabditis elegans1 | CCD63118.1 | [36] |
Capra hircus | XP_017903600.1 | [37] |
Danio rerio | AAN03678.2 | [38] |
Haemonchus contortus | ACQ84508.1 | [27] |
Heligmosomoides polygyrus | ACR27076.1 | [27] |
Homo sapiens | NP_005802.1 | [39] |
Mus musculus | NP_034402.1 | [40] |
Nippostrongylus brasiliensis | ACR27077.1 | [27] |
Parastrongyloides trichosuri | ABQ10586.1 | [23] |
Strongyloides stercoralis | AAV84743.1 | [24] |
Strongyloides ratti | AAT79346.1 | [23] |
Teladorsagia circumcincta | ACR27078.1 | [27] |
Toxocara canis | KHN71899.1 | [41] |
Trichinella spiralis | KRY30333.1 | [42] |
1 Sequence was used as the outgroup for phylogenetic analyses. |
Transcriptional analyses by real-time PCR
Total RNA was isolated from individual developmental stages of H. contortus (eggs, L1, L2, iL3, male and female L4s, male and female adults) using TRIzol (Life Technologies, USA). RNA integrity and yields were verified by electrophoresis and spectrophotometric analysis (NanoDrop Technologies). Complementary DNA (cDNA) was synthesised from RNA (1 µg) employing the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time; Takara, Japan). Nucleic acids were stored at -80 °C until use.
According to the isolated coding sequence of Hc-tgh-2 [27](GenBank Accession no. FJ391183), one set of primers Hc-tgh-2-rtF/R (Additional file 1: Table S1) were designed to detect the transcriptional level of Hc-tgh-2 in eight developmental stages of H. contortus by real-time PCR under the protocol as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s and 72 °C for 20 s. Hc-tub8-9 was set as a reference in all samples (in triplicate) [43] employing a set of specific primers Hc-tub8-9-rtF/R (Additional file 1: Table S1). The data of the real-time PCR were analyzed by comparing with the relative quantities of egg (egg = 1) using the 2−△△Ct method [44]. This assay was repeated three times.
Prokaryotic expression of Hc-TGH-2 and the localization of Hc-TGH-2 in H. contortus
According to the coding sequence of Hc-tgh-2 [27](GenBank Accession no. FJ391183), one set of primers Hc-tgh-2-eF/R were designed to amplify the partial cDNA (151–732 bp) of Hc-tgh-2 under the PCR cycling protocol: 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 40 s and 72 °C for 20 s; and then 72 °C/5 min. The amplicon was inserted into the expression vector pET-28a and the construct was transformed into E. coli Rosseta-DE3, then the truncated protein Hc-TGH-2 (51–244 aa) was expressed and purified, followed by SDS-PAGE detection. Purified recombinant Hc-TGH-2 protein (500 µg) was injected subcutaneously into two rabbits with multiple sites (4 immunizations, two weeks interval between each immunization). A pre-bleed was taken from each rabbit prior to first injection while a final bleed was taken one week after the last immunization. Serum was treated according to a standard procedure [45]. All the sera were analyzed by Western Blot using the total protein extracted from adults of H. contortus with the Total Protein Extraction Kit (Bestbio Company, China).
The serum was used to detect the expression patterns of Hc-TGH-2 in adult males and females of H. contortus by immunohistochemistry, respectively, as previously described [29]. In brief, approximately 50 H. contortus adult males or females were fixed in 4% paraformaldehyde (Biosharp, China) at 4 °C, respectively. Then the single worm was dehydrated in a series of graded ethanol (75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h once and 100% twice for 30 min) sequentially, followed by embedding in paraffin. Sections (4 µm) were cut and flattened on polysine slides, followed by paraffinating (xylene treated twice for 20 min) and rehydrating in a series of graded ethanol (100% twice for 10 min; 95% once for 5 min, 90% once for 5 min, 80% once for 5 min, 70% once for 5 min each), then washed with phosphate buffer solution (PBS) for three times (5 min). Antigens were recovered by the microwave, then endogenous catalase was eliminated by 3% hydrogen peroxide. The sections were washed with PBS for three times (5 min), then blocked with 5% bovine serum albumin (BSA) for 20 min in a humidified chamber. The sections were incubated with approximately 50 µl polyclonal anti-Hc-TGH-2 antibody (positive serum) or negative serum (each at 1:100 dilution) at 4 °C overnight, respectively. The serum was removed and the sections were washed with PBS for three times (5 min). Then the sections were incubated at 37 °C for 50 min in anti-rabbit immunoglobulin (IgG) (raised in sheep) conjugated with fluorescein (Aspen, China) in dark. The sections were then washed with PBS for three times (5 min) to remove the secondary antibody. After that, the sections were incubated at room temperature for 5 min in 4, 6-diamidino-2-phenylindole (DAPI) solution in dark. The sections were washed in PBS three times (5 min) again and then assessed in detail using an epifluorescence microscope (Olympus CX-21, Japan). All images were processed using Photoshop CS 6.0.
Preparation of double-stranded RNA, and RNA interference (RNAi) in H. contortus
Two sets of specific PCR primers (Hc-tgh-2-sF1/sR1 and Hc-tgh-2-sF2/sR2) were designed to amplify the coding sequences of Hc-TGH-2 functional domain (864 bp) for constructing two plasmids that were used to synthesize specific dsRNA (Additional file 1: Table S1) as previously described [28, 29]. One set of primers Hc-tgh-2-sF1/sR1 were tagged with T7 promoter site in forward direction and restriction enzyme BamH I site in reverse direction, respectively. Another set of primers Hc-tgh-2-sF2/sR2 were tagged with restriction enzyme BamH I site in forward direction and T7 promoter site in reverse direction, respectively. The sequences of Hc-TGH-2 functional domain (864 bp) were amplified by these two sets of specific PCR primers under the same following cycling conditions: 95 °C/5 min, followed by 95 °C/30 s, 55.4 °C/30 s; 72 °C/2 min for 35 cycles; and then 72 °C/5 min, and inserted into pTOPO-Blunt Simple vector (Aidlab Biotechnologies Co., Ltd) by a ClonExpress™ II One Step Cloning Kit (Vazyme Biotech Co., Ltd), respectively. The irrelevant control was a cry1Ac gene from Bacillus thuringiensis (Bt-cry1Ac, GenBank accession no. GU322939.1), which has no homology with any H. contortus gene [28, 29]. The sequence of Bt-cry1Ac was cloned using two sets of specific PCR primers Bt-cry1Ac-sF1/sR1 and Bt-cry1Ac-sF2/sR2 (Additional file 1: Table S1) under the following cycling protocol: 95 °C/5 min; followed by 35 cycles of 95 °C/30 s, 55 °C/30 s, 72 °C/1 min; then 72 °C/5 min, and inserted into pTOPO-Blunt Simple vector by the same way. All constructs were extracted by plasmid Maxi Kit (Aidlab Biotechnologies Co., Ltd) and then were estimated spectrophotometrically (NanoDrop Technologies) and stored at -20 °C until use, respectively. The restriction enzyme BamH I was used to linearize the constructs containing Hc-tgh-2 or Bt-cry1Ac fragments, respectively, and the linearized templates were verified by electrophoresis and spectrophotometric analysis (NanoDrop Technologies) and used to synthesize single-stranded RNA (ssRNA) by RNA large-scale T7 production system according to the instruction manual of MEGAscript® T7 Transcription Kit (Ambion, USA). Equal quantity (~ 500 µg) of sense ssRNA and antisense ssRNA were used to synthesize dsRNA with the treatment of 5 × annealing buffer at room temperature for 2 hours. The quality and yields of ssRNA and dsRNA were verified by electrophoresis and spectrophotometric analysis (NanoDrop Technologies), respectively. All RNA was immediately frozen and stored at -80 °C until use.
The RNA interference assay was performed as previously described [28]. Briefly, H. contortus L3s collected from faecal culture were exsheathed in 0.1% sodium hypochlorite/PBS for 30 min at 38 °C, followed by washing twice in sterile PBS by centrifugation at 600 g (5 min) at 23 °C and four times in PBS containing Antibiotic-Antimycotic solution (Gibco, USA). Then, the exsheathed L3s (xL3s) were suspended in Earle’s Balanced Salt Solution (EBSS, Sigma, USA; pH adjusted to 5.2) with Antibiotic-Antimycotic solution (Gibco, USA) with a final concentration at 33,000 xL3/mL and incubated with different dsRNA (1 mg/mL). Before incubation, the Hc-tgh-2 dsRNA, Bt-cry1Ac-specific dsRNA (irrelevant control) or nuclease-free water (blank control) were pre-incubated (separately) with RNasin (8 U) and Lipofectin Reagent (Invitrogen, USA) for 10 min at 25 °C (room temperature), respectively, and added into 30 µL of EBSS cultures containing xL3s. After incubation at 37 °C in 20% CO2 for 24 h, 300 larvae were transferred to 100 µL of new EBSS (in triplicate) and cultured for another 7 days and the replicate cultures were examined by microscopy to count the numbers of xL3s and L4s according to the morphological changes of the buccal capsule [28, 46, 47]. The remaining larvae were collected to isolate RNA for detecting the transcriptional change of the gene Hc-tgh-2 by real-time PCR with one set of primers Hc-tgh-2-rtF/R (Additional file 1: Table S1). The 18S rRNA was used as a reference marker [48] and the sequences of primers Hc-18 s-rtF/R were shown in Additional file 1: Table S1. This PCR cycling protocol was: 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s and 72 °C for 20 s. The efficiency of the PCR was calculated using an established formula and the real-time PCR data was subjected to analysis using the 2−△△Ct method [44].