Bioinformatic analyses
The coding sequences of Hcdaf5 were conceptually translated into predicted amino acids using DNA star software (http://www.dnastar.com/). The predicted amino acid sequence of HcDAF5 was compared with sequences in non-redundant protein databases using BLASTp from the National Center for Biotechnology Information (NCBI) to confirm the homologous sequences.
The structural domains (Dach boxes and SDS) of HcDAF5 were aligned with its homologues from 11 species (Ancylostoma ceylanicum, Brugia malayi, Caenorhabditis briggsae, C. elegans, Danio rerio, Drosophila melanogaster, Equus caballus, Homo sapiens, Mus musculus, Nippostrongylus brasiliensis, Toxocara canis) (Additional file 1, Table S2) using the program Bioedit. For HcDAF5, the coiled coil region is predicted by Expasy (https://embnet.vital-it.ch/software/COILS_form.html).
The homologues from eight species (C. briggsae, C. elegans, D. rerio, D. melanogaster, H. sapiens, M. musculus, N. brasiliensis, T. canis) (Additional file 1, Table S2) were aligned and used for phylogenetic analyses using the neigubour-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. Confidence limits were assessed using a bootstrap procedure employing 1000 pseudo-replicates in MEGA [6].
Transcriptional analyses of Hcdaf5 in different stages of H. contortus by real-time PCR
Real-time PCR was carried out using specific primers Hc-daf-5-qF/Hc-daf-5-qR (Additional file 1, Table S1) to determine the mRNA levels in different developmental stages of H. contortus including eggs, L1s, L2s, L3s and both sexes of L4s and adult worms. Total RNA of each stage was isolated with TRizol reagents according to the manufacture’s instruction and treated with DNase I to remove gDNA before synthesis of cDNA. The real-time PCR reaction condition included: one cycle at 50°C/2 min, 95°C/30 s; 40 cycles at 95°C/15 s, 60°C/15 s, 72°C/20 s; followed by a cycle at 60 ºC/1 min, 95 ºC/15 s, 60 ºC/15 s. Each sample was tested in triplicate, employing a β-tubulin of H. contortus (GenBank: M76493) as a reference gene (using specific primers Hc-tubulin-qF and Hc-tubulin-qR; Additional file 1, Table S1), and average threshold (Ct) was taken to compare the relative quantities with Am (Am = 1) using 2-△△Ct method [7]. This assay was repeated three times. Statistical analysis was carried out using one-way ANOVA in GraghPad Prism 6. P-values were calculated using the Turkey’s post-hoc test; values of < 0.05 were considered statistically significant.
Production of polyclonal antibody against recombinant HcDAF5 and immunoblot analysis
A pair of specific primers Hc-daf-5-pF/Hc-daf-5-pR (Additional file 1, Table S1) containing double restriction sites were designed according to the CDS of Hcdaf5 and used in PCR to amplify the CDS of Hcdaf5. Then amplified sequence was cloned into the prokaryotic expression vector to create the expression plasmid pET-28a-Hc-daf-5. Then this plasmid was transformed into BL21 (DE3) cells of Escherichia coli, followed by 1 mM isopropy β-D-1-thiogalactopyranoside (IPTG) induction at 16°C for 12 h to produce recombinant rHcDAF5. The recombinant rHcDAF5 was purified using a Ni Sepharose column system (GE, USA) according to the manufacturer’s protocol. Then purified rHcDAF5 was used in immunizing New Zealand white rabbit to produce anti-HcDAF5 polyclonal antibody. The titer and specificity of anti-HcDAF5 polyclonal antibody were determined by Enzyme-linked immunosorbent assay (ELISA) and Western blot.
Western blot was performed as follows. Proteins were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon®-PSQ membrane (Merck Millipore Ltd). The immunoblot membranes were blocked with blocking buffer (1% (w/v) BSA (BioFROXX, China) in PBS, 20% Tween-20; PBST) for 6 h at 4°C, washed 3 times with PBST and incubated with the HcDAF5 antiserum (1:1000 in PBST) overnight at 4°C. Samples were washed 6x in PBST and subsequently incubated with an HRP-conjugated goat anti-rabbit antibody (1:1000, Beyotime Biotechnology, China) for 2 h at 37°C, followed by 5x additional washes. Immunodetection was performed by chemiluminescence (WesternBright ECL kit; K-12045-D10, China), and images were acquired by ChemiDoc XRS + system (Bio-Rad, USA).
Immunofluorescent assay to evaluate protein expression in H. contortus
Fresh male and female adults of H. contortus were fixed in 4% paraformaldehyde at 4°C for 24 h and then were consecutively dehydrated in an ethanol series (75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h, and 100% for 30 min twice). Then dehydrated worms were incubated in xylene: absolute ethanol (1:1) solution for 5 min and xylene for 10 min, then embedded in paraffin. Paraffin sections of 4 µm were subjected to immunofluorescence staining. For immunofluorescent assay, slices were treated with EDTA buffer at 100°C for 10 min. After blocking with 5% BSA for 20 min, anti-HcDAF5 polyclonal antibody and goat anti-rabbit IgG diluted 1:100 was sequentially added and incubated at 4°C overnight and 37°C for 50 min. Then sections were stained with 4’6-diamidino-2-phenylindole (DAPI) for 5 min at 37°C in the dark place. After washed by PBS toughly, sections were observed under fluorescence microscopy (Olympus CX-21, Japan).
RNA interference in H. contortus by soaking in siRNA
The CDS of Hcdaf5 was used to design the siRNA sequences with the siRNA Design Tools program, and siRNA oligos (Additional file1, Table S3) were synthesized chemically by Shanghai GenePharma Co. Ltd.
For RNAi experiment, L3s were exsheathed and washed five times in 0.9% NaCl solution followed by centrifuged at 600 g with DEPC-treated water for further three times. For each silencing assay, 50 µl of nematode suspension (about 5,000 larvae) was placed into a 96-well plate. 10 µl Lipofectamine 2000 (Thermo Fisher, USA) were incubated with 5 µl EBSS (pH 5.2) containing 2.5 µg/µl amphotericin, 100 µg/µl streptomycin and 100 IU/ml of penicillin (Gibco, USA) at 25°C for 5 min. The RNAsin (0.2 U) and siRNA solution were added and incubated for 15 min. Three siRNAs of Hc-daf-5 were mixed with equal amounts, and the final concentration of each siRNA was 1 µM, while final concentration of negative control siRNA was 3 µM, and nuclease-free water was set as blank control.
The knock down experiments were carried out as previously described [8]. In brief, three groups each consisting of 5,000 xL3s kept in 80 µl EBSS (pH 5.2) supplemented with the respective siRNA were soaked for 72 h. Approximately, 100 larvae of each group were transferred to fresh EBSS culture medium for another 5 d to assess their development. The remaining larvae were subjected to RNA extraction.
All RNA extractions were performed with the Trizol according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Takara, China). The same amount of cDNA for each sample was applied to a 10 µl reaction. The 18S gene of H. contortus was served as the endogenous control using the primer set of Hc-18S-qF/Hc-18S-qR (Additional file 1, Table S1). Primers (Hc-daf-5-qF/Hc-daf-5-qR) used in detecting transcriptional profiles were also used here for detection the transcriptional changes of Hcdaf5 in worms treated with siRNA. Amplification efficiency of the primers was tested by a standard curve assay and linear regression analysis showed similar slopes for all tested primers. The real-time PCR was performed on an ABI 7100 thermal cycler (Applied Biosystems, Germany) using the following conditions: one cycle at 50°C/2 min, 95°C/30 s; 40 cycles at 95°C/15 s, 60°C/15 s, 72°C/20 s; followed by a cycle at 60°C/1 min, 95°C/15 s, 60°C/15 s. Fold change expression of Hcdaf5 after RNAi calculated by 2-ΔΔCt method. ΔΔCt = [(Ct RNAi,Hcdaf5)-(Ct RNAi,Hc18S)]-[(Ct Blank,Hcdaf-5)-(Ct Blank,Hc18S)] [9].
Assessing the interaction between HcDAF5 and HcDAF3 by BiFC
The BHK21 cells were grown and maintained in DMEM (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The plasmid pbJun-HA-KN151 and pbFos-Myc-LC151 were used as original plasmids for plasmid construction. In brief, the CDS fragment of Hcdaf3 was amplified with primer sets of Hc-daf-3-fF/Hc-daf-3-fR (additional file 1, Table S1) and cloned into pbJun-HA-KN151 between NheI and XhoI sites to replace bJun, generating the pHcDAF3-HA-KN151 plasmid, while the fragment of Hcdaf5 gene was amplified with primer sets of Hc-daf-5-fF/Hc-daf-5-fR (additional file 1, Table S1) and cloned into pbFos-Myc-LC151 between NheI and PvuI sites to replace bFos, generating pHcDAF5-Myc-LC151 plasmid. BHK21 cells were seeded onto 6-well plates and grown to 70 ~ 80% confluency for transfection. To examine the interactions between HcDAF3 and HcDAF5, the plasmids pHcDAF3-HA-KN151 and pHcDAF5-Myc-LC151 were co-transfected using Lipofectamine 2000 (Thermo Fisher, USA). Then cells were incubated at 37°C with 5% CO2 for further 20 h, subsequently fluorescent was detected within a range from 580 to 680 nm and imaged.
In addition to the full-length constructs of HcDAF3, a DNA fragment encoding the HcDAF3-MH2 domain region (645 bp; 484–697 aa) was amplified with primer sets of Hc-daf-3-MH2-F/Hc-daf-3-fR (additional file 1, Table S1), sequenced and cloned into pHcDAF3-MH2-HA-KN151. Similarly, a DNA fragment encoding the HcDAF5-SDS domain region (900 bp; 1-300 aa) was also amplified with primer sets of Hc-daf-5-fF/Hc-daf-5-SDS-R (additional file 1, Table S1), sequenced and cloned into pHcDAF5-SDS-Myc-LC151. Plasmids pbFos-Myc-LC151 and pbJun-HA-KN151, which express bJun and bFos, respectively, were co-transfected as positive control, while pHA-KN151 and pHcDAF5-SDS-Myc-LC151 were performed as negative control.