Animals were treated in strict accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. All animal procedures were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
Parasites and antigen
T. spiralis (ISS534) ML were isolated from infected mice at 35 dpi by artificial digestion with pepsin-HCl. The protocols of isolation of Ad from infected rats intestines and NBL from female adults were followed as previous study . Soluble antigens of T. spiralis and excretory/secretory antigen were prepared according to the method reported .
Cloning of TsCF1 gene
Key words “Trichinella spiralis AND cathepsin F” were input for Gene database of GenBank search. Three cathepsin F-like genes (Tsp_02382, Tsp_04256 and Tsp_02383) were acquired and named TsCF1, TsCF2 and TsCF3 respectively. Total RNA was extracted from the ML at 35 dpi, Ad and NBL of T. spiralis using Trizol (Invitrogen). Primer PR1 (5′-GCGAATTCTGCAGGATCCAACTTTTTTTTTTTTTTTTTT-3′) was used as the reverse transcription primer. Primer 1 (5′-CATCATTATGGTTTCCGTA-3′) and primer 2 (5′-GCGAATTCTGCAGGATCCAAC-3′) were used to amplify TsCF1 gene and study the transcription at different developmental stages. The PCR products were purified, ligated into pMD18-T vector (Takara Bio, Japan) and sequenced. Analysis of the deduced amino acid sequence was conducted with DNASTAR (DNASTAR, Madison, WI, USA) and http://www.expasy.org/tools/.
Molecular modeling of TsCF1
Homology modeling of the mature domain of TsCF1 was performed with MODELLER v9.5 . In terms of the improvement in model quality, an advanced modeling approach that is based on multiple templates of MODELLER was chosen. Thus, 1M6D (Crystal structure of human cathepsin F), 2P86 (cathepsin L protease from T. brucei rhodesiense) and 3BCN (Crystal structure of a papain-like cysteine protease) were selected as templates for homology modeling. Alignment between the target sequence and templates were performed using SALIGN  in MODELLER. New models of target sequence were built based on the multiple templates alignment. The structure of TsCF1 generated by MODELLER was improved by energy minimization using the Discovery Studio 2.5 (Accelrys Inc., San Diego, CA, USA). After energy minimization, structures were submitted to the website of PDBsum Server (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=index.html) (PROCHECK program) and the Structural Analysis and Verification Server (http://services.mbi.ucla.edu/SAVES/) (ERRAT program), and generated a full set of structural analysis for models.
Ligands E64 and K11777 were docked into the activity pocket of the receptor TsCF1 using AUTODOCK 4.0 . Lamarckian genetic algorithm (LGA) was used to search for the optimized conformation. The maximum number of generations and energy evaluations were set to 2.7 × 105 and 2.5 × 106, respectively. The conformations with lowest binding free energy of all docking results were selected and hypothesized to be a representative binding mode of ligand and receptors. Energy minimizations were performed to produce a series of more meaningful complex conformations for analysis. The complex structures for analysis were minimized with the steepest descent and conjugate gradient by Discovery Studio 2.5.
Phylogenetic analysis of TsCF1
To explore the phylogenetic relationships involving T. spiralis TsCFs and cathepsin Fs of other helminths, a rooted Bayesian (BI) phylogenetic tree was constructed by MrBayes v3.2.2 (https://www.phylo.org/) under the best-fit model WAG + I + G [24, 25]. The best-fit model (WAG + I + G) for amino acid substitution was selected using ProtTest v2.4 with discrete gamma distribution in four categories . For each model, the MCMC was run for 2 million steps and sampled every 500 steps. The first 500,000 steps of each run are discarded as burn-in. The tree was visualized using a FigTree program v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).
Expression, purification and refolding of recombinant TsCF1
The TsCF1 prodomain and the entire mature domain were amplified using the specific primers carrying Xho I and Bam HI restriction enzyme sites: PF2 (5′-CGCGGATCCTTGCCAATGAAGCAAAAGAGA-3′) and PR2 (5′- CGCCTCGAGTTAATCAATCACAACTGA-3′) with pMD18T-TsCF1 plasmid as the template. Then the amplified PCR product was ligated into pET-30a vector (Novagen, USA) which was digested by Xho I and Bam HI and the recombinant plasmid was transformed into E. coli BL21 (DE3) cells (Transgen). The expression of the recombinant TsCF1 protein (rTsCF1) was induced by adding isopropyl-1-thio-β-d-galactopyranoside (IPTG) to a final concentration of 1 mM and analyzed on Coomassie-stained SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels. After SDS-PAGE, protein bands were excised from SDS-PAGE zymography gels and stored in ultrapure water. MALDI -TOF/TOF-MS/MS was performed by 4800 Plus MALDI TOF/TOF TM Analyzer (Applied Biosystems, Foster City, USA) in Shanghai Applied Protein Technology Co., Ltd (Shanghai). The MS data were used to search against the non-redundant protein database (nr database, NCBI).
To purify rTsCF1, the cells were harvested at 4 h after incubation with IPTG at 37 °C, and then the pellet of cells from 2,000 ml of culture were suspended in 8 M urea lysis buffer. The rTsCF1 was purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Merck, Germany), and the purity was analyzed by SDS-PAGE. Refolding of the purified rTsCF1 was performed as follows: Ni-NTA affinity purified rTsCF1 (10 mg) was slowly added into 1 L of 100 mM Tris–HCl (pH 8.0) containing 1 mM EDTA, 250 mM l-arginine, 5 mM reduced glutathione, 1 mM oxidised glutathione and gently stirred at 4 °C for 20 h as described previously .
Production of rabbit polyclonal antibody and Western blot analysis
Two New Zealand rabbits were used to produce the polyclonal antibodies against rTsCF1. Rabbits were primarily immunized subcutaneously at two locations with 1 mg rTsCF1 mixed with Complete Freund′ Adjuvant. Booster immunizations were performed at 3 week intervals with 1 mg rTsCF1 emulsion of incomplete Freund′ Adjuvant. The blood samples were collected prior to each immunization, and 7 days after the last immunization, the antibody titers were checked by ELISA.
The extracts from T. spiralis Ad, ML and E/S antigens as well as rTsCF1 were separated on 12.5 % SDS-PAGE before being electrophoretically transferred onto Hybond C extra membranes (Amersham, USA). The membrane were then blocked and incubated with 1:200 diluted anti-rTsCF1 rabbit sera. After washing, the membranes were incubated with goat anti-rabbit immunoglobin G (IgG) alkaline phosphatase (AP) conjugate (1:4,000). Finally, the bands were detected using NBT/BCIP (Promega, USA).
ML of T. spiralis were recovered from infected mice by the acid-pepsin digestion of striated muscles at 35 dpi. The parasites were fixed in cold acetone (−20 °C) for 10 min and then centrifuged for 5 min at 1000 g, followed by washing three times in PBS. After treated with 0.1 % Triton X-100 for 10 min at room temperature, the parasites were blocked with 5 % normal goat serum in PBS and then incubated in a moist chamber at 37 °C for 1 h with a 1: 10 dilution of immune and normal rabbit sera. After washing three times in PBS, the larvae were incubated with a 1: 20 dilution of FITC-labeled goat anti-rabbit IgG (Sigma-Aldrich, USA) and 0.01 % Evan’s blue for 1 h, washed five times in PBS, then transformed to slides and added 90 % Buffered Glycerol then observed under a fluorescent microscope (Leica, Germany).
Enzyme activity assay and inhibitory test
Enzyme activity was assayed fluorometrically as the hydrolysis of Z-Phe-Arg-AMC (Peptide Institute, Osaka, Japan). Briefly, 0.2 μg of enzyme was added to 190 μl of sodium acetate buffer (pH 5.5) containing 2 μM Z-Phe-Arg-AMC and 10 mM DTT, and the release of fluorescence (excitation 355 nm, emission 460 nm) over 20 min at room temperature was assessed with a SpectraMax M5 (Molecular Device Corporation, Florida, USA).
For evaluation of the inhibitory kinetic features of rTsCF1, a known inhibitor E64, was selected for assay . The 50 % inhibitory concentration (IC50) values of E64 of rTsCF1 was measured in a fluorescence endpoint assay using Z-Phe-Arg-AMC as a substrate . The assay was carried out in black 96-well plates at 37 °C in a 50 mM sodium acetate buffer at pH 5.5 containing 10 mM DTT, 5 mM EDTA, and 2 μM Z-Phe-Arg-AMC. Prior to the addition of substrate, different concentration of the inhibitor ranging from 0.1 to 200 nM were preincubated for 30 min with the enzyme to allow the establishment of the enzyme-inhibitor complex. The reaction was started by the addition of the substrate and stopped after a 20 min reaction at 37 °C according to the previously described method. All values used were mean values from three independent assays to produce statistically significant results.