The ethics statement for this study
All animal experiments were conducted under the ethical guideline and regulations issued by the Animal Ethics Committee of Xinxiang Medical University (Reference No. 2015016), which guided the researchers to make all efforts to alleviate various sufferings of the animals during experiments. In this study, the infected mice were euthanized at the humane endpoints when the mice appeared moribund. Euthanasia was executed by confining the animals in a closed space with 60–70% CO2 for five minutes.
Preparation of experimental animals and T. vaginalis
Six weeks old BALB/c female mice and SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and bred under a specific pathogen-free (SPF) environment.
The strain of T. vaginalis used in this study was isolated from the vaginal discharges of the patients with the diagnosis of trichomoniasis, placed in TYM medium containing 50 mg/mL ciprofloxacin, 100 mg/mL ceftriaxone, 2.5 mg/mL amphotericin B and 10% calf serum, and cultured in a humidified chamber containing 5% CO2 with the temperature of 37 °C. For further experiments, 2×106 parasites were harvested at their stationary phase, which was confirmed as the actin genotype E strain by PCR-restriction fragment length polymorphism (PCR-RFLP).
Preparation of soluble proteins from T. vaginalis trophozoites
Approximate 5×107 T. vaginalis trophozoites were harvested and washed by the centrifugation three times with the speed of 2500 rpm for ten minutes with 0.1 M PBS ( pH 7.2). After the pellet of trophozoites was re-suspended in 2 mL PBS, the mixed resuspend underwent repeated freeze-thaw cycles three times at the temperatures of -20 and 4 °C to disintegrate the parasite plasma membranes. For purifying the parasite protein, the lysed parasitic mixture was first sonicated on the ice at a speed of 60 W/s, then centrifuged at 12000 rpm for 30 min at 4 °C, and thereafter, the concentration of the protein in the supernatant was measured with the Bradford method, and finally, the soluble proteins from T. vaginalis were aliquoted and stored at -70 °C for future uses.
Total RNA extraction from T. vaginalis
The E.Z.N.A.TM Total RNA Kit I (OMEGA, Zhengzhou, China) was used to extract total RNA from T. vaginalis trophozoites, and the extracted RNA was re-suspended with the DEPC-treated water, which was further treated with ribonuclease inhibitor (TaKaRa, Dalian, China), and RNase-free DNase I (TaKaRa) to rid contamination of the genomic DNA for conducting reverse transcription. Purified RNA with the ratio of OD260/OD280 in between 1.9 and 2.0 was considered to reach the required purity.
Trophozoite cDNA was obtained by RT-PCR, and then the open reading frame (ORF) of TvAP65 (GenBank accession no. U35243.1) was amplified from the cDNA with a BamHI-anchored forward primer (5ʹ-CGCGGATCCATGCTCGCATCT TCAGTCGC-3ʹ) and XhoI-anchored reverse primer (5ʹ-CCGCTCGAGTTAGTA GAGTTGCTCGTATTCAGCC-3ʹ), and cloned into the pMD19-T vector (TaKaRa). Afterward, the cloned recombinant pMD19-T-TvAP65 was sequenced and then transformed in E. coli (DH5a) competent cells purchased from Yi Fei Xue Biotechnology (Nanjing, China) for the future amplification of TvAP65. The online sequence check (http://www.ncbi. nlm.nih.gov/BLAST/) was blasted to verify the sequence homolog between the fragment of rTvAP65 and the sequence in the GenBank.
Bioinformatics analysis of sequences
The homology between the cloned TvAP65 and AP65 in the Genebank was completed through BLASTX and BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the amino acid sequence of TvAP65 underwent the alignment analysis with CLUSTALW1.8, and the putative motifs, possible secondary structures, and potential signal peptides were predicted by applying the online analysis tools and programs aspreviously reported .
Protein purification of TvAP65 and pET-32a
TvAP65 fragment was successfully sub-cloned into pET-32a (+) expression vector system (Novagen, USA) from the recombinant plasmid pMD19-T-TvAP65, and subsequently confirmed to be inserted in the right place of the recombined plasmid. Afterward, sequencing confirmed recombinant plasmid pET-32a-TvAP65 was introduced into competent E. coli BL21 (DE3), and when OD 600 of the culture reached 0.6 at 37°C, isopropyl-b-D-thiogalactopyranoside (IPTG; Sigma–Aldrich, USA) was added into the bacterial growth culture media to induce the recombinant protein expression. Then, following five more hours incubation with IPTG at 37°C, the bacteria were harvested and further lysed with 10 mg/mL lysozyme (Sigma–Aldrich, USA). Lastly, the extracts from lysed bacteria were run through 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The recombined TvAP65 protein was then purified through a Ni2+-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare, USA), and post-purification purity was determined by a 12% SDS-PAGE gel . After measuring the concentration, the rTvAP65 protein was stored at -20°C for future experiments. Additionally, through the same process, the pET-32a proteins with 109 amino acid residues composed of 6 histidines and Trx•TagTM thioredoxin protein were obtained for future uses.
Production of Anti- rTvAP65 serums
SD rats were inoculated by subcutaneously injecting a combo of Freund’s complete adjuvant with 0.3 mg purified rTvAP65 protein at the ratio of 1:1 to different sites on the rats for producing antigen-specific polyclonal antibodies. 14 days later, the rats were boosted then 4 rounds of injections with the combo of Freund’s incomplete adjuvant with 0.3 mg purified rTvAP65 protein as above at a 7-day interval. After completing the immunization, the polyclonal serums were made and stored for the use in the next experiments. The serums used as negative control were made before the first injection above . Moreover, for generating antiserum against T. vaginalis, the parasites were injected into mice, and serums were collected ten days post-infection.
Immunoblot analysis of TvAP65
SDS–PAGE separated the proteins containing recombinant TvAP65 as well as soluble trophozoite proteins of T. vaginalis, and the separated proteins were then transferred to the nitrocellulose membrane (Millipore, Shanghai, China). Following the transfer, the membranes were incubated with either mouse (recombinant TvAP65) or rodent antiserums (soluble trophozoite proteins of T. vaginalis) as primary antibodies at the concentration of 1:100 or 1:200 respectively for one hour; afterward, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rat IgG (Sigma, Shanghai, China) as the secondary antibodies were added in the incubation accordingly for another 1 hour. All the incubations were carried out at 370C environment. The proteins were detected with 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB) as the chromogen (Boster Bio-Technology, Wuhan, China).
Localization of TvAP65 in trophozoites of T. vaginalis
Harvested T. vaginalis trophozoite cells were smeared on a poly-L-lysine coated glass slides for 15 minutes drying. The mounted slides underwent the 10-minute fixation with 4% paraformaldehyde PBS at room temperature, after that, the parasites on the slides were permeabilized with 1% Triton X-100 solution for 10 minutes. Finally, the slides were blocked at 37 °C with PBST containing 4% (w/v) BSA for one hour. For the immunofluorescence study, sequentially the slides with the parasites were first incubated either with the rat anti-TvAP65 serum or the control serums at the dilution ratio of 1:100 for overnight at 4 °C; further with goat anti-rat IgG antibody labeled with Cy3 (Beyotime, Shanghai, China) at dilution ratio 1:1,000 in the dark for 40 minutes; then with DAPI (Beyotime) to stain the nuclei for 5 min; and lastly with fluorescent mounting medium (Beyotime). During the staining procedure, PBS was continuously used to wash the slides. Finally, the processed slides were visualized under laser confocal microscopy (Nikon, Beijing, China).
Immunization and challenge infection
80 BALB/c mice with the age of 6 weeks old were randomized into 4 groups of 20 each, and then immunization combos were made including the rTvAP65 mixture of 100 μg of rTvAP65 with Freund adjuvant at the ratio of 1: 1, the pET-32a mixture of 100 μg of pET-32a protein with Freund adjuvant at the ratio of 1:1, and the adjuvant mixture of Freund adjuvant alone. For testing the immunogenicity of rTvAP65, the mice in the first three groups were injected subcutaneously either with the rTvAP65 mixture, or pET-32a mixture, or adjuvant mixture, while the fourth group of mice was designated as the blank control without the inoculation of any kind. The procedure was carried out as previously reported . Ten days after the last vaccination, the mice all were given the intraperitoneal injection of 1×107 trophozoites of T. vaginalis, and then under surveillance on the infectious and survival status throughout the entire period after challenge. If any animal presented with the infectious symptoms caused by T. vaginalis, they were euthanized by CO2.
Thirty days after the challenge with T. vaginalis, the survival rate of mice was calculated with the following formula: the number of survived mice after immunization / the total number of mice before immunization×100%.
Determination of antibody levels in serums
The blood samples from mice in each group (n = 5) were collected at 0, 2, 4, and 6 weeks. Serum was isolated from the blood sample and stored at -20°C for evaluating antibodies and measuring cytokines. IgG isotypes and anti-TvAP65 antibodies in the serums were quantitatively studied with indirect ELISA . Briefly, the wells of microtiter plates (Costar, New York, NY, United States) were first coated by rTvAP65 (2.5 μg/mL, 100 μL/well) in the carbonate buffer with a pH value of 9.6 at 4°C for overnight, and then blocked with 4% BSA at 37°C for 2 hours. Mice serums were prepared by dilution with the addition of PBS at a ratio of 1 to 10. Afterward, the diluted mice serums were added into the plates for 2 hour incubation at 37 °C, thereafter, the plates were rinsed with PBST three times and sequentially treated with the HRP-conjugated secondary antibodies goat anti-mouse IgG2a, IgG1, and IgG, (SouthernBiotech, Birmingham, AL, United States). For conducting ELISA, 100 μL of 3, 3, 5, 5-tetramethylbenzidine was pipetted into each well of the plates, the reaction was terminated with100 μL (2 M) sulfuric acid. The 450 nm absorption was used to read the plates by an automatic ELISA reader (MULTISKANFC, Thermo Scientific, Waltham, MA, United States), and all plates were read in triplicate.
Measurement of the secretory levels of various cytokines
The secretory levels of pro-inflammatory cytokine were determined in the serum from all experimental rodents. Interferon-gamma (IFN-ɣ), and interleukin-2, 4, 10, 17 were measured using commercially available ELISA kits (Boster, Wuhan, China) with the recombinant IFN-ɣ, IL-17, 10, 4, and 2 as the corresponding controls for quantification. The data obtained from three individual experiments were further analyzed.
One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was performed to analyze the differences among different experimental groups. The survival-related data were analyzed using the Kaplan–Meier method. SPSS for Windows 16 (SPSS Inc., Chicago, IL) was used for all the statistical analyses, and P <0.05 indicated statistical significance.