The B. microti strain was obtained from the American Type Culture Collection (ATCC, PRA-99^TM) and was injected into Kunming mice. B. microti was maintained in purified mice erythrocytes and the parasite was isolated when the erythrocyte infection rate reached 30–40%, which was confirmed with Giemsa-stained thin-blood smears.

Total RNA of B. microti was extracted using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, United States) and dissolved in RNase-free water. RNA integrity was verified by gel electrophoresis, and quantity was determined using a NanoVue spectrophotometer (GE Healthcare Life Sciences, Pittsburg, PA, United States). cDNA library construction and Illumina sequencing of the samples were performed at Genomics Institute (BGI, Shenzhen, China). RNA sequence libraries were constructed according to Illumina manufacturer’s instructions for 150-bp paired-ends and then sequenced using Illumina HiSeq 2000 (Illumina, United States). A total of 3,024 genes were obtained from transcriptome assembly.

Based on the open reading frame (ORF) sequence of BmTPx-2 downloaded from GenBank (XM_012792398.1), we designed a pair of specific primers: BmTPx-2-F: 5′-TT CCATGG CA ATG CGG TAC ATA ACA CTT TTA-3′, BmTPx-2-R: 5′-TT CTCGAG AGA AGT TTG GCC AAA AGC CTC-3′, NcoI and XhoI sites are underlined. We amplified the ORF full sequence using the cDNA library as template. Then, the purified PCR products were ligated onto a pMD-18T vector (TaKaRa, Japan) and sequenced. Multiple sequence alignment of BmTPx-2 was performed with 2-Cys TPx-2 of Plasmodium falciparum (Pf: GenBank Accession number BAA97121.1), 2-Cys TPx-2 of Theileria parva (Tp: XP_765704.1), and 2-Cys TPx-2 of Tamarix hispida (Th: FH74407.1), TPx-2 of Toxoplasma gondii (Tg: AG25678.2).

To obtain the full sequence of the 3′ full sequence and 5′ full sequence of BmTPx-2, the 5′ and 3′ regions of the mRNA were amplified using a SMARTER RACE cDNA amplification kit (Clontech, San Jose, CA, United States), following the manufacturer’s instructions. The primers were designed based on the ORF sequence earlier obtained. The primer sequences were as follows: GSP1: 5′-GACAGCCATGTCACCTCCCT-3′, GSP2: 5′-CAAGTATGTGCCGTATATTTCCAAA-3′, and GSP3: 5′-GCGAGTTTGTAAAGTGAATGTATGG-3′ for 5′ rapid amplification of cDNA ends (RACE); GSP1: 5′-TGGGTAGGAGTGTGGATGAGAC-3′ and GSP2: 5′-AAGGGTAATAAGGGAATGGCGGC-3′ for 3′ RACE. The resulting PCR products were subsequently purified and ligated onto the pMD-18T vector (TaKaRa, Japan) and sequenced. RACE analyses were performed according to the manufacturer’s recommendations for using the 3′ RACE system for rapid amplification of cDNA ends and 5′ RACE system for rapid amplification of cDNA Ends (Invitrogen, United States).

We cloned the ORF of BmTPx-2 into the prokaryotic expression vector pET-28a. Then, to generate the fusion protein, the recombinant vector pET-28a-BmTPx-2 was transformed into Escherichia coli BL21 cells at 16°C by 1 mM isopropylthio-β-D-galactoside (IPTG) induction. The fusion protein was purified with NI-NTA agarose beads under native conditions (Merck, United States).

Next, to evaluate the antioxidant activity of the recombinant proteins, a mixed-function oxidation (MFO) assay was performed to determine the ability of BmTPx-2 in protecting DNA from damage in oxidative conditions: the MFO mix reaction buffer contained 8 mM FeCl₃, 40 mM dithiothreitol (DTT), 20 mM EDTA, 25 mM HEPES (pH 7), 180 ng pBluescript plasmid DNA, and various concentrations of the purification fusion protein rBmTPx-2 (335, 167, 83, and 41 μg/mL). After mixing, the reaction buffer was pre-incubated at 37°C for 1 h, 180 ng pBluescript plasmid DNA was then added, and the mixture was incubated at 37°C for another 3 h. Nicking of the supercoiled plasmids was detected by 1% agarose gel electrophoresis and stained with ethidium bromide.

A standard experiment to identify the antioxidant activity of rBmTPx-2 was performed in a 200-μL mixture buffer that contained 375 μM of NADPH, 1 mM of H₂O₂, 6.4 μM Trx, 0.14 μM TrxR, HEPES-NaOH (pH 7), and 100 μg/mL rBmTPx-2. The negative control group contained H₂O instead of rBmTPx-2 in the assay system. NADPH oxidation was monitored for 30 min at 30°C by measuring the absorbance at a wavelength of 340 nm using a spectra Max M5 (MD, United States).

Eight-week-old male mice (Slac, China) were used for the production of antiserum against rBmTPx-2. For primary immunization, a mixture of 100 μg of BmTPx-2 and an equal volume of Freund’s complete adjuvant (Sigma, United States) was intraperitoneally injected into the mice. After 2 weeks, boost immunization was performed in a similar manner as the primary immunization. Serum was collected 20 days after boost immunization. All animal experimental procedures were conducted in accordance with the Guiding Principles of Animal Experimentation in the Shanghai Veterinary Research Institute.

Babesia microti-infected RBCs were collected when the infection reached 40–50%. RBCs were pelleted by centrifugation and hemolyzed with a red cell lysis buffer (TianGen, China), shaken repeatedly, and incubated at room temperature for 5 min. After centrifugation, the pellet was washed thrice with PBS until the supernatant was clear, and freeze-thaw cycles in liquid nitrogen and water bath were performed thrice. The RBC lysate was mixed with an equal volume of SDS-PAGE loading buffer and heated to 100°C for 10 min, and the protein was separated by SDS-PAGE (15% gel) and subsequently transferred onto a polyvinylidene difluoride membrane (Immobilon; Millipore). The membranes were blocked with 5% skim milk diluted in PBS containing 0.05% Tween (PBST) for 2 h at 37°C, and then was washed with 1× PBST thrice. The membranes were reacted with anti-BmTPx-2 mouse serum at 1:100 at 37°C for 1 h, followed by goat anti-mouse IgG (1:2,000). The signals were developed with an Immobilon western chemiluminescence HRP substrate.

To examine the expression of BmTPx-2 at different stages of infection, 30 6-week-old male mice (Slac, China) were injected with RBCs infected with B. microti, and then the mice RBCs were detected by quantitative real-time PCR (qRT-PCR) using the expression of actin mRNA as internal control. We divided 30 mice into 10 groups following B. microti injection, and blood was collected from three mice/group from the 2nd–11th day after injection. Total RNA of B. microti was extracted using the TRIzol method, and cDNA was synthesized according to the PrimeScript RT reagent kit with gDNA eraser (TaKaRa, Japan). qRT-PCR was performed using cDNA as template and a pair of specific primers, following the recommendations provided in the SYBR premix ExTaq (TaKaRa, Japan) with StepOnePlus PCR system. The experimental values were expressed as relative amounts, and analysis was performed with the 2^-ΔΔc_t method.

To detect BmTPx-2 gene transcription at the tick stage, five 6-week-old male mice (Slac, China) were injected with RBCs infected with B. microti. Two days after injection, Haemaphysalis longicornis larval ticks were placed on the mice. A total of 54 engorged larval ticks were collected, which were cultivated in a 24°C incubator until these exuviated into nymphs. The nymph ticks were placed on the mice, engorged nymph ticks were then collected and cultivated to the adult phase. Next, 24 adult ticks were harvested for DNA extraction with a DNA extraction kit (Shanghai SBS Genetech, China), and the 18S RNA of B. microti was detected by qRT-PCR. Then, the 30 other adult ticks were divided into 10 groups for RNA extraction, the 10-group RNA for BmTPx-2 transcription analysis.

The present work was approved by the Animal Ethics Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS) (No. SHVRI-Mo-0158). The protocol was approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, CAAS.

The full-length cDNA of BmTPx-2 was 826 bp, and the ORF length was 756 bp (GenBank Accession Number MF459654), encoding 251 amino acid residues without a signal peptide. The protein had a molecular weight of 28 kDa, and a pI of 8.231. BLASTp (NCBI) analysis determined that BmTPx-2 belongs to the 2-Cys Prx2 family. Compared to other known 2-Cys Prxs, we found that BmTPx-2 also contained two conserved Cys VCP motif residues at Cys¹⁰¹ and Cys²²¹ (Figure 1). The black boxes with black letters indicate similar residues. The two conserved VCP motifs are underlined.

A fragment of the BmTPx-2 ORF was amplified by PCR, and the product was cloned into a pET-28a vector and transformed into E. coli BL21(DE3). The fusion protein was expressed at 20°C for 8 h and purified using Ni-NTA agarose beads. The fusion and purification of the recombinant proteins was identified by SDS-PAGE and standard Coomassie brilliant blue staining. The results indicated that rBmTPx-2 had a molecular mass of about 28 kDa (Figure 2).

Both FeCl₃ and DTT of the MFO system produced hydroxyl radicals that can damage the plasmid, and the molecular weight of the nicked plasmid apparently increased (Figure 3A). However, the presence of rBmTPx-2 protected the plasmid from hydroxyl radical-induced damage. When the concentration of rBmTPx-2 was 335 μg/mL, about half of the DNA was undamaged, but when the concentration of BmTPx-2 was reduced to 41 μg/mL, nearly all the DNA was damaged. These findings indicated an inverse relationship between the concentration of rBmTPx-2 and the quantity of the nicked plasmid. These results suggest that the antioxidant activity of rBmTPx-2 was concentration-dependent (Figure 3A).

The Trx-TrxR assay system showed a decrease in the absorbance at a wavelength of 340 nm; however, the degree of the decline was not significant, in which after 40 min, the absorbance decreased about 0.32. The absorbance of the negative control group decreased to about 0.08 compared to that of the BmTPx-2 group (Figure 3B).

Babesia microti native BmTPx-2 protein extracted from RBCs of mice infected with B. microti was detected using an anti-BmTPx-2 mouse antiserum, whereas the negative control showed no specific bands (Figure 4). The results demonstrated that the molecular weight of BmTPx-2 of B. microti were about 28 or 24 kDa.

The level of BmTPx-2 mRNA expression in erythrocytes was maintained at a relatively stable level from the 2nd to 7th day (Figure 5). BmTPx-2 expression peaked on the 8th day and then decreased on the 9th day. On the other hand, no B. microti 18S DNA was detected in 24 adult ticks by using RT-PCR, and we also could not detect the BmTPx-2 transcription of B. microti among 10 adult RNA.