Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries

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Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries Nan-Nan Liu a,1 , Zeng-Shan Liu a,1 , Shi-Ying Lu a,1 , Pan Hu a , Yan-Song Li a , Xiao-Li Feng a , Shou-Yin Zhang b , Nan Wang b , Qing-Feng Meng c , Yong-Jie Yang a , Feng Tang d , Yun-Ming Xu e , Wen-Hui Zhang f , Xing Guo a , Xiao-Feng Chen a , Yu Zhou a , Hong-Lin Ren a,∗ a Key Laboratory of Zoonosis Research, Ministry of Education/Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Xi An Da Lu 5333, Changchun 130062, China b Jilin Provincial Center for Animal Disease Control and Prevention, Changchun 130062, China c Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun 130062, China d College of Animal Husbandry and Veterinary, Liaoning Medical University, Jinzhou 121001, China e Department of Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong 212400, China f Fujian Institute of Subtropical Botany, Xiamen 361006, China

a r t i c l e

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Article history: Received 17 August 2014 Received in revised form 13 January 2015 Accepted 14 January 2015 Keywords: Ovis aries Peroxiredoxin 6 Monoclonal antibodies Expression analysis Brucellosis

a b s t r a c t Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2 . In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753 bp containing a 5 -untranslated region (UTR) of 93 bp, a 3 -UTR of 985 bp with a poly(A) tail, and an open reading frame (ORF) of 675 bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07 kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. © 2015 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +86 431 87835735; fax: +86 431 87836703. E-mail address: [email protected] (H.-L. Ren). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.vetimm.2015.01.006 0165-2427/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Liu, N.-N., et al., Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.01.006

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1. Introduction Reactive oxygen species (ROS) such as superoxide anions (O2 •− ), hydrogen peroxide (H2 O2 ) and hydroxyl radicals (OH• ) are produced during many physiological processes of normal cell metabolism, immune responses and stressful on environmental stimuli (Nikapitiya et al., 2009). Furthermore, they are utilized in immunity, cell proliferation and differentiation, signal transduction and ion transport (Aguirre et al., 2005). However, high levels of ROS may cause oxidative stresses, protein oxidation, lipid peroxidation, DNA strand breaks, DNA base modifications and cell death (Nikapitiya et al., 2009), finally resulting in disease states (Mates et al., 1999). Cellular defense mechanisms have evolved to keep the homeostasis between ROS and ROS-removing antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), thioredoxin (TRX), peroxiredoxins (Prdxs), and glutaredoxins (GRXs) (Henkler et al., 2010; Kinnula et al., 2004). The peroxiredoxin (Prdx) family includes ubiquitous antioxidant enzymes that prevent oxidative damage (Rhee et al., 2001, 2005a), and is used as an effective medicine for ROS-related diseases (Arockiaraj et al., 2012). It was reported that all members of Prdxs contain the conserved catalytically active cysteine (Robinson et al., 2010), however the functions of Prdx family members are diversified (Radyuk et al., 2001). The mammalian Prdx family has six isoforms which are classified into two sub-groups including five 2-Cys Prdxs (from Prdx1 to Prdx5) and one 1-Cys Prdx (Prdx6), and the 2-Cys Prdxs are divided into two classes called ‘typical’ and ‘atypical’ 2-Cys Prdxs depending on whether the conserved cysteine residues form intermolecular or intramolecular disulfide bridges respectively (Manevich and Fisher, 2005; Wood et al., 2003). The expression and role of Prdxs were reported against bacterial infections (Bacano Maningas et al., 2008; Zhang et al., 2007), and it is implicated that Prdxs take part in immune responses against viral or bacterial infection (De Zoysa et al., 2012; Yang et al., 2007). In mammals, the 1Cys Prdx referred to as Peroxiredoxin 6 (Prdx6) contains the sole conserved cysteine residue at the molecular catalytic site (Rhee et al., 2005b). Prdx6 mRNA is firstly cloned from a ciliary body of bovine, and widely expressed in various tissues and organs such as lung, brain, heart, liver, kidney, spleen and intestine, especially lung with particularly high expression levels (Kim et al., 1998; Kinnula et al., 2002; Shichi and Demar, 1990). Prdx6 is a bifunctional enzyme that shows the activities of peroxidase and phospholipase A2 (PLA2 ) (Kang et al., 1998; Kim et al., 1997). Up to date, some genes or cDNAs of Prdx6 have been cloned from some vertebrates and invertebrates (Chae et al., 1994; Fatma et al., 2001; Fujii et al., 2001; Singh and Shichi, 1998), and the biological activities of recombinant Prdx6 proteins from some organisms have been characterized (Fujii et al., 2001; Radyuk et al., 2001; Wang et al., 2008; Zhang et al., 2007). As reported, Prdx6 is an important antioxidant enzyme and has a major role in lung phospholipid metabolism (Manevich and Fisher, 2005). Recently, more and more reports showed that Prdx6 is associated with diseases such as carcinomas (Huang

et al., 2011), cataract (Pak et al., 2006) and atherogenesis (Wang et al., 2004), and could be considered as a molecular candidate to predict or treat some diseases in the future (Huang et al., 2011). Prdx6 is also likely to be involved in protective responses during host immune defense against bacterial infection (Zheng et al., 2010). On the other hand, bacterial 1-Cys Prdx is able to limit the extent of oxidative damage to the pathogen, so it could be a survival factor for pathogenic microbes (Dubbs and Mongkolsuk, 2007; Nevalainen, 2010). At present, there are no reports published about the relationship between Prdx6 and brucellosis, which is the most prevalent bacterial zoonosis worldwide. To date, the full-length cDNAs of Prdx6 from Brucella or sheep (Ovis aries) have not been identified. In our previous experiment, a suppression subtractive hybridization (SSH) cDNA library of buffy coat from Brucella-infected sheep was constructed, and a partial cDNA sequence containing a 3 -UTR of the differentially expressed peroxiredoxin 6 (OaPrdx6) gene of sheep (O. aries) was screened and sequenced. In this study, we identified the full-length cDNA of OaPrdx6 for the first time, and then the recombinant protein OaPrdx6 was expressed and its DNA protection activity was analyzed. Using real-time PCR and Western blot methods, the expression patterns of OaPrdx6 were observed respectively at mRNA and protein levels. The results from this study may facilitate further study on the functions of OaPrdx6 in host responses to infection of different virulent Brucella. 2. Materials and methods 2.1. Experimental animals and cell lines Small Tail Han Sheep (O. aries) free of the most relevant sheep infectious diseases (paratuberculosis, chlamydiosis, mycoplasmosis, scrapie and salmonellosis) purchased from brucellosis free and unvaccinated flocks of Sangang farm (Jilin Province, China) were raised with a natural daylight cycle and normal feed (Tang et al., 2012). Female BALB/c mice were fed in cages with a natural daylight, food and water available at any time, and at the ambient temperature of 20 ± 2 ◦ C while relative humidity of 50 ± 5% (Feng et al., 2013). All the animal experiments were carried out abiding by the provisions of EU animal management practices (1986.11.24). Myeloma Cells SP2/0, Escherichia coli DH5␣ competent cells and E. coli BL21-Codonplus competent cells were reserved by Key Laboratory of Zoonosis Research, Ministry of Education, Jilin University (China). 2.2. Cloning and sequence analysis of the full-length OaPrdx6 cDNA 2.2.1. Amplification and sequence assembly Twenty-five milliliter blood of O. aries was sampled into a centrifuge tube of 50 mL containing 3 mL anticoagulant solution, and centrifuged at 800 × g for 15 min at 4 ◦ C to collect the buffy coat (Ren et al., 2009). Total RNA from buffy coat of O. aries was extracted using Trizol Reagent

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Table 1 Primers used in this study. Primer

Object

Sequence (5 –3 )

GSP1 NGSP1 GSP2 NGSP2 (23)KLS (23)KLWHA (23)KLHisA ␤-actin-S ␤-actin-A OaPrdx6S OaPrdx6A

5 -RACE 5 -RACE 5 -RACE 5 -RACE ORF amplification ORF amplification ORF amplification RT-PCR RT-PCR RT-PCR RT-PCR

5 -TCACGCCTTCTGCTACACCTCTCTG-3 5 -GATTCTCTCCCCAGGCTCATCTTTC-3 5 -CTGTCCCCATTCTTCCAGTCAACCG-3 5 -AAACACCACACGAGCAGTCACAGGC-3 5 -CATATGCCCGGAGGTCTCCTCCTCG-3 5 -CTCGAGCTATGGCTGGGGTGTGTAGCGG-3 5 -CTCGAGTGGCTGGGGTGTGTAGCGGAG-3 5 -CCCAAGGCCAACCGTGAGAAGATGA-3 5 -CGAAGTCCAGGGCCACGTAGCAGAG-3 5 -GCTCGTGTGGTGTTTATTTTTGGTC-3 5 -GGGTGTG TAGCGGAGGTATTTCTTG-3

The letters marked by the single underline in the primer sequences stood for the restriction sites of Nde I (CATATG) and Xho I (CTCGAG).

(Invitrogen, USA) according to the manufacturer’s instructions and cleaned up using recombinant DNase I (RNase-free) (QIAGEN, Germany) and RNeasy MinElute Cleanup Kit (QIAGEN, Germany) following the manufacturer’s recommendations. Two sets of gene-specific primers (GSP1/NGSP1 and GSP2/NGSP2) described in Table 1 were designed for nested PCR amplification of 5 rapid amplification of cDNA end (5 -RACE) according to the partial cDNA sequence of OaPrdx6 obtained from SSH cDNA library of buffy coat from Brucella-infected sheep. The 5 -RACE reaction was carried out using the purified RNA, the two sets of primers and SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) following the manufacturer’s instructions. The RACE products were analyzed by 1% agarose gel stained with ethidium bromide (EB) and purified using Axy Prep Gel Extraction Kit (Axygen, USA), and then DNA fragments were ligated into pMD18T vector following the instructions (TaKaRa, Japan). The recombinant plasmids were transformed into E. coli DH5␣ competent cells and confirmed by PCR with M13 forward and reverse primers. The confirmed recombinant plasmids were sequenced by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. The full-length cDNA sequence of OaPrdx6 was assembled by the sequences of two cloned fragments of 5 -RACE amplification and the partial cDNA sequence screened from SSH cDNA library of sheep buffy coat.

2.2.2. Sequence analysis The full-length sequence of OaPrdx6 was identified with NCBI BLAST search programs (http://blast.ncbi. nlm.nih.gov/Blast.cgi). Both nucleic acid and predicted protein sequences of OaPrdx6 were analyzed using DNAman software. The NCBI BLAST program was used to search for nucleotide and protein sequences similar to OaPrdx6. The signal peptide was predicted through the SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). Characteristic domains or motifs were identified using the InterPro internet program (http://www.ebi.ac.uk/interpro/scan.html). Pair-wise and multiple sequence alignments of the predicted OaPrdx6 protein with the known Prdx6 proteins were analyzed using ClustalW version 1.83. The phylogenetic tree was constructed using the

Neighbor-Joining method and plotted with MEGA version 4.1 program. 2.3. Expression and purification of recombinant OaPrdx6 protein 2.3.1. Cloning and expression of OaPrdx6 coding sequence For ligation with pET-30a expression vectors, the coding sequence of OaPrdx6 was amplified by two sets of primers containing the corresponding restriction sites of Nde I and Xho I respectively at their 5 -ends, as shown in Table 1. The forward primer (23)KLS and the reverse primer (23)KLWHA were designed for no His6 -tag fused at the C-terminal of the recombinant OaPrdx6 protein (OaPrdx6W), meanwhile the forward primer (23)KLS and the reverse primer (23)KLHisA were designed for fusing a His6 -tag at the C-terminal of the recombinant OaPrdx6 protein (OaPrdx6H). In brief, a PCR reaction with a total volume of 20 ␮L was performed with 0.5 units of Ex Taq polymerase (TaKaRa, Japan), 2 ␮L of 10× Ex Taq buffer, 2 ␮L of dNTP Mix (2.5 mM each), 1 ␮L of each primer (10 ␮M) and 2 ␮L of plasmids containing the whole opening read frame (ORF) of OaPrdx6. The amplification reactions were carried out with an initial denaturation at 95 ◦ C for 1 min; 30 cycles of 30 s denaturation at 95 ◦ C, 40 s of annealing at 62 ◦ C and 1 min of elongation at 72 ◦ C; then the last extension at 72 ◦ C for 5 min. After analyzing the PCR product by 1% agarose gel stained with EB, the DNA fragments purified using the Axy Prep Gel Extraction Kit (Axygen, USA) were ligated into pMDTM 18-T Simple vectors following the instructions (TaKaRa, Japan) and transformed into E. coli DH5␣ competent cells. The sequenced recombinant plasmids containing the correct coding sequences of OaPrdx6 and pET-30a vectors were digested with the same set of restriction enzymes (Nde I and Xho I), and ligated together at 16 ◦ C over night using DNA ligation kit Ver.2.0 (TaKaRa, Japan) according to the manufacturer’s instructions. The two recombinant expression plasmids (pET-30a-Prdx6-cpw plasmid for OaPrdx6W and pET30a-Prdx6-cph plasmid for OaPrdx6H) were respectively transformed into E. coli BL21-Codonplus cells, and proteins of OaPrdx6H (His6 -tagged OaPrdx6) and OaPrdx6W (OaPrdx6 without His6 -tag) were expressed with induction

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of 1.5 mM isopropyl-␤-d-1-thiogalactopyranoside (IPTG) at 37 ◦ C for 5.5 h in 5 mL of LB broth. Finally, the cells were harvested by centrifugation at 12 000 × g for 1 min and resuspended with 100 ␮L 0.01 M PBS, and then 10 ␮L aliquot of bacterial suspension was analyzed by 12% SDSPAGE. 2.3.2. Purification of recombinant OaPrdx6 proteins The resulting pET-30a-Prdx6-cpw cells cultured in 200 mL LB broth under induction of 1.5 mM IPTG at 37 ◦ C for 5.5 h were collected at centrifugation of 12 000 × g for 5 min at 4 ◦ C, and then the bacterial pellets were resuspended in ratio of 10 mL 0.01 M PBS per gram bacterial pellets and crushed by sonication (Power setting 700 Watts, 6 s constant pulse, and 6 s pause for a 10 min total process time). The total proteins were run on 10% SDS-PAGE for separation. The OaPrdx6W protein was cut from 10% SDSPAGE gel, and collected through horizontal electrophoresis for 45 min after placed in the dialysis bag. 0.01 M PBS and polyethylene glycol (PEG) 20 000 (Sigma, USA) were respectively used to dialyze and concentrate the proteins. The purified OaPrdx6W proteins dissolved in 0.01 M PBS were stored at −80 ◦ C until use for preparing the monoclonal antibody (mAb) against the OaPrdx6 protein. The pelleted pET-30a-Prdx6-cph cells (cultured as above) were lysed in binding buffer (20 mM sodium phosphate, 30 mM imidazole, 0.5 M NaCl, pH 7.4) by sonication and clarified by centrifugation of 12 000 × g for 30 min at 4 ◦ C. The supernatant containing His6 -tagged OaPrdx6H proteins were loaded onto a HisTrapTM FF Crude (GE Healthcare, Germany). Then, the purified OaPrdx6H proteins collected in the elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) through washing and eluting were dialyzed in 0.01 M PBS (pH 8.0) and stored at −80 ◦ C until use for characterization analyses. The purified recombinant OaPrdx6 proteins were analyzed by 12% SDS-PAGE to check the molecular mass and purity of proteins. 2.4. Preparation and immunoassay specificity analysis of the mAb against OaPrdx6 2.4.1. Preparation and purification of the mAb In order to determine the expression level of OaPrdx6, the mAb against OaPrdx6 was produced. The female BALB/c mice used for immunization were 8–10 weeks old while 12 weeks old for ascites production. There were no special requirements for the environment of keeping the mice except for the adequate food and water. The purified OaPrdx6W protein was emulsified with equal volume of complete Freund’s adjuvant (CFA, Sigma, USA) or incomplete Freund’s adjuvant (IFA, Sigma, USA) following the method reported (Zhou et al., 2009). According to the method of cell fusion described previously (Feng et al., 2013), the spleen cells of the mice immunized with OaPrdx6W were fused with myeloma cells (SP2/0) at a ratio of 5–10:1 under the effect of PEG 1000 (Sigma, USA), then the fused cells were cultured in the 96-well cell culture plates with complete medium (RPMI 1640 (Gibco, USA) with 20% (v/v) FBS (Hyclone, USA)/HAT (Sigma, USA) medium). The positive hybridoma cells secreting mAbs

against OaPrdx6 were screened by ELISA, and cloned at least three times by limited dilution. The ascites from mice injected with positive hybridoma cells were prepared and purified by a method of caprylic/ammonium sulfate precipitation (CA-AS) (Zhou et al., 2010). The subclass of the antibody was identified according to the previous method (Feng et al., 2013) and the concentration of the purified ascites was determined using the Microplate Spectrophotometer (Epoch, BioTek, USA). 2.4.2. Immunologic specificity analysis of the mAb Immunologic specificity assay was carried out using Western blot. To obtain the whole protein lysates, 0.10 g frozen lung tissues from healthy sheep (O. aries) bought from the livestock market in Changchun (Jilin Province, China) were homogenized in 0.75 mL RIPA lysis buffer (Beyotime, China) for 10 min in ice water bath. Then the homogenates were centrifuged at 5000 rpm for 15 min at 4 ◦ C and the supernatant containing total proteins of lung was collected. Protein samples from the lung tissue lysates and the expression bacteria of recombinant OaPrdx6 were separated on 12% SDS-PAGE, followed by transferring to a PVDF membrane (Millipore, USA). Then, the PVDF membrane with protein samples was probed by anti-Prdx6 mAbs prepared above at a dilution of 1:5000, and the second antibodies of appropriate horseradish peroxidase labeled goat anti-mouse IgG (dilution at 1:2000; BOSTER, China) was added for binding with the first anti-Prdx6 mAb. The results were detected using Enhanced Chemiluminescence Substrate Reagent (BeyoECL Plus, Beyotime, China) by the ECL Detection System (Microchemi 4.2, DNR, Israel). 2.5. Analysis of supercoiled DNA protection activity of OaPrdx6 by metal-catalyzed oxidation assay in vitro Metal-catalyzed oxidation (MCO) assay was conducted using supercoiled DNAs of pUC19 plasmids as reaction substrates to determine the DNA protection effect of OaPrdx6 according to the reference (De Zoysa et al., 2008). Briefly, 50 ␮L of reaction mixtures containing 33 ␮M FeCl3 , 3.3 mM DTT, and different concentrations of the purified recombinant OaPrdx6H (6.25–200 ␮g/mL) were incubated at 37 ◦ C for 2 h. Then, 300 ng of supercoiled pUC19 DNAs were added to each reaction mixture with further incubating at 37 ◦ C for 2.5 h. The DNA protection effect was assessed by running 10 ␮L reaction mixtures on 1.0% agarose gel electrophoresis. Bovine serum albumin (BSA) was assayed separately in the same reaction conditions as negative control. 2.6. Differential expression analysis of OaPrdx6 2.6.1. Sampling, Rose Bengal Test, total protein extraction and cDNA synthesis For differential expression analysis of OaPrdx6 at the translation level, tissue samples including heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node, and white blood cells were collected from 3 healthy sheep (O. aries). While for that responding to Brucella infection at the transcription level, the serum and buffy coat

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samples respectively from 3 sheep treated with sterile 0.85% NaCl (the normal group), 3 sheep infected with a virulent field strain (BmF) of Brucella melitensis (the BmFchallenged group) and 3 sheep inoculated with a vaccine strain S2 of Brucella suis (the S2-challenged group) were obtained at 14, 40 and 60 days post-challenge (dpc) with a total challenge dose of 2.2 × 109 cfu bacteria per sheep, as shown in our previous report (Tang et al., 2012). To test the immune responses to Brucella from the sheep challenged with Brucella, Rose Bengal Test (RBT), a common serological diagnostic method for brucellosis, was conducted to detect antibodies against Brucella in sera according to the previous report (Diaz et al., 2011). Briefly, 30 ␮L of sera and 30 ␮L of RBT antigens (Harbin pharmaceutical Group Bio-vaccine Co., Ltd. China) were mixed on a white glossy glass tile by a toothpick. Then, after the tile was rocked at room temperature for 4 min, any visible agglutination and/or typical rim was considered as a positive result of successful challenge with Brucella. Whole proteins and total RNAs from each sample were prepared using the same method as described in Sections 2.4.2 and 2.2.1, respectively. Synthesis of the first strand cDNA was performed from 1 ␮g total RNA using PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) following the manufacturer’s instructions. 2.6.2. Expression analysis of OaPrdx6 in different tissues Differential expression of OaPrdx6 in different tissues was investigated using Western blot as described in Section 2.4.2. Whole protein lysates of heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node, and white blood cells from 3 healthy sheep were prepared, and their concentrations were determined using BCA Protein concentration Assay Kit (Sangon, China). Protein samples were separated on 12% SDS-PAGE, then transferred to a PVDF membrane. The expression level of ␤-actin was used as a reference for the semi-quantification assay. Optical densities and relative quantification of OaPrdx6 compared to the reference of ␤-actin were calculated using Quantity One software (Bio-Rad, USA). Statistical analysis of differences was conducted by one-way analysis of variance (ANOVA) using SPSS 13.0 software. 2.6.3. Differential transcription analysis of OaPrdx6 mRNA in buffy coat after challenged with virulent and avirulent Brucella by real-time PCR Differentially expressed OaPrdx6 transcripts from whole white blood cells (buffy coat) of O. aries after challenge with different virulent Brucella were determined at 14, 40 and 60 dpc by relative quantitative real time PCR (qPCR) using a pair of gene specific primers (OaPrdx6S and OaPrdx6A) (Table 1), and ␤-actin (GenBank accession number: U39357) was considered as an internal housekeeping gene control. The relative qPCR was carried out in a total volume of 20 ␮L containing 10 ␮L SYBR® Premix Ex taq (2×) (TaKaRa, Japan), 0.4 ␮L of ROX Reference Dye II (50×), 2.0 ␮L of the 1:10 diluted cDNA, 0.4 ␮L 10 ␮M each of primers. Real time PCR cycling was 95 ◦ C for 30 s; 40 cycles of 30 s at 95 ◦ C and 62 ◦ C for 34 s. Each sample was operated by the triple. The baseline was set by Applied Biosystems 7500 SDS Software automatically to

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maintain consistency. Relative expression of OaPrdx6 from buffy coat of O. aries was calculated using a 2−CT method (Livak and Schmittgen, 2001). Statistical analysis of the differences was conducted by one-way ANOVA using SPSS 13.0 software. 3. Results 3.1. Cloning and sequence analysis of the full-length cDNA of OaPrdx6 Total RNA of buffy coat was isolated as the template of reverse transcription to synthesize the 5 -Ready-cDNA. According to the partial cDNA sequence of OaPrdx6 screened from SSH cDNA library of O. aries, 3 -end of OaPrdx6 cDNA has been sequenced, and 5 -end of OaPrdx6 cDNA was amplified using 5 RACE technology. The assembled full-length sequence of OaPrdx6 cDNA was identified and deposited in GenBank with the accession number KC342239. The full-length cDNA of OaPrdx6 was 1753 bp containing 675 bp of ORF predicted to code OaPrdx6 protein with the deduced molecular weight of 25.07 kDa (Fig. 1). A putative polyadenylation signal (AATAAA) was identified before the poly(A) tail. There was no signal peptide in OaPrdx6 protein by SignalP prediction. Moreover, the predicted OaPrdx6 protein displayed unique cysteine residue (C47 ) of the peroxidase motif (45 PVCTTE50 ) and catalytic serine residue (S32 ) of the lipase motif (30 GDSWG34 ) at the N-terminus (Fig. 1), both of which were the essential amino acid residues of active sites for activities of the peroxidase and the PLA2 , respectively. The multiple sequence alignments revealed that the deduced amino acid sequence of OaPrdx6 showed significant homology with other known Prdx6s, of which the greatest identity with the Prdx6 of Bos Taurus (GenBank GI accession number gi27807167) was 100%. The amino acid sequence of ‘PVCTTE’ was highly conserved (Fig. 2). The phylogenetic tree showed that there were close genetic relations of Prdx6 between O. aries and other mammals, especially for B. Taurus (Fig. 3). 3.2. Expression and purification of recombinant OaPrdx6 proteins The recombinant OaPrdx6W and OaPrdx6H proteins were over-expressed in E. coli BL21-CodonPlus cells with IPTG induction, as shown in Fig. 4. It was clear that OaPrdx6 was highly induced (lane 2 for OaPrdx6W; lane 3 for OaPrdx6H) compared to un-induced cells (lane 1) as described in Fig. 4A. Then OaPrdx6W and OaPrdx6H were purified as shown in lane 2 of Fig. 4B and C, respectively. 3.3. Characteristics of the mAb against OaPrdx6 Four hybridoma cell lines secreting mAbs against OaPrdx6 were obtained and named as D41G4, F1F1, B11B9, 3RD5, respectively. The isotype of the first three mAbs belonged to IgG1, while the other one was IgG 2a. The concentration of the purified F1F1 mAb was 34.57 mg/mL with titer of 1.024 × 106 (data not shown). The result of specificity assay indicated that the purified anti-OaPrdx6 mAb

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Fig. 1. The full-length cDNA and the deduced amino acid sequence of OaPrdx6 from O. aries. The letters in bold indicated the start codon (ATG) and stop codon (TAG). The polyadenylation signal sequence (AATAAA) was marked in bold and underlined. The lipase motif was bold and shaded showing catalytic serine residue (S32 ) of the lipase motif in box. The peroxidase motif was only shaded with the conserved catalytic cysteine residue (C47 ) framed.

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Fig. 2. Multiple alignment of Prdx6s from O. aries and other known species. The conserved amino acid residues of Prdx6s were indicated by asterisks (*)

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Fig. 3. Phylogenetic analysis of amino acid sequences. The tree distances were generated according to the ClustalW algorithm, and the tree was constructed using MEGA 4.1.

could specifically bind with the recombinant and natural OaPrdx6 proteins (Fig. 5).

3.4. DNA protection activity of OaPrdx6 by MCO assay in vitro The DNA nicking assay was conducted to determine the DNA protection activity of recombinant OaPrdx6 proteins in terms of shifting in gel mobility of pUC19 plasmids after the treatment with MCO system and the recombinant OaPrdx6. Full degradation of the supercoiled DNA was observed in complete MCO system without OaPrdx6 (Fig. 6 lane 3), and similar DNA degradation in BSA group was also observed (Fig. 6 lane 4). With the concentration of OaPrdx6

increasing from 6.25 to 25 ␮g/mL (Fig. 6 lanes 5–7), the nicked form of DNA increased in a dose dependent manner. When the concentration of OaPrdx6 increased from 50 to 200 ␮g/mL (Fig. 6 lanes 8–10), the nicked form of DNA reduced and the supercoiled DNA increased in a dose dependent manner. The results indicated the recombinant OaPrdx6 protein had protective ability to minimize the DNA damage.

3.5. Differential expression analysis of OaPrdx6 Through Western blotting, significant expression differences at the protein level were observed among the whole proteins extracted from heart, liver, spleen, lung, kidney,

above the column. Conserved substitutions were showed by colons (:) and dots (.) for indicating the semi-conserved extent. The letters in box respectively indicated the lipase motif ‘GDSWG’ and the peroxidase motif ‘PVCTTE’. GenBank GI Numbers of Prdx6 protein sequences were showed as follows: Arenicola marina Prdx6 (gi68348727); Bombyx mori Prdx6 (gi157313403); Bos Taurus Prdx6 (gi27807167); Columba livia Prdx6 (gi379645435); Cristaria plicata Prdx6 (gi306846415); Danaus plexippus Prdx6 (gi357609745); Danio rerio Prdx6 (gi41387146); Eriocheir sinensis Prdx6 (gi209363473); Gallus gallus Prdx6 (gi86129578); Heterocephalus glaber Prdx6 (gi351695627); Homo sapiens Prdx6 (gi4758638); Ictalurus punctatus Prdx6 (gi318124171); Macaca mulatta Prdx6 (gi388453487); Oncorhynchus mykiss Prdx6 (gi259089135); Oplegnathus fasciatus Prdx6 (gi299507658); Ovis aries Prdx6 (gi469664895); Pongo abelii Prdx6 (gi197099987); Pteropus alecto Prdx6 (gi431916013); Rattus norvegicus Prdx6 (gi16758348); Saccostrea glomerata Prdx6 (gi260841345); Salmo salar Prdx6 (gi209732278); Scophthalmus maximus Prdx6 (gi299892750); Scylla paramamosain Prdx6 (gi213556919); Sparus aurata Prdx6 (gi298361180); Sus scrofa Prdx6 (gi47523870); Sepiella maindroni Prdx6 (gi336309249); Tupaia chinensis Prdx6 (gi444730526); Xenopus laevis Prdx6 (gi148233854).

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Fig. 4. Expression and purification analyses of the recombinant OaPrdx6 over-expressed in E. coli BL21-codonplus cells by SDS-PAGE. (A) Expression of the recombinant OaPrdx6. M: protein marker (Sangon, China); lane 1: total proteins from uninduced pET-30a-Prdx6-cpw cells; lane 2: total proteins from induced pET-30a-Prdx6-cpw cells (expressing OaPrdx6 with no His6 -tag); lane 3: total proteins from induced pET-30a-Prdx6-cph cells (expressing OaPrdx6 with His6 -tag). (B) Purification of the recombinant OaPrdx6 with no His6 -tag. M: protein marker (Sangon, China); lane 1: total proteins from induced pET-30a-Prdx6-cpw cells; lane 2: purified recombinant protein OaPrdx6W (with no His6 -tag). (C) Purification of the His6 -tagged recombinant OaPrdx6. M: protein marker (TaKaRa, Japan); lane 1: total proteins from induced pET-30a-Prdx6-cph cells; lane 2: purified recombinant protein OaPrdx6H (with His6 -tag). As shown, the recombinant OaPrdx6 was expressed in E. coli BL21-codonplus cells and purified by the methods of cut-gel and His6 -tag affinity chromatography.

Fig. 5. Immunoassay specificity of the purified monoclonal antibody F1F1. The Western blot was used in analyzing the immunoassay specificity of the purified mAb F1F1. M: protein marker (Thermo, USA); lane 1: total proteins extracted from uninduced pET-30a-Prdx6-cpw cells (expressing OaPrdx6 with no His6 -tag); lane 2: total proteins extracted from induced pET-30a-Prdx6-cpw cells; lane 3: total proteins extracted from uninduced pET-30a-Prdx6-cph cells (expressing OaPrdx6 with His6 -tag); lane 4: total proteins extracted from induced pET-30a-Prdx6-cph cells; lane 5: total proteins extracted from lung of O. aries.

stomach, intestine, muscle, lymph node, and white blood cells of healthy sheep (Fig. 7). However, OaPrdx6 could be detected in all tissues with the highest expression in the lung. As Fig. 8 shown, sera from all normal sheep and ones challenged with Brucella at 14 dpc were observed as negative results by the standard RBT. However, sera from the Brucella-challenged sheep at 40 dpc and 60 dpc showed highly positive, and there were no significant differences

between sheep challenged with avirulent strain S2 of Brucella and those challenged with virulent field strain of Brucella observed by naked eyes. So, RBT could not differ the sheep vaccinated against brucellosis from those infected with virulent Brucella. As shown in Fig. 9, the mRNA transcription level of OaPrdx6 in buffy coat of O. aries challenged with virulent and avirulent Brucella was quantified by qPCR using ␤-actin as internal control. There was no statistically significant difference between normal sheep and those challenged with virulent field strain of B. melitensis (BmF) at 14 dpc (p > 0.05), but at 40 dpc and 60 dpc, moderate up-regulation of OaPrx6 transcription compared with the normal group was detected in the BmF-infected group with significant differences. Correspondingly in the S2-inoculated group, the transcription of OaPrdx6 was down-regulated in contrast to the other groups (the normal control and the BmF-infected group) with statistical difference. As a result, OaPrdx6 from buffy coat of sheep showed different expression patterns in response to challenge with different virulent strain of Brucella. 4. Discussion In the present study, a novel full-length cDNA of OaPrdx6 gene from O. aries was isolated and characterized. The recombinant OaPrdx6 protein was produced and the DNA protection activity of the recombinant OaPrdx6 against metal-catalyzed oxidation was identified. The expression of OaPrdx6 in different tissues was investigated. Further, the mRNA transcription of OaPrdx6 gene was determined in buffy coat of sheep challenged with the virulent and avirulent Brucella. According to the partial OaPrdx6 cDNA containing the 3 -end sequence screened from SSH cDNA library of O. aries, OaPrdx6 cDNA with the 5 -end was obtained by 5 RACE. Each nested PCR including two steps was performed

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Fig. 6. Protection of nicking supercoiled DNAs by the recombinant OaPrdx6 in metal-catalyzed oxidation (MCO) assay. Lane 1: pUC19 (300 ng) without any treatment; lane 2: pUC19 + H2 O; lane 3: pUC19 + master mix (3.3 mM of DTT + 33 ␮M of FeCl3 ); lane 4: pUC19 + master mix + BSA (4 mg/mL); lanes 5–10: pUC19 + master mix + OaPrdx6 (6.25, 12.5, 25, 50, 100, and 200 ␮g/mL, respectively). NF: the nicked form of the plasmid; SF: the supercoiled form of the plasmid.

for cloning partial cDNA fragments of OaPrdx6 with two pairs of gene-specific primers designed in terms of the obtained partial cDNA sequences. After the first round of nested PCR using the first set of gene-specific primers GSP1 and NGSP1, there was not a single-specific band appeared. However, a 1.4 kb of specific bright band was

Fig. 7. Tissue distribution of OaPrdx6 from healthy sheep (O. aries). The tissue distribution analysis of OaPrdx6 was carried out by Western blot. Each sample was collected from 3 sheep. OaPrdx6 protein levels in tissues were normalized with the expression level of ␤-actin. The results were calculated in a measure of the relative quantification regarding the expression of OaPrdx6 in heart as a value of 100%. Statistical analysis of differences among heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node, and white blood cell groups were done by one-way analysis of variance (ANOVA) using SPSS 13.0 software. Data are presented as the mean relative expression ± SD (n = 3). An asterisk indicated the statistically significant difference (p < 0.05).

obtained by 1% agarose gel electrophoresis after the second round of PCR. Via sequencing and analysis, this specific DNA fragment did not contain the full-length 5 -end cDNA of OaPrdx6. As a result, according to the new extended cDNA sequence, we designed another set of gene-specific primers (GSP2 and NGSP2), so as to clone the full-length 5 end cDNA. As observed in the first nested PCR, only in the second round of PCR amplification was the bright specific band shown. Through assembling these partial sequences of OaPrdx6 cDNA, the novel full-length cDNA of Prdx6 gene from O. aries was first cloned and the coding amino acid sequence was deduced. The predicted OaPrdx6 showed 100% identity of the amino acid sequence with Prdx6 of B. Taurus (GenBank GI accession number gi27807167). Moreover, OaPrdx6 possessed the conserved peroxidase catalytic center (45 PVCTTE50 ) of a cysteine peroxiredoxin domain, which is characteristic of peroxiredoxin family and highly conserved in Prdx6 members (Wood et al., 2003). Meanwhile, OaPrdx6 also had a lipase catalytic center (30 GDSWG34 ), which is another typical structural feature of Prdx6 members. The phylogenetic tree showed that there was a close genetic relationship between OaPrdx6 and 1Cys Prdx from other mammals, especially B. Taurus (Fig. 3). The characteristics of OaPrdx6 cDNA mentioned above confirmed that OaPrdx6 obtained in our study belonged to 1-Cys Prdx sub-group of Prdx gene family. Our previous research results showed that there was no significant expression of the recombinant OaPrdx6 in E. coli BL21(DE3) cells by IPTG-driven induction (data not shown). So, E. coli BL21-Codonplus cells were used to produce the recombinant OaPrdx6 proteins. The relatively higher expression of recombinant OaPrdx6 proteins in E. coli BL21Codonplus cells could imply that E. coli BL21-Codonplus cells as host were suitable for the expression of OaPrdx6. It was reported that recombinant Prdx6 proteins from various sources display the DNA protection activity (De Zoysa et al., 2012; Sharapov et al., 2009). In this study, we identified the DNA protection activity of the recombinant OaPrdx6 was in a dose dependent manner. Results showed that OaPrdx6 could protect the nicked plasmid DNAs at

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Fig. 8. The Rose Bengal Test (RBT) for serological diagnosis of O. aries challenged with Brucella. N: the normal sheep (N1–N3) injected with sterile 0.85% NaCl; S: the sheep (S1–S3) challenged with an avirulent vaccine strain S2 of B. suis; B: the sheep (B1–B3) challenged with a virulent field strain of B. melitensis.

low concentrations and the supercoiled plasmid DNAs at high concentrations between 0.05 mg/mL and 0.20 mg/mL (Fig. 6). Brucellosis is a zoonosis caused by Gram-negative Brucella spp., which infects numerous species, widely distributing all over the world (Boschiroli et al., 2001; Mantecon Mde et al., 2008). At present, serological tests are used to detect antibodies in sera for the diagnosis of brucellosis, however they are not able to distinguish the brucellosis-vaccine-inoculated sheep from the virulent-Brucella-infected ones, as confirmed in Fig. 8. The differential diagnosis identifying between the vaccineinoculated animals and the Brucella-infected ones is crucial to eliminate the diseased animals for preventing and controlling brucellosis (Tang et al., 2012). Prdx6 is associated with host responses against invasion of bacteria and virus

Fig. 9. Differential transcription of OaPrdx6 from buffy coat of O. aries challenged with virulent and avirulent Brucella. Each treated group contained three sheep. Analysis of OaPrdx6 mRNA transcription was carried out using relative quantitative real-time PCR. N: the normal sheep group; S: the avirulent Brucella S2-challenged group; B: the virulent field Brucella BmF-challenged group. The expression fold was calculated by the 2−CT method regarding O. aries ␤-actin as a reference gene. The relative expression of OaPrdx6 at each time point was compared to that of 0.85% NaCl-injected control (Normal). Statistical analysis of differences among the normal control group, the avirulent Brucella S2-inoculated group and the virulent Brucella BmF-challenged group were done by one-way analysis of variance (ANOVA) using SPSS 13 software. Data were presented as mean relative expression ± SE (n = 3). ‘a’ indicated the statistically significant difference between the normal control and the Brucella (S2 or BmF) challenged group (p < 0.05). ‘b’ indicated the statistically significant difference between the avirulent Brucella S2-inoculated group and the virulent Brucella BmF-challenged group (p < 0.05).

(De Zoysa et al., 2012; Zheng et al., 2010), and may be a candidate of molecular biomarkers and treatment targets for some diseases (Huang et al., 2011). However, the expression of peroxiredoxin proteins exhibits circadian oscillations in cells (Edgar et al., 2012). As a result, the characteristics of OaPrdx6 as a biomarker need further to be validated in the future. In this study, it was observed that virulent Brucella induced the up-regulated expression of OaPrdx6 in white blood cells compared to the normal sheep, while avirulent Brucella induced the downregulated expression of OaPrdx6. The close relationship was firstly described between Prdx6 expression and Brucella infection. The results suggested that OaPrdx6 in white blood cells might be a potential candidate of molecular biomarkers to discern between sheep infected with virulent Brucella and ones inoculated with vaccines against brucellosis. In summary, the full-length cDNA of OaPrdx6 from O. aries was identified and characterized. OaPrdx6 gene was widely expressed in various tissues from sheep, and the mRNA transcription of OaPrdx6 from sheep infected with a virulent field strain of B. melitensis and ones inoculated with a vaccine strain S2 of B. suis was respectively up-regulated and down-regulated compared to the normal sheep, suggesting that OaPrdx6 might play an important physiological role and be associated with immune responses against invasion of Brucella. In light of our current findings, it was speculated that OaPrdx6 would be used as a potential biomarker to assess Brucella infection and facilitate preventing brucellosis.

Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (No. 30901070) and the Science & Technology Development Project of Jilin Province, China (No. 20150204078NY).

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Please cite this article in press as: Liu, N.-N., et al., Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.01.006