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Molecular cloning and characterization of a novel bovine IFN-ε
Molecular Cloning and Characterization of a novel bovine IFN- Yongli Guo, Mingchun Gao, Jun Bao, Xiuxin Luo, Ying Liu, Dong An, Haili Zhang, Bo Ma, Junwei Wang PII: DOI: Reference:
S0378-1119(14)01427-9 doi: 10.1016/j.gene.2014.12.031 GENE 40145
To appear in:
Gene
Received date: Revised date: Accepted date:
11 July 2014 10 November 2014 14 December 2014
Please cite this article as: Guo, Yongli, Gao, Mingchun, Bao, Jun, Luo, Xiuxin, Liu, Ying, An, Dong, Zhang, Haili, Ma, Bo, Wang, Junwei, Molecular Cloning and Characterization of a novel bovine IFN-, Gene (2014), doi: 10.1016/j.gene.2014.12.031
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ACCEPTED MANUSCRIPT Molecular Cloning and Characterization of a novel bovine IFN-ε Yongli Guo1, Mingchun Gao1*, Jun Bao2, 3, Xiuxin Luo1, Ying Liu1, Dong An1, Haili Zhang1,
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Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast
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BoMa1, Junwei Wang1, 3*
Agricultural University, Harbin, Heilongjiang 150030, P. R. China 2
College of Animal Science and Technology, Northeast Agricultural University, Harbin,
Synergetic Innovation Center Of Food Safety and Nutrition, Northeast Agricultural University,
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3
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Heilongjiang 150030, P. R. China
Harbin, 150030, P. R. China
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*Correspondence address: Tel: +86-451-55190385; Fax: +86-451-55191672;
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Email: [email protected], [email protected]
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Abstract: A bovine IFN-ε (BoIFN-ε) gene was amplified from bovine liver genomic DNA consisting of a 463bp partial 5’UTR, 582bp complete ORF and 171bp partial 3’UTR, which
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encodes a protein of 193 amino acids with 21-amino acid signal peptide and shares 61 to 87% identity with other species IFN-ε. Then BoIFN-ε gene was characterized, and it can be transcribed in EBK cells at a high level after infected by VSV. Recombinant proteins were expressed in E. coli and the antiviral activity was determined in vitro, which revealed bovine IFN-ε has less antiviral activity than bovine IFN-α. In addition, an immunofluorescence assay indicated that BoIFN-ε expressed in MDBK cells could be detected by polyclonal antibody against BoIFN-ε. Furthermore, the BoIFN-ε gene can be constitutively expressed in liver, thymus, kidney, small intestine and testis, but not in the heart. This study revealed that BoIFN-ε has the typical characteristics of type I interferon and can be expressed constitutively in certain tissue, which not 1
ACCEPTED MANUSCRIPT only can be a likely candidate for a novel, effective therapeutic agent, but also facilitate further research on the role of bovine IFN system.
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Keywords: Bovine; Interferon-ε; Type I interferon; mRNA transcription; Antiviral activity;
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Tissue distribution.
Introduction
The interferons (IFNs) are a large family of multifunctional secreted protein involved in
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antiviral defense, cell growth regulation, and immune activation. Among them, mammalian type I
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IFNs represents a large family of related proteins, mainly virus-inducible, divided in at least 8 distinct subfamilies named α, β, ω, δ, ɛ, ν, τ and κ (Krause and Pestka, 2005). Through a feedback
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loop of production and action, type I IFNs not only activate antiviral responses by autocrine means
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but also function systemically to induce an antiviral state in surrounding and distal cells. This
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antiviral activity, therefore, provides an avenue for the therapeutic application of type I IFN, is crucial in homeostasis and host defense.
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IFN-ε has been classified into the type I IFN family due to the same receptor type and gene locus, similarity of protein structure (Hardy et al., 2004; Kontsek et al., 2003; Peng et al., 2005). Structurally, the human IFN-ε consists of 192 amino acids, and the analysis of its protein structure has indicated that IFN-ε has overall similarity to IFN-β (Hardy et al., 2004). Unlike other type I IFNs, IFN-ε has found to be expressed constitutively in the lung, brain, small intestine, and reproductive tissues (Demers et al., 2014; Fung et al., 2013; Hardy et al., 2004; Hermant et al., 2013), which has less antiviral, anti-proliferative, and natural killer cell enhancing activities compared with IFN-α2b (Peng et al., 2007), but it is considered as playing a role in reproductive function, in either viral protection or early placental development in placental mammals (Fung et 2
ACCEPTED MANUSCRIPT al., 2013; Hermant et al., 2013; Matsumiya et al., 2007; Xi et al., 2012). By far, most information on type I IFN system is derived from studies of humans and mice,
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IFN-α and IFN-β have been the best studied, as well as IFN-ε (Conklin et al., 2001; Kontsek et al.,
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2003; Matsumiya et al., 2007; Peng et al., 2005), while IFN-α, IFN-β, IFN-ω in bovine have been studied, especially IFN-α and IFN-β. . With the increased awareness of viral disease in bovine, knowledge about bovine IFN-ε (BoIFN-ε) is crucial and these data can provide new opportunities
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to targeting viruses, such as FMDV. The human, murine, swine, and canine’s IFN-ε have been
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reported (Hardy et al., 2004; Sang et al., 2010; Yang et al., 2013), while not much work has been done on bovine IFN-ε. Here, we present the cloning and characterization of a novel gene encoding
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BoIFN-ε, which was demonstrated to have moderate antiviral activity in vitro and the gene can be
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constitutively expressed in certain tissue. This knowledge will contribute to the future use of type I
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IFN.
Materials and methods
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Cells and viruses
Liver was collected from Holstein bovine at dairy farm in Harbin, Heilongjiang, in Northeast China. MDCK cells, Vesicular stomatitis virus (VSV) were purchased from the China Institute of Veterinary Drug Control. MDBK, PK-15 cells, primary bovine testicular (BT) cells and primary embryo bovine kidney (EBK) cells were kindly provided by Dr. Li Yu. Cloning of the gene encoding BoIFN-ε Genomic DNA was extracted from the liver of bovine (Sambrook and Russell, 2008). First, we used human IFN-ε gene (GenBank: NM_176891.4) to locate the bovine IFN-ε gene in the genome of bovine（GenBank: GCA_000003205.4）. Then a pair of specific primers BoIFNES and 3
ACCEPTED MANUSCRIPT BoIFNEA (Table. 1) were designed according to the bovine genomic sequence containing the BoIFN-ε. Bovine genomic DNA was then applied as template in PCR amplification with the
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following thermal profile: initial denaturation at 94°C for 5 min, 30 amplification cycles of 94°C
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for 30 s, 52°C for 30 s, and 72°C for 90 s, followed by a final extension at 72°C for 10 min. The PCR product obtained was cloned into the pEASY Blunt-T Vector (TransGen, Beijing, China) and sequenced. At the same time, we performed RT-PCR with cDNAs prepared from EBK cells
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stimulated by VSV. Then total cellular mRNA was extracted with Trizol reagent (Sigma-Aldrich,
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St. Louis, MO) and treated with DNase I (Takara, Japana). The forward primer BoNE-RNAS2 and reverse primer BoNE-RNAA1, BoNE-RNAA2 (Table. 1) were designed according to the DNA
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sequence cloned here. The DNase-treated RNA was then reverse-transcribed to cDNA using a
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reverse transcription kit (Takara) with the specific primer BoNE-RNAA1. PCR was conducted
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with primers BoNE-RNAS2 and BoNE-RNAA2. RT-PCR products were cloned into the pEASY Blunt-T Vector and sequenced.
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Sequence characterization analysis of BoIFN-ε The potential open reading frame (ORF) was searched by the ORF Finder algorithm (http://www.ncbi.nlm.nih.gov/gorf/). The BoIFN-ε DNA sequence was analyzed using the Editseq and Megalign programs of the Lasergene 11 package (DNAStar, Inc., USA) and the putative amino acid sequence was compared with its counterparts of other animals by using the program ClustalX. Multiple alignments and phylogenetic tree were performed with ClustalX and MEGA 5.0 using the method of UPGMA, and the availability of branch stem was verified with the Bootstrap by the parameters 500. The glycosylation sites were analyzed by NetGlycate 1.0 Server （ http://www.cbs.dtu.dk/services/NetGlycate/ ） online. Secondary structure elements were 4
ACCEPTED MANUSCRIPT predicted using the algorithms available from NPS (Combet et al., 2000) (http: //www.npsa-pbil.ibcp.fr.).
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Tissue expression analysis of BoIFN-ε gene
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RNA was isolated from tissues, reverse-transcribed and subjected to RT-PCR as above, GAPDH was used as internal reference. The DNase-treated RNA was then reverse-transcribed to cDNA with the specific primer BoNE-RNAA1, BoNA-A and BoGAPDH-A. PCR was conducted
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with primers BoNE-RNAS2 and BoNE-RNAA2, BoNA-S and BoNA-A, BoGAPDH-S and
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BoGAPDH-A (Table. 1) separately for amplification for the gene of BoIFN-ε, BoIFN-αA and internal reference. The reactions for BoIFN-ε, BoIFN-αA and GAPDH were always run in
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separate tubes on the same plate with the appropriate negative controls.
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Protein expression and identification of BoIFN-ε
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pET32a and pET30a (Invitrogen, CA, USA) were used as the expression vector. In order to obtain the protein with His tag and non-His tag, the mature peptide were amplified by forward
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primer BoNEORFEI and BoNEORFNI, which shared the same reverse primer BoNEORFXI (Table. 1). The BoIFN-ε gene was cloned into vector pET-32a and pET-30a separately to yield pET32a-BoIFNε and pET30a-BoIFNε. The recombinant protein His-BoIFNε and rBoIFNε were induced with IPTG (Sigma-Aldrich) and the expression products were analyzed by SDS-PAGE. Then the recombinant protein His-BoIFNε was purified on a nickel chelated column (GenScript) according to the manufacturer’s instructions. After denaturation and renaturation, the soluble homogeneous protein concentration was measured by BCA kit (Beyotime, Beijing, China), which was used for measuring the biologically active of BoIFN-ε. At the same time, the recombinant protein rBoIFNε was purified by SDS-PAGE gel extraction and the product was used as 5
ACCEPTED MANUSCRIPT immunogen to prepared polyclonal antibody (PAb) (Coligan et al., 2005; Wang et al., 2012). BoIFN-ε mature peptide sequence was amplified by PCR using the
primers
vector
pcDNA3.1(+)
(Invitrogen).
The
recombinant
expression
vector
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expression
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pcDNA3.1-BoNES and BoNEORFXI(Table. 1). The PCR product was cloned into the eukaryotic
pcDNA3.1-BoNE and the negative control vector pcDNA3.1(+) were transfected into MDBK according to the DMRIE-C Reagent (Invitrogen) protocol. The cells were then incubated with
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PAb against BoIFN-ε (diluted at 1:100) prepared above for 2 h at 37℃. After washed three times
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with PBS, the plates were incubated with FITC-conjugated goat anti-rabbit IgG (ZSGB, Beijing, China, diluted at 1:200) for 1 h at 37℃. After washed three times by PBST, the cells treated with
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PAb were analyzed with fluorescence microscope.
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Antiviral activity analysis of BoIFN-ε in vitro
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First, VSV titers were determined by an endpoint dilution assay and the titers were expressed as the tissue culture infectious dose 50 (TCID50) per milliliter using the Reed-Muench method
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(Cheng et al., 2006; Sun et al., 2008). Virus titers were calculated by determining the dilution giving 50% of wells containing cells that displayed cytopathic effect. Antiviral activity was determined in a standard cytopathic effect assay. Briefly, monolayers of MDBK cells seeded in 96-well plates were treated with 100μL of 4-fold serial dilutions of His-BoIFNε for 24h and challenged with VSV (100TCID50/well), and the capacity of His-BoIFNε to inhibit the cytopathic effect was determined (Rubinstein et al., 1981). The wells without viruses were taken as cell controls and the wells without His-BoIFNε were used as virus controls. The plate was then re-incubated under the conditions of 37℃ in a humidified 5% CO2 atmosphere for 18~24h. One antiviral activity unit was defined as 50% reduction in the destruction of the cell 6
ACCEPTED MANUSCRIPT monolayer. In addition, the antiviral activity of BoIFN-ε were also conducted on EBK cells, BT cells, PK15 cells, MDCK cells and BHK21 cells.
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The specificity of BoIFN-ε antiviral protective activity was analyzed by antiviral activity
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blocking assay. MDBK cells were treated with His-BoIFNε neutralized for 24 h by PAb against BoIFN-ε (diluted 1:20) for 4 h at 37℃, and the controls were untreated for 24 h. Then, all cells were challenged with 100 pfu VSV.
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Results
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Characterization of BoIFN-ε gene
The BoIFN-ε gene fragments were obtained containing the 5'UTR, 3'UTR and an ORF
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(Fig.1). What’s more, BoIFN-ε gene can be expressed at a high level in EBK cells after infection
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with VSV, and the size of the expected amplimer was 368 bp (Fig. 4A). The putative protein with
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193 amino acids were obtained, encoding a signal peptide of 21 amino acids and a mature protein of 172 amino acids , and molecular mass of 22.694 kDa and theoretical isoelectric point of 8.34
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were estimated. Alignment of the sequences shows striking overall conservation of IFN-εs, with 71%–87% identity to human, swine and murine at the amino acid level (Fig.2A). What’s more, the ORF of the BoIFN-ε sequence has been submitted to the GenBank databases under the accession number KJ778878. The amino acid residues Ser-38, Glu-107 and Ile-167 are conserved residues among IFN-εs (Conklin et al., 2001). There are three cysteine residues at positions 53, 163 and 175 of the mature peptide conserved in IFN-εs, and two of them (Cys-53 and Cys-175) can be formed of the disulphide bonds that benefit for maintaining the natural structure of BoIFN-ε. The investigation of secondary structure indicates that BoIFN-ε has five putative alpha helices which were labeled as A to E according to the division of human and murine (Table.2). The predicted 7
ACCEPTED MANUSCRIPT three-dimensional structure reveal that model of BoIFN-ε is consistent with interferon molecular that analyzed by crystal diffraction (Fig. 2B), such as the recombinant murineIFN-β, ovineIFN-τ,
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humanIFN-β, humanIFN-α2b, who has five putative alpha helices.
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Putative transcription factor binding sites were conserved among the human, the mouse and bovine sequence. These conserved motifs include the two IRF binding sites (also designed VRE or ISRE) and progesterone receptor response element (PRE) (Fig.1) (Hardy et al., 2004) There
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are two regions of continuous “GT” tandem repeated (9 and 6, respectively) at the 3’UTR (Fig.1),
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but the functional significance of this motif is unknown.
A phylogenetic tree constructed from the amino acid sequences of BoIFN-ε was compared to
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other animals IFN sequence. The tree shows that BoIFN-ε is more similar to IFN-ε of other
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animals, but not with the other IFNs of bovine (Fig.2C), suggesting that IFN-ε gene arose from an
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ancestral duplication of a Type I IFN gene that was distinct from other bovine IFNs. The tree constructed indicated that IFN enumerated in Fig.2B belong to three distinct branches: type I, type
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II and type III, and BoIFN-ε belong to type IFN. Analysis of putative glycation sites in BoIFN-ε predicted seven potential glycation sites. It is possible that these glycation sites may act as spacers and protect the interferon molecule from protease-mediated hydrolysis, thereby playing an important role in maintaining effector functions by contributing to the processes of folding, oligomerization, and stability (Hosoi et al., 1988). The identification of such sites raised the possibility that BoIFN-ε cloned here may form a glycoprotein. Protein expression and identification of BoIFN-ε SDS-PAGE analysis revealed that the recombinant protein His-BoIFNε was mainly 8
ACCEPTED MANUSCRIPT expressed as the inclusion body form with an apparent molecular weight of 35 kDa (Fig.4B) and rBoIFNε was at the molecular weight of 20 kDa (Fig.4C). The protein concentration of purified
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His-BoIFNε was 0.192mg/ml and rBoIFNε was 0.278mg/ml.
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The results of immunofluorescence assay showed that cells transfected with recombinant expression vector pcDNA3.1-BoNE has green fluorescence, while there was no fluorescence with
expression of BoIFN-ε.
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Antiviral activities of BoIFN-ε in vitro
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cells in control group (Fig.4D), which showed the PAb against BoIFN-ε can identify the
In order to determine whether BoIFN-ε has the biological activities, cytopathic inhibition
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assays in vitro was used to analyze its antiviral activity. The results show that BoIFN-ε has a
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protective effect on MDBK, EBK, BT and PK-15 cells, but not on MDCK and BHK21 cells.
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What’s more, the antiviral activity against VSV of BoIFN-ε was lower than rBoIFN-αA on MDBK, EBK, BT and PK-15 cells (Table. 3).
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The results of antiviral activity blocking assay suggest that the antiviral activity of His-BoIFNε was completely abrogated by PAb against BoIFN-ε at a dilution of 1:16, and when the antibody dilution was 1:128, the blocking activity was nearly non-existent, while no antiviral activity was observed by addition of preimmune rabbit serum.
Discussion Type I IFNs are central to innate and adaptive immunity and comprise many functional subgroups, such as IFN-α, β, ɛ, δ, ω and κ, and these IFNs have different antiviral activities. IFN-α may play the role of the “soldier” IFN, as some subtypes of IFN-α possess the highest specific antiviral activity of all the type I IFNs as well as the greatest cytotoxic activity (Pestka et al., 1987; 9
ACCEPTED MANUSCRIPT Pestka, 1997, 2000). IFN-κ is an interferon with relatively weak specific antiviral activity expressed predominantly by keratinocytes (Nardelli et al., 2000). Some IFN-ω and IFN-δ subtypes,
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IFN-ε, and IFN-β differed in their antiviral activity based on target cells and viruses (Krause and
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Pestka, 2005).
IFN-ε is expressed in a tissue-specific fashion, by eptithelial cells of the female reproductive tract, which serves a specific role in reproductive tissues either in viral protection or early
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placental development (Fung et al., 2013). Here, we present the cloning and characterization of a
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novel gene encoding Bovine IFN-ε. In general, type I IFNs can be cloned from genome because of the typical feature of lacking intron in mammals and birds (Lowenthal et al., 2001), so we could
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clone the BoIFN-ε gene from genomic DNA, then it was verified by RT-PCR that BoIFN-ε gene
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can be transcribed in EBK cells after stimulation by VSV. Tissue distribution analysis suggested
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BoIFN-ε could be express in liver, thymus, kidney, small intestine and testis but not in heart. As we took tissue samples from bull, the expression in female reproductive tract were not analyzed
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here. There are some differences in the tissue expression of IFN-ε gene measured in different species, but the IFN-ε gene tend to be expressed on the mucosal tissue. In human, the expression of IFN-ε was not detectable at significant levels in any organ with the notable exception of the uterus, cervix, vagina and ovary (Fung et al., 2013). Mouse IFN-ε gene mRNA was expressed in ovaries, uterus, fibroblasts, lung, brain, adrenal glands, and heart but was not detected in thymus, spleen, testis, liver, and kidney (Hardy et al., 2004). While in rhesus macaques, IFN-ε can be expressed highly in cervix, ovary and testis, the heart, thymus, intestine, kidney, brain and spinal cord also can be detected the expression of IFN-ε (Demers et al., 2014). The swine IFN-ε gene can be detected in all the tested tissues such as skin, intestine, mesenteric lymph nodes, testis, spleen 10
ACCEPTED MANUSCRIPT and PMC, and highly expressed in intestine, mesenteric lymph nodes and skin (Sang et al., 2010). The tissue distribution of IFN-ε gene in different species is different from each other.
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The characterization of the BoIFN-ε was analyzed by bioinformatics software. First, in
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alignments of the inferred amino acid sequences, it showed the highest similarity with swine IFN-ε, at 87.1% identity. Second, BoIFN-ε was found to contain cysteines at the same positions as those in IFN-εs, which may play an important role in maintaining the secondary structure of IFN.
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The relative position of BoIFN-ε gene within the IFN-εs are also conserved between humans and
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mice. Third, seven potential glycosylation sites were predicted in BoIFN-ε, which may play an important role in maintaining effector functions, raising the possibility for BoIFN-ε to form a
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glycoprotein. Fourth, homology modeling showed the BoIFN-ε has the typical characterization of
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IFN, which has five alpha helices. Fifth, phylogenetic analysis showed that BoIFN-ε has a closer
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genetic relationship to IFN-ε than to any other IFN analyzed, suggesting that the IFN-ε gene of swine, human, murine, cat, monkey and wolf may have arisen during evolution by duplication of a
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common ancestor. What’s more, the PAb prepared with rBoNE can be used to detect bovine IFN-ε in MDBK cells.
According to the preceding analysis, it is novel bovine IFN-ε. To see whether BoIFN-ε possesses biological function, we expressed the BoIFN-ε in Escherichia coli andmeasured the antiviral activity using CPE inhibition assays in vitro. These results demonstrated that BoIFN-ε protected MDBK, BT, EBK and PK-15 cells from challenge with VSV, indicating that bovine IFN-ε has certain antiviral activity on homologous cells, as well as the cells derived from the species that have near relative, which is different from canine IFN-ε (Yang et al., 2013). In addition, bovine IFN-ε has lower antiviral activity than BoIFNαA as described above, which is 11
ACCEPTED MANUSCRIPT similar to the human and canine IFN-ε (Peng et al., 2007; Yang et al., 2013). At the same time, the
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PAb against BoIFN-ε in the dose dependent manner (Li et al., 2006).
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antiviral activity of BoIFN-ε can be inhibited under certain experimental conditions such as by
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IFN-ε expression patterns in the reproductive tract is related to expression of estrogen (Fung et al., 2013), and there are progesterone receptor response element (PRE) existing in the gene promoter region (Hardy et al., 2004), suggesting that IFN-ε is regulated by hormones. IFN-ε was
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constitutively expressed by epithelial cells of the female reproductive tract and hormonally
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regulated in human and murine (Fung et al., 2013). In our study, IRF1 and IRF2 were predicted in the upstream of BoIFN-ε coding region, and BoIFN-ε expression patterns may regulated by virus
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stimulation in some non-reproductive tract tissues. Canine IFN-ε can be obtained from MDCK
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cells after stimulated by polyI:C, and polyI:C can effectively simulate the double-stranded RNA
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viruses (Yang et al., 2013). Mouse IFN-ε1 mRNA was constitutively expressed and when the levels of the mRNA transcripts encoding IFN-ε1 were quantified relative to GAPDH the levels
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increased only three fold 12 h after infection by SFV (Hardy et al., 2004). These suggest IRF binding sites may exert the role in induction of IFN. EBK cells can induced more BoIFN-ε specifically after stimulated by VSV, which also confirmed that BoIFN-ε can be transcribed in vivo.. In this study, a novel gene encoding BoIFN-ε was first reported, then the tissue distribution and antiviral activity were analyzed, which can enrich knowledge
of bovine type I IFNs.
Consequently, there is much discussion on the significance of the existence of bovine IFN-ε. It is speculated that bovine IFN-ε may have particular importance during certain viral infections and may be optimally active in specific cell types. Further studies are required to address these 12
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Acknowledgements
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We thank Dr. Li Yu (Harbin Veterinary Research Institute, Chinese Academy of Agricultural
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Sciences, Harbin, China) for kindly providing MDBK, PK-15 and EBK cells. This study is supported by the Earmarked Fund for China Agriculture Research System (No. CARS-37), Synergetic Innovation Center of Food Safety and Nutrition, the sub-project of National 12th
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5-year Support Key Projects (2012BAD12B03, 2012BAD12B05) and the Key Technologies
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Research and Development Program of Heilongjiang province (GA09B302). We would like to express our sincere appreciation to the reviewers for their insightful recommendations, which have
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greatly aided us in improving the quality of the paper.
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References:
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Cheng, G., Chen, W., Li, Z., Yan, W., Zhao, X., Xie, J., Liu, M., Zhang, H., Zhong, Y., Zheng, Z.,
SC R
2006. Characterization of the porcine alpha interferon multigene family. Gene 382, 28-38. Coligan, J., Bierer, B., Margulies, D., 2005. Short Protocols in Immunology. John Wiley & Sons, Inc., New Jersey.
NU
Combet, C., Blanchet, C., Geourjon, C., Deleage, G., 2000. NPS@: network protein sequence analysis.
MA
Trends Biochem Sci 25, 147-150.
Conklin, D.C., Grant, F.J., Rixon, M.W., Kindsvogel, W., 2001. INTERFERON-EPSILON., C07H
D
21/02 ed, p. 64.
TE
Demers, A., Kang, G., Ma, F., Lu, W., Yuan, Z., Li, Y., Lewis, M., Kraiselburd, E.N., Montaner, L. Li,
CE P
Q. 2014. The mucosal expression pattern of interferon-epsilon in rhesus macaques. J Leukoc Biol Fung, K.Y., Mangan, N.E., Cumming, H., Horvat, J.C., Mayall, J.R., Stifter, S.A., De Weerd, N.,
AC
Roisman, L.C., Rossjohn, J., Robertson, S.A., Schjenken, J.E., Parker, B., Gargett, C.E., Nguyen, H.P., Carr, D.J., Hansbro, P.M., Hertzog, P.J., 2013. Interferon-epsilon protects the female reproductive tract from viral and bacterial infection. Science 339, 1088-1092. Hardy, M.P., Owczarek, C.M., Jermiin, L.S., Ejdeback, M., Hertzog, P.J., 2004. Characterization of the type I interferon locus and identification of novel genes. Genomics 84, 331-345. Hermant, P., Francius, C., Clotman, F., Michiels, T., 2013. IFN-epsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines. PLoS One 8, e71320. Hosoi, K., Utsumi, J., Kitagawa, T., Shimizu, H., Kobayashi, S., 1988. Structural characterization of 14
ACCEPTED MANUSCRIPT fibroblast human interferon-beta 1. J Interferon Res 8, 375-384. Kontsek, P., Karayianni-Vasconcelos, G., Kontsekova, E., 2003. The human interferon system:
IP
T
characterization and classification after discovery of novel members. Acta Virol 47, 201-215.
friends and relatives. Pharmacol Ther 106, 299-346.
SC R
Krause, C.D., Pestka, S., 2005. Evolution of the Class 2 cytokines and receptors, and discovery of new
Li, H.T., Ma, B., Mi, J.W., Jin, H.Y., Xu, L.N., Wang, J.W., 2006. Cloning, in vitro expression and
NU
bioactivity of goose interferon-alpha. Cytokine 34, 177-183.
MA
Lowenthal, J.W., Staeheli, P., Schultz, U., Sekellick, M.J., Marcus, P.I., 2001. Nomenclature of avian interferon proteins. J Interferon Cytokine Res 21, 547-549.
D
Matsumiya, T., Prescott, S.M., Stafforini, D.M., 2007. IFN-epsilon mediates TNF-alpha-induced
CE P
J Immunol 179, 4542-4549.
TE
STAT1 phosphorylation and induction of retinoic acid-inducible gene-I in human cervical cancer cells.
Nardelli, B., LaFleur, D. W., Chinchilla, D., Tsareva, T., Guoxian, C., Mather, D., et al., 2000.
AC
IFN-κ, a novel type I interferon. Eur Cytokine. Peng, F.W., Duan, Z.J., Zheng, L.S., Xie, Z.P., Gao, H.C., Zhang, H., Li, W.P., Hou, Y.D., 2007. Purification of recombinant human interferon-epsilon and oligonucleotide microarray analysis of interferon-epsilon-regulated genes. Protein Expr Purif 53, 356-362. Peng, F.W., Gao, H.C., Xie, Z.P., Zhang, H., Li, Q.M., Duan, Z.J., Hou, Y.D., 2005. [Biological activities of recombinant human interferon Epsilon]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 19, 227-231.
Pestka, S., Langer, J. A., Zoon, K. C., & Samuel, C. E., 1987. Interferons and their actions. Annu Rev Biochem 56, 727-777 15
ACCEPTED MANUSCRIPT Pestka, S., 1997. The human interferon-a species and hybrid proteins. Semin Oncol 24(3 Suppl. 9), S9.
IP
T
Pestka, S., 2000. The human interferon a species and receptors. Biopolymers 55(4), 254-287
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Rubinstein, S., Familletti, P.C., Pestka, S., 1981. Convenient assay for interferon. J. Virol. 37, 755-758. Sambrook, J., Russell, D.W., 2008. Molecular Cloning: A Laboratory Manual. Science Press, Beijing. Sang, Y., Rowland, R.R., Hesse, R.A., Blecha, F., 2010. Differential expression and activity of the
NU
porcine type I interferon family. Physiol Genomics 42, 248-258.
MA
Sun, Y., Li, W.,H. Li, M., Gao, M., Wei, W., Wei, S., Wang, J., 2008. Expression and purification of IFN-α gene from bovine. Chinese Journal of Preventive Veterinary Medicine 30, 889-892.
D
Wang, J., Guo, Y., Gao, M., 2012. Preparationof polyclonal antibody specific for light chain of goose
TE
immunoglobulin using recombinant protein. Journal of Northeast Agricultural University 43, 60-63.
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Xi, Y., Day, S.L., Jackson, R.J., Ranasinghe, C., 2012. Role of novel type I interferon epsilon in viral infection and mucosal immunity. Mucosal Immunol 5, 610-622.
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Yang, L., Xu, L., Li, Y., Li, J., Bi, Y., Liu, W., 2013. Molecular and functional characterization of canine interferon-epsilon. J Interferon Cytokine Res 33, 760-768.
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Figure legends:
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Fig.1 Nucleotide sequence and inferred amino acid sequences of BoIFN-ε gene. The nucleotide
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and amino acid sequences are numbered on the left and right sides, respectively. The signal peptide is underlined, and the conserved cysteines are indicated by shaded circles, while the conserved amino acids Ser-38, Glu-107 and Ile-167 are indicated by gray-shaded boxes. In the
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5’UTR, the virus-responsive elements are boxed in gray and the continuous repeat regions in
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3’UTR are italic and bold.
Fig.2 A. IFN-ε amino acid alignment of Bovine, Human, Murine and Canine. B. Predicted
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three-dimensional structures of bovine IFN-ε. The positions of helices A to E are labeled. C.
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Phylogenetic tree of BoIFN-ε amino acid sequences of several mammals IFN. The bar scale
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represents the genetic distance. The credibility value for each node is shown. B. Fig.3 Tissue distribution expression of IFN-ε. Bovine organs were harvested and IFN-ε expression
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(A), IFN-αA expression (B) and GAPDH (C) expression in heart, liver, thymus, kidney, small intestine and testis were measured by RT-PCR. Fig.4 A. RT-PCR analysis BoIFN-ε mRNA transcripts in EBK cells infected with VSV. Lane M: Trans2K DNA marker; Lane 1: PCR--amplified with EBK cells infected with VSV; Lane 2: Negative control; Lane 3: PCR product at 9h. B. SDS-PAGE analysis of the expressed and purified His-BoIFNε, Lane M: Unstained Protein Marker; Lane 1: Negative control of empty vector pET-32a (+); Lane 2: His-BoIFNε after induction; Lane 3: Sedimentations of His-BoIFNε; Lane 4: Supernatants of His-BoIFNε; Lane 5: Purified His-BoIFNε. C. SDS-PAGE analysis of the purified rBoIFNε, Lane M: Unstained Protein Marker; Lane 1: rBoIFNε after induction; Lane 2: 17
ACCEPTED MANUSCRIPT Purified rBoIFNε. D. Immunofluorescence micrographs analysis of the BoIFNε overexpressed in
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MDBK cells.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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ACCEPTED MANUSCRIPT Table 1. Nucleotide Sequence of the PCR primers used in this study Sequence(5’-3’)
BoIFNES BoIFNEA BoNE-RNAS2 BoNE-RNAA1 BONEBoNE-RNAA2 BoIFNA-S BoIFNA-A BoGAPDH-S BoGAPDH-A BoNEORFEI BoNEORFNI BoNEORFXI pcDNA3.1-BoNES
GGTGCTGCTAAGCCTCTGA CAGTCGGCTGTTCCTTCAC AGACCTCGTCAGTTCAGCAG TCAAGTTTCCATGCCCTGT GACAATAGTCCAGGCACAGC TGGTCCTTCCTGCTATCCCT CTGACAACCTCCCAGGCACA TTGGCATCGTGGAGGGACT GAGTGAGTGTCGCTGTTGAAGT TGTTGAATTC EcoR I CAAGAGCTGAAACTGGTT CTGTTCATATGNde ICAAGAGCTGAAACTGGTT TCACTCGAG Xho I TCAAGTTTCCATGCCCTGT ACTGAATTCEcoR IATGCAAGAGCTGAAACTGGTT
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Primer
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Note: The restriction enzyme sites that were introduced in primers are underlined.
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ACCEPTED MANUSCRIPT Table 2. Comparison of the IFN-ε structural feature among human, murine and bovine Amino Acid Residues
Amino Acid Residues
Amino Acid Residues
Interferon-epsilon
of Human
of Murine
of Bovine
Signal sequence
1-21
1-21
Helix A
27-48
27-47
AB Loop
49-75
Helix B
76-94
BC Loop
95-102
Helix C
103-123
CD Loop
124-138
Helix D
139-158
DE Loop
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Structural Feature of
1-21 27-48 49-75
75-93
76-94
94-101
95-102
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48-74
103-123
123-137
124-138
138-157
139-158
159-162
158-161
159-162
Helix E
163-184
162-183
163-184
C-terminus
185-208
184-192
185-193
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102-122
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ACCEPTED MANUSCRIPT Table 3. Comparison of antiviral activity against VSV between BoIFN-ε and BoIFN-αA
rBoIFN-αA
MDBK cells
6.7×103
5.95×104
EBK cells
1.13×104
BT cells
2.27×104
8.25×106
PK-15 cells
3.53×102
3.90×105
MDCK cells
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rBoIFN-ε
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Antiviral activity against VSV（U/mg）
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BHK21 cells
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2.90×106
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ACCEPTED MANUSCRIPT Abbreviations IFN-ε, interferon-episilon; BoIFN-ε, bovine interferon-episilon; ORF, open reading frame; VSV,
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vesicular stomatitis virus; UTR, untranslated regions; FITC goat-anti-rabbit IgG, fluorescein
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isothiocyanate-conjugated goat anti-rabbit IgG; cDNA, DNA complementary to RNA; MDBK cells, Madin-Darby bovine kidney cells; EBK cells, Embryonic Bovine Kidney cells; PAb, polyclonal antibody; IPTG, isopropyl β-D-thiogalactoside; MDCK cells, Madin-Darby canine
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kidney cells; BHK21 cells, Baby Hamster Syrian Kidney cells; VRE, virus-responsive elements;
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Cys, cysteine residues; PRE, progesterone receptor response element.
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ACCEPTED MANUSCRIPT Highlights
Bovine IFN-ε was first cloned here and the characterization of bovine IFN-ε was analyzed.
The mature peptide was highly expressed in prokaryotic expression system, and it could be
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overexpressed in MDBK cells.
Bovine IFN-ε has moderate antiviral activity on MDBK, BT, EBK and PK-15cells, but not on
Bovine IFN-ε gene can be constitutively expressed in liver, thymus, kidney, small intestine
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and testis, but not in the heart.
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BHK-21 and MDCK cells.
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S0378-1119(14)01427-9 doi: 10.1016/j.gene.2014.12.031 GENE 40145
To appear in:
Gene
Received date: Revised date: Accepted date:
11 July 2014 10 November 2014 14 December 2014
Please cite this article as: Guo, Yongli, Gao, Mingchun, Bao, Jun, Luo, Xiuxin, Liu, Ying, An, Dong, Zhang, Haili, Ma, Bo, Wang, Junwei, Molecular Cloning and Characterization of a novel bovine IFN-, Gene (2014), doi: 10.1016/j.gene.2014.12.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Molecular Cloning and Characterization of a novel bovine IFN-ε Yongli Guo1, Mingchun Gao1*, Jun Bao2, 3, Xiuxin Luo1, Ying Liu1, Dong An1, Haili Zhang1,
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Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast
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BoMa1, Junwei Wang1, 3*
Agricultural University, Harbin, Heilongjiang 150030, P. R. China 2
College of Animal Science and Technology, Northeast Agricultural University, Harbin,
Synergetic Innovation Center Of Food Safety and Nutrition, Northeast Agricultural University,
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3
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Heilongjiang 150030, P. R. China
Harbin, 150030, P. R. China
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*Correspondence address: Tel: +86-451-55190385; Fax: +86-451-55191672;
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Email: [email protected], [email protected]
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Abstract: A bovine IFN-ε (BoIFN-ε) gene was amplified from bovine liver genomic DNA consisting of a 463bp partial 5’UTR, 582bp complete ORF and 171bp partial 3’UTR, which
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encodes a protein of 193 amino acids with 21-amino acid signal peptide and shares 61 to 87% identity with other species IFN-ε. Then BoIFN-ε gene was characterized, and it can be transcribed in EBK cells at a high level after infected by VSV. Recombinant proteins were expressed in E. coli and the antiviral activity was determined in vitro, which revealed bovine IFN-ε has less antiviral activity than bovine IFN-α. In addition, an immunofluorescence assay indicated that BoIFN-ε expressed in MDBK cells could be detected by polyclonal antibody against BoIFN-ε. Furthermore, the BoIFN-ε gene can be constitutively expressed in liver, thymus, kidney, small intestine and testis, but not in the heart. This study revealed that BoIFN-ε has the typical characteristics of type I interferon and can be expressed constitutively in certain tissue, which not 1
ACCEPTED MANUSCRIPT only can be a likely candidate for a novel, effective therapeutic agent, but also facilitate further research on the role of bovine IFN system.
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Keywords: Bovine; Interferon-ε; Type I interferon; mRNA transcription; Antiviral activity;
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Tissue distribution.
Introduction
The interferons (IFNs) are a large family of multifunctional secreted protein involved in
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antiviral defense, cell growth regulation, and immune activation. Among them, mammalian type I
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IFNs represents a large family of related proteins, mainly virus-inducible, divided in at least 8 distinct subfamilies named α, β, ω, δ, ɛ, ν, τ and κ (Krause and Pestka, 2005). Through a feedback
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loop of production and action, type I IFNs not only activate antiviral responses by autocrine means
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but also function systemically to induce an antiviral state in surrounding and distal cells. This
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antiviral activity, therefore, provides an avenue for the therapeutic application of type I IFN, is crucial in homeostasis and host defense.
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IFN-ε has been classified into the type I IFN family due to the same receptor type and gene locus, similarity of protein structure (Hardy et al., 2004; Kontsek et al., 2003; Peng et al., 2005). Structurally, the human IFN-ε consists of 192 amino acids, and the analysis of its protein structure has indicated that IFN-ε has overall similarity to IFN-β (Hardy et al., 2004). Unlike other type I IFNs, IFN-ε has found to be expressed constitutively in the lung, brain, small intestine, and reproductive tissues (Demers et al., 2014; Fung et al., 2013; Hardy et al., 2004; Hermant et al., 2013), which has less antiviral, anti-proliferative, and natural killer cell enhancing activities compared with IFN-α2b (Peng et al., 2007), but it is considered as playing a role in reproductive function, in either viral protection or early placental development in placental mammals (Fung et 2
ACCEPTED MANUSCRIPT al., 2013; Hermant et al., 2013; Matsumiya et al., 2007; Xi et al., 2012). By far, most information on type I IFN system is derived from studies of humans and mice,
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IFN-α and IFN-β have been the best studied, as well as IFN-ε (Conklin et al., 2001; Kontsek et al.,
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2003; Matsumiya et al., 2007; Peng et al., 2005), while IFN-α, IFN-β, IFN-ω in bovine have been studied, especially IFN-α and IFN-β. . With the increased awareness of viral disease in bovine, knowledge about bovine IFN-ε (BoIFN-ε) is crucial and these data can provide new opportunities
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to targeting viruses, such as FMDV. The human, murine, swine, and canine’s IFN-ε have been
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reported (Hardy et al., 2004; Sang et al., 2010; Yang et al., 2013), while not much work has been done on bovine IFN-ε. Here, we present the cloning and characterization of a novel gene encoding
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BoIFN-ε, which was demonstrated to have moderate antiviral activity in vitro and the gene can be
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constitutively expressed in certain tissue. This knowledge will contribute to the future use of type I
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IFN.
Materials and methods
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Cells and viruses
Liver was collected from Holstein bovine at dairy farm in Harbin, Heilongjiang, in Northeast China. MDCK cells, Vesicular stomatitis virus (VSV) were purchased from the China Institute of Veterinary Drug Control. MDBK, PK-15 cells, primary bovine testicular (BT) cells and primary embryo bovine kidney (EBK) cells were kindly provided by Dr. Li Yu. Cloning of the gene encoding BoIFN-ε Genomic DNA was extracted from the liver of bovine (Sambrook and Russell, 2008). First, we used human IFN-ε gene (GenBank: NM_176891.4) to locate the bovine IFN-ε gene in the genome of bovine（GenBank: GCA_000003205.4）. Then a pair of specific primers BoIFNES and 3
ACCEPTED MANUSCRIPT BoIFNEA (Table. 1) were designed according to the bovine genomic sequence containing the BoIFN-ε. Bovine genomic DNA was then applied as template in PCR amplification with the
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following thermal profile: initial denaturation at 94°C for 5 min, 30 amplification cycles of 94°C
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for 30 s, 52°C for 30 s, and 72°C for 90 s, followed by a final extension at 72°C for 10 min. The PCR product obtained was cloned into the pEASY Blunt-T Vector (TransGen, Beijing, China) and sequenced. At the same time, we performed RT-PCR with cDNAs prepared from EBK cells
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stimulated by VSV. Then total cellular mRNA was extracted with Trizol reagent (Sigma-Aldrich,
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St. Louis, MO) and treated with DNase I (Takara, Japana). The forward primer BoNE-RNAS2 and reverse primer BoNE-RNAA1, BoNE-RNAA2 (Table. 1) were designed according to the DNA
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sequence cloned here. The DNase-treated RNA was then reverse-transcribed to cDNA using a
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reverse transcription kit (Takara) with the specific primer BoNE-RNAA1. PCR was conducted
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with primers BoNE-RNAS2 and BoNE-RNAA2. RT-PCR products were cloned into the pEASY Blunt-T Vector and sequenced.
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Sequence characterization analysis of BoIFN-ε The potential open reading frame (ORF) was searched by the ORF Finder algorithm (http://www.ncbi.nlm.nih.gov/gorf/). The BoIFN-ε DNA sequence was analyzed using the Editseq and Megalign programs of the Lasergene 11 package (DNAStar, Inc., USA) and the putative amino acid sequence was compared with its counterparts of other animals by using the program ClustalX. Multiple alignments and phylogenetic tree were performed with ClustalX and MEGA 5.0 using the method of UPGMA, and the availability of branch stem was verified with the Bootstrap by the parameters 500. The glycosylation sites were analyzed by NetGlycate 1.0 Server （ http://www.cbs.dtu.dk/services/NetGlycate/ ） online. Secondary structure elements were 4
ACCEPTED MANUSCRIPT predicted using the algorithms available from NPS (Combet et al., 2000) (http: //www.npsa-pbil.ibcp.fr.).
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Tissue expression analysis of BoIFN-ε gene
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RNA was isolated from tissues, reverse-transcribed and subjected to RT-PCR as above, GAPDH was used as internal reference. The DNase-treated RNA was then reverse-transcribed to cDNA with the specific primer BoNE-RNAA1, BoNA-A and BoGAPDH-A. PCR was conducted
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with primers BoNE-RNAS2 and BoNE-RNAA2, BoNA-S and BoNA-A, BoGAPDH-S and
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BoGAPDH-A (Table. 1) separately for amplification for the gene of BoIFN-ε, BoIFN-αA and internal reference. The reactions for BoIFN-ε, BoIFN-αA and GAPDH were always run in
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separate tubes on the same plate with the appropriate negative controls.
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Protein expression and identification of BoIFN-ε
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pET32a and pET30a (Invitrogen, CA, USA) were used as the expression vector. In order to obtain the protein with His tag and non-His tag, the mature peptide were amplified by forward
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primer BoNEORFEI and BoNEORFNI, which shared the same reverse primer BoNEORFXI (Table. 1). The BoIFN-ε gene was cloned into vector pET-32a and pET-30a separately to yield pET32a-BoIFNε and pET30a-BoIFNε. The recombinant protein His-BoIFNε and rBoIFNε were induced with IPTG (Sigma-Aldrich) and the expression products were analyzed by SDS-PAGE. Then the recombinant protein His-BoIFNε was purified on a nickel chelated column (GenScript) according to the manufacturer’s instructions. After denaturation and renaturation, the soluble homogeneous protein concentration was measured by BCA kit (Beyotime, Beijing, China), which was used for measuring the biologically active of BoIFN-ε. At the same time, the recombinant protein rBoIFNε was purified by SDS-PAGE gel extraction and the product was used as 5
ACCEPTED MANUSCRIPT immunogen to prepared polyclonal antibody (PAb) (Coligan et al., 2005; Wang et al., 2012). BoIFN-ε mature peptide sequence was amplified by PCR using the
primers
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pcDNA3.1(+)
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expression
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pcDNA3.1-BoNES and BoNEORFXI(Table. 1). The PCR product was cloned into the eukaryotic
pcDNA3.1-BoNE and the negative control vector pcDNA3.1(+) were transfected into MDBK according to the DMRIE-C Reagent (Invitrogen) protocol. The cells were then incubated with
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PAb against BoIFN-ε (diluted at 1:100) prepared above for 2 h at 37℃. After washed three times
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with PBS, the plates were incubated with FITC-conjugated goat anti-rabbit IgG (ZSGB, Beijing, China, diluted at 1:200) for 1 h at 37℃. After washed three times by PBST, the cells treated with
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PAb were analyzed with fluorescence microscope.
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Antiviral activity analysis of BoIFN-ε in vitro
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First, VSV titers were determined by an endpoint dilution assay and the titers were expressed as the tissue culture infectious dose 50 (TCID50) per milliliter using the Reed-Muench method
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(Cheng et al., 2006; Sun et al., 2008). Virus titers were calculated by determining the dilution giving 50% of wells containing cells that displayed cytopathic effect. Antiviral activity was determined in a standard cytopathic effect assay. Briefly, monolayers of MDBK cells seeded in 96-well plates were treated with 100μL of 4-fold serial dilutions of His-BoIFNε for 24h and challenged with VSV (100TCID50/well), and the capacity of His-BoIFNε to inhibit the cytopathic effect was determined (Rubinstein et al., 1981). The wells without viruses were taken as cell controls and the wells without His-BoIFNε were used as virus controls. The plate was then re-incubated under the conditions of 37℃ in a humidified 5% CO2 atmosphere for 18~24h. One antiviral activity unit was defined as 50% reduction in the destruction of the cell 6
ACCEPTED MANUSCRIPT monolayer. In addition, the antiviral activity of BoIFN-ε were also conducted on EBK cells, BT cells, PK15 cells, MDCK cells and BHK21 cells.
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The specificity of BoIFN-ε antiviral protective activity was analyzed by antiviral activity
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blocking assay. MDBK cells were treated with His-BoIFNε neutralized for 24 h by PAb against BoIFN-ε (diluted 1:20) for 4 h at 37℃, and the controls were untreated for 24 h. Then, all cells were challenged with 100 pfu VSV.
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Results
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Characterization of BoIFN-ε gene
The BoIFN-ε gene fragments were obtained containing the 5'UTR, 3'UTR and an ORF
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(Fig.1). What’s more, BoIFN-ε gene can be expressed at a high level in EBK cells after infection
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with VSV, and the size of the expected amplimer was 368 bp (Fig. 4A). The putative protein with
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193 amino acids were obtained, encoding a signal peptide of 21 amino acids and a mature protein of 172 amino acids , and molecular mass of 22.694 kDa and theoretical isoelectric point of 8.34
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were estimated. Alignment of the sequences shows striking overall conservation of IFN-εs, with 71%–87% identity to human, swine and murine at the amino acid level (Fig.2A). What’s more, the ORF of the BoIFN-ε sequence has been submitted to the GenBank databases under the accession number KJ778878. The amino acid residues Ser-38, Glu-107 and Ile-167 are conserved residues among IFN-εs (Conklin et al., 2001). There are three cysteine residues at positions 53, 163 and 175 of the mature peptide conserved in IFN-εs, and two of them (Cys-53 and Cys-175) can be formed of the disulphide bonds that benefit for maintaining the natural structure of BoIFN-ε. The investigation of secondary structure indicates that BoIFN-ε has five putative alpha helices which were labeled as A to E according to the division of human and murine (Table.2). The predicted 7
ACCEPTED MANUSCRIPT three-dimensional structure reveal that model of BoIFN-ε is consistent with interferon molecular that analyzed by crystal diffraction (Fig. 2B), such as the recombinant murineIFN-β, ovineIFN-τ,
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humanIFN-β, humanIFN-α2b, who has five putative alpha helices.
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Putative transcription factor binding sites were conserved among the human, the mouse and bovine sequence. These conserved motifs include the two IRF binding sites (also designed VRE or ISRE) and progesterone receptor response element (PRE) (Fig.1) (Hardy et al., 2004) There
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are two regions of continuous “GT” tandem repeated (9 and 6, respectively) at the 3’UTR (Fig.1),
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but the functional significance of this motif is unknown.
A phylogenetic tree constructed from the amino acid sequences of BoIFN-ε was compared to
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other animals IFN sequence. The tree shows that BoIFN-ε is more similar to IFN-ε of other
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animals, but not with the other IFNs of bovine (Fig.2C), suggesting that IFN-ε gene arose from an
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ancestral duplication of a Type I IFN gene that was distinct from other bovine IFNs. The tree constructed indicated that IFN enumerated in Fig.2B belong to three distinct branches: type I, type
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II and type III, and BoIFN-ε belong to type IFN. Analysis of putative glycation sites in BoIFN-ε predicted seven potential glycation sites. It is possible that these glycation sites may act as spacers and protect the interferon molecule from protease-mediated hydrolysis, thereby playing an important role in maintaining effector functions by contributing to the processes of folding, oligomerization, and stability (Hosoi et al., 1988). The identification of such sites raised the possibility that BoIFN-ε cloned here may form a glycoprotein. Protein expression and identification of BoIFN-ε SDS-PAGE analysis revealed that the recombinant protein His-BoIFNε was mainly 8
ACCEPTED MANUSCRIPT expressed as the inclusion body form with an apparent molecular weight of 35 kDa (Fig.4B) and rBoIFNε was at the molecular weight of 20 kDa (Fig.4C). The protein concentration of purified
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His-BoIFNε was 0.192mg/ml and rBoIFNε was 0.278mg/ml.
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The results of immunofluorescence assay showed that cells transfected with recombinant expression vector pcDNA3.1-BoNE has green fluorescence, while there was no fluorescence with
expression of BoIFN-ε.
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Antiviral activities of BoIFN-ε in vitro
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cells in control group (Fig.4D), which showed the PAb against BoIFN-ε can identify the
In order to determine whether BoIFN-ε has the biological activities, cytopathic inhibition
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assays in vitro was used to analyze its antiviral activity. The results show that BoIFN-ε has a
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protective effect on MDBK, EBK, BT and PK-15 cells, but not on MDCK and BHK21 cells.
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What’s more, the antiviral activity against VSV of BoIFN-ε was lower than rBoIFN-αA on MDBK, EBK, BT and PK-15 cells (Table. 3).
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The results of antiviral activity blocking assay suggest that the antiviral activity of His-BoIFNε was completely abrogated by PAb against BoIFN-ε at a dilution of 1:16, and when the antibody dilution was 1:128, the blocking activity was nearly non-existent, while no antiviral activity was observed by addition of preimmune rabbit serum.
Discussion Type I IFNs are central to innate and adaptive immunity and comprise many functional subgroups, such as IFN-α, β, ɛ, δ, ω and κ, and these IFNs have different antiviral activities. IFN-α may play the role of the “soldier” IFN, as some subtypes of IFN-α possess the highest specific antiviral activity of all the type I IFNs as well as the greatest cytotoxic activity (Pestka et al., 1987; 9
ACCEPTED MANUSCRIPT Pestka, 1997, 2000). IFN-κ is an interferon with relatively weak specific antiviral activity expressed predominantly by keratinocytes (Nardelli et al., 2000). Some IFN-ω and IFN-δ subtypes,
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IFN-ε, and IFN-β differed in their antiviral activity based on target cells and viruses (Krause and
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Pestka, 2005).
IFN-ε is expressed in a tissue-specific fashion, by eptithelial cells of the female reproductive tract, which serves a specific role in reproductive tissues either in viral protection or early
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placental development (Fung et al., 2013). Here, we present the cloning and characterization of a
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novel gene encoding Bovine IFN-ε. In general, type I IFNs can be cloned from genome because of the typical feature of lacking intron in mammals and birds (Lowenthal et al., 2001), so we could
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clone the BoIFN-ε gene from genomic DNA, then it was verified by RT-PCR that BoIFN-ε gene
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can be transcribed in EBK cells after stimulation by VSV. Tissue distribution analysis suggested
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BoIFN-ε could be express in liver, thymus, kidney, small intestine and testis but not in heart. As we took tissue samples from bull, the expression in female reproductive tract were not analyzed
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here. There are some differences in the tissue expression of IFN-ε gene measured in different species, but the IFN-ε gene tend to be expressed on the mucosal tissue. In human, the expression of IFN-ε was not detectable at significant levels in any organ with the notable exception of the uterus, cervix, vagina and ovary (Fung et al., 2013). Mouse IFN-ε gene mRNA was expressed in ovaries, uterus, fibroblasts, lung, brain, adrenal glands, and heart but was not detected in thymus, spleen, testis, liver, and kidney (Hardy et al., 2004). While in rhesus macaques, IFN-ε can be expressed highly in cervix, ovary and testis, the heart, thymus, intestine, kidney, brain and spinal cord also can be detected the expression of IFN-ε (Demers et al., 2014). The swine IFN-ε gene can be detected in all the tested tissues such as skin, intestine, mesenteric lymph nodes, testis, spleen 10
ACCEPTED MANUSCRIPT and PMC, and highly expressed in intestine, mesenteric lymph nodes and skin (Sang et al., 2010). The tissue distribution of IFN-ε gene in different species is different from each other.
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The characterization of the BoIFN-ε was analyzed by bioinformatics software. First, in
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alignments of the inferred amino acid sequences, it showed the highest similarity with swine IFN-ε, at 87.1% identity. Second, BoIFN-ε was found to contain cysteines at the same positions as those in IFN-εs, which may play an important role in maintaining the secondary structure of IFN.
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The relative position of BoIFN-ε gene within the IFN-εs are also conserved between humans and
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mice. Third, seven potential glycosylation sites were predicted in BoIFN-ε, which may play an important role in maintaining effector functions, raising the possibility for BoIFN-ε to form a
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glycoprotein. Fourth, homology modeling showed the BoIFN-ε has the typical characterization of
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IFN, which has five alpha helices. Fifth, phylogenetic analysis showed that BoIFN-ε has a closer
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genetic relationship to IFN-ε than to any other IFN analyzed, suggesting that the IFN-ε gene of swine, human, murine, cat, monkey and wolf may have arisen during evolution by duplication of a
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common ancestor. What’s more, the PAb prepared with rBoNE can be used to detect bovine IFN-ε in MDBK cells.
According to the preceding analysis, it is novel bovine IFN-ε. To see whether BoIFN-ε possesses biological function, we expressed the BoIFN-ε in Escherichia coli andmeasured the antiviral activity using CPE inhibition assays in vitro. These results demonstrated that BoIFN-ε protected MDBK, BT, EBK and PK-15 cells from challenge with VSV, indicating that bovine IFN-ε has certain antiviral activity on homologous cells, as well as the cells derived from the species that have near relative, which is different from canine IFN-ε (Yang et al., 2013). In addition, bovine IFN-ε has lower antiviral activity than BoIFNαA as described above, which is 11
ACCEPTED MANUSCRIPT similar to the human and canine IFN-ε (Peng et al., 2007; Yang et al., 2013). At the same time, the
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PAb against BoIFN-ε in the dose dependent manner (Li et al., 2006).
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antiviral activity of BoIFN-ε can be inhibited under certain experimental conditions such as by
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IFN-ε expression patterns in the reproductive tract is related to expression of estrogen (Fung et al., 2013), and there are progesterone receptor response element (PRE) existing in the gene promoter region (Hardy et al., 2004), suggesting that IFN-ε is regulated by hormones. IFN-ε was
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constitutively expressed by epithelial cells of the female reproductive tract and hormonally
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regulated in human and murine (Fung et al., 2013). In our study, IRF1 and IRF2 were predicted in the upstream of BoIFN-ε coding region, and BoIFN-ε expression patterns may regulated by virus
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stimulation in some non-reproductive tract tissues. Canine IFN-ε can be obtained from MDCK
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cells after stimulated by polyI:C, and polyI:C can effectively simulate the double-stranded RNA
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viruses (Yang et al., 2013). Mouse IFN-ε1 mRNA was constitutively expressed and when the levels of the mRNA transcripts encoding IFN-ε1 were quantified relative to GAPDH the levels
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increased only three fold 12 h after infection by SFV (Hardy et al., 2004). These suggest IRF binding sites may exert the role in induction of IFN. EBK cells can induced more BoIFN-ε specifically after stimulated by VSV, which also confirmed that BoIFN-ε can be transcribed in vivo.. In this study, a novel gene encoding BoIFN-ε was first reported, then the tissue distribution and antiviral activity were analyzed, which can enrich knowledge
of bovine type I IFNs.
Consequently, there is much discussion on the significance of the existence of bovine IFN-ε. It is speculated that bovine IFN-ε may have particular importance during certain viral infections and may be optimally active in specific cell types. Further studies are required to address these 12
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Acknowledgements
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We thank Dr. Li Yu (Harbin Veterinary Research Institute, Chinese Academy of Agricultural
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Sciences, Harbin, China) for kindly providing MDBK, PK-15 and EBK cells. This study is supported by the Earmarked Fund for China Agriculture Research System (No. CARS-37), Synergetic Innovation Center of Food Safety and Nutrition, the sub-project of National 12th
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5-year Support Key Projects (2012BAD12B03, 2012BAD12B05) and the Key Technologies
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Research and Development Program of Heilongjiang province (GA09B302). We would like to express our sincere appreciation to the reviewers for their insightful recommendations, which have
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greatly aided us in improving the quality of the paper.
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References:
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Cheng, G., Chen, W., Li, Z., Yan, W., Zhao, X., Xie, J., Liu, M., Zhang, H., Zhong, Y., Zheng, Z.,
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2006. Characterization of the porcine alpha interferon multigene family. Gene 382, 28-38. Coligan, J., Bierer, B., Margulies, D., 2005. Short Protocols in Immunology. John Wiley & Sons, Inc., New Jersey.
NU
Combet, C., Blanchet, C., Geourjon, C., Deleage, G., 2000. NPS@: network protein sequence analysis.
MA
Trends Biochem Sci 25, 147-150.
Conklin, D.C., Grant, F.J., Rixon, M.W., Kindsvogel, W., 2001. INTERFERON-EPSILON., C07H
D
21/02 ed, p. 64.
TE
Demers, A., Kang, G., Ma, F., Lu, W., Yuan, Z., Li, Y., Lewis, M., Kraiselburd, E.N., Montaner, L. Li,
CE P
Q. 2014. The mucosal expression pattern of interferon-epsilon in rhesus macaques. J Leukoc Biol Fung, K.Y., Mangan, N.E., Cumming, H., Horvat, J.C., Mayall, J.R., Stifter, S.A., De Weerd, N.,
AC
Roisman, L.C., Rossjohn, J., Robertson, S.A., Schjenken, J.E., Parker, B., Gargett, C.E., Nguyen, H.P., Carr, D.J., Hansbro, P.M., Hertzog, P.J., 2013. Interferon-epsilon protects the female reproductive tract from viral and bacterial infection. Science 339, 1088-1092. Hardy, M.P., Owczarek, C.M., Jermiin, L.S., Ejdeback, M., Hertzog, P.J., 2004. Characterization of the type I interferon locus and identification of novel genes. Genomics 84, 331-345. Hermant, P., Francius, C., Clotman, F., Michiels, T., 2013. IFN-epsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines. PLoS One 8, e71320. Hosoi, K., Utsumi, J., Kitagawa, T., Shimizu, H., Kobayashi, S., 1988. Structural characterization of 14
ACCEPTED MANUSCRIPT fibroblast human interferon-beta 1. J Interferon Res 8, 375-384. Kontsek, P., Karayianni-Vasconcelos, G., Kontsekova, E., 2003. The human interferon system:
IP
T
characterization and classification after discovery of novel members. Acta Virol 47, 201-215.
friends and relatives. Pharmacol Ther 106, 299-346.
SC R
Krause, C.D., Pestka, S., 2005. Evolution of the Class 2 cytokines and receptors, and discovery of new
Li, H.T., Ma, B., Mi, J.W., Jin, H.Y., Xu, L.N., Wang, J.W., 2006. Cloning, in vitro expression and
NU
bioactivity of goose interferon-alpha. Cytokine 34, 177-183.
MA
Lowenthal, J.W., Staeheli, P., Schultz, U., Sekellick, M.J., Marcus, P.I., 2001. Nomenclature of avian interferon proteins. J Interferon Cytokine Res 21, 547-549.
D
Matsumiya, T., Prescott, S.M., Stafforini, D.M., 2007. IFN-epsilon mediates TNF-alpha-induced
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J Immunol 179, 4542-4549.
TE
STAT1 phosphorylation and induction of retinoic acid-inducible gene-I in human cervical cancer cells.
Nardelli, B., LaFleur, D. W., Chinchilla, D., Tsareva, T., Guoxian, C., Mather, D., et al., 2000.
AC
IFN-κ, a novel type I interferon. Eur Cytokine. Peng, F.W., Duan, Z.J., Zheng, L.S., Xie, Z.P., Gao, H.C., Zhang, H., Li, W.P., Hou, Y.D., 2007. Purification of recombinant human interferon-epsilon and oligonucleotide microarray analysis of interferon-epsilon-regulated genes. Protein Expr Purif 53, 356-362. Peng, F.W., Gao, H.C., Xie, Z.P., Zhang, H., Li, Q.M., Duan, Z.J., Hou, Y.D., 2005. [Biological activities of recombinant human interferon Epsilon]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 19, 227-231.
Pestka, S., Langer, J. A., Zoon, K. C., & Samuel, C. E., 1987. Interferons and their actions. Annu Rev Biochem 56, 727-777 15
ACCEPTED MANUSCRIPT Pestka, S., 1997. The human interferon-a species and hybrid proteins. Semin Oncol 24(3 Suppl. 9), S9.
IP
T
Pestka, S., 2000. The human interferon a species and receptors. Biopolymers 55(4), 254-287
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Rubinstein, S., Familletti, P.C., Pestka, S., 1981. Convenient assay for interferon. J. Virol. 37, 755-758. Sambrook, J., Russell, D.W., 2008. Molecular Cloning: A Laboratory Manual. Science Press, Beijing. Sang, Y., Rowland, R.R., Hesse, R.A., Blecha, F., 2010. Differential expression and activity of the
NU
porcine type I interferon family. Physiol Genomics 42, 248-258.
MA
Sun, Y., Li, W.,H. Li, M., Gao, M., Wei, W., Wei, S., Wang, J., 2008. Expression and purification of IFN-α gene from bovine. Chinese Journal of Preventive Veterinary Medicine 30, 889-892.
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Wang, J., Guo, Y., Gao, M., 2012. Preparationof polyclonal antibody specific for light chain of goose
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immunoglobulin using recombinant protein. Journal of Northeast Agricultural University 43, 60-63.
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Xi, Y., Day, S.L., Jackson, R.J., Ranasinghe, C., 2012. Role of novel type I interferon epsilon in viral infection and mucosal immunity. Mucosal Immunol 5, 610-622.
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Yang, L., Xu, L., Li, Y., Li, J., Bi, Y., Liu, W., 2013. Molecular and functional characterization of canine interferon-epsilon. J Interferon Cytokine Res 33, 760-768.
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Figure legends:
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Fig.1 Nucleotide sequence and inferred amino acid sequences of BoIFN-ε gene. The nucleotide
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and amino acid sequences are numbered on the left and right sides, respectively. The signal peptide is underlined, and the conserved cysteines are indicated by shaded circles, while the conserved amino acids Ser-38, Glu-107 and Ile-167 are indicated by gray-shaded boxes. In the
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5’UTR, the virus-responsive elements are boxed in gray and the continuous repeat regions in
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3’UTR are italic and bold.
Fig.2 A. IFN-ε amino acid alignment of Bovine, Human, Murine and Canine. B. Predicted
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three-dimensional structures of bovine IFN-ε. The positions of helices A to E are labeled. C.
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Phylogenetic tree of BoIFN-ε amino acid sequences of several mammals IFN. The bar scale
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represents the genetic distance. The credibility value for each node is shown. B. Fig.3 Tissue distribution expression of IFN-ε. Bovine organs were harvested and IFN-ε expression
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(A), IFN-αA expression (B) and GAPDH (C) expression in heart, liver, thymus, kidney, small intestine and testis were measured by RT-PCR. Fig.4 A. RT-PCR analysis BoIFN-ε mRNA transcripts in EBK cells infected with VSV. Lane M: Trans2K DNA marker; Lane 1: PCR--amplified with EBK cells infected with VSV; Lane 2: Negative control; Lane 3: PCR product at 9h. B. SDS-PAGE analysis of the expressed and purified His-BoIFNε, Lane M: Unstained Protein Marker; Lane 1: Negative control of empty vector pET-32a (+); Lane 2: His-BoIFNε after induction; Lane 3: Sedimentations of His-BoIFNε; Lane 4: Supernatants of His-BoIFNε; Lane 5: Purified His-BoIFNε. C. SDS-PAGE analysis of the purified rBoIFNε, Lane M: Unstained Protein Marker; Lane 1: rBoIFNε after induction; Lane 2: 17
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MDBK cells.
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Fig. 1
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Fig. 2
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ACCEPTED MANUSCRIPT Table 1. Nucleotide Sequence of the PCR primers used in this study Sequence(5’-3’)
BoIFNES BoIFNEA BoNE-RNAS2 BoNE-RNAA1 BONEBoNE-RNAA2 BoIFNA-S BoIFNA-A BoGAPDH-S BoGAPDH-A BoNEORFEI BoNEORFNI BoNEORFXI pcDNA3.1-BoNES
GGTGCTGCTAAGCCTCTGA CAGTCGGCTGTTCCTTCAC AGACCTCGTCAGTTCAGCAG TCAAGTTTCCATGCCCTGT GACAATAGTCCAGGCACAGC TGGTCCTTCCTGCTATCCCT CTGACAACCTCCCAGGCACA TTGGCATCGTGGAGGGACT GAGTGAGTGTCGCTGTTGAAGT TGTTGAATTC EcoR I CAAGAGCTGAAACTGGTT CTGTTCATATGNde ICAAGAGCTGAAACTGGTT TCACTCGAG Xho I TCAAGTTTCCATGCCCTGT ACTGAATTCEcoR IATGCAAGAGCTGAAACTGGTT
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Primer
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Note: The restriction enzyme sites that were introduced in primers are underlined.
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ACCEPTED MANUSCRIPT Table 2. Comparison of the IFN-ε structural feature among human, murine and bovine Amino Acid Residues
Amino Acid Residues
Amino Acid Residues
Interferon-epsilon
of Human
of Murine
of Bovine
Signal sequence
1-21
1-21
Helix A
27-48
27-47
AB Loop
49-75
Helix B
76-94
BC Loop
95-102
Helix C
103-123
CD Loop
124-138
Helix D
139-158
DE Loop
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Structural Feature of
1-21 27-48 49-75
75-93
76-94
94-101
95-102
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48-74
103-123
123-137
124-138
138-157
139-158
159-162
158-161
159-162
Helix E
163-184
162-183
163-184
C-terminus
185-208
184-192
185-193
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102-122
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ACCEPTED MANUSCRIPT Table 3. Comparison of antiviral activity against VSV between BoIFN-ε and BoIFN-αA
rBoIFN-αA
MDBK cells
6.7×103
5.95×104
EBK cells
1.13×104
BT cells
2.27×104
8.25×106
PK-15 cells
3.53×102
3.90×105
MDCK cells
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rBoIFN-ε
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Antiviral activity against VSV（U/mg）
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BHK21 cells
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2.90×106
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0
0
ACCEPTED MANUSCRIPT Abbreviations IFN-ε, interferon-episilon; BoIFN-ε, bovine interferon-episilon; ORF, open reading frame; VSV,
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vesicular stomatitis virus; UTR, untranslated regions; FITC goat-anti-rabbit IgG, fluorescein
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isothiocyanate-conjugated goat anti-rabbit IgG; cDNA, DNA complementary to RNA; MDBK cells, Madin-Darby bovine kidney cells; EBK cells, Embryonic Bovine Kidney cells; PAb, polyclonal antibody; IPTG, isopropyl β-D-thiogalactoside; MDCK cells, Madin-Darby canine
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kidney cells; BHK21 cells, Baby Hamster Syrian Kidney cells; VRE, virus-responsive elements;
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Cys, cysteine residues; PRE, progesterone receptor response element.
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ACCEPTED MANUSCRIPT Highlights
Bovine IFN-ε was first cloned here and the characterization of bovine IFN-ε was analyzed.
The mature peptide was highly expressed in prokaryotic expression system, and it could be
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overexpressed in MDBK cells.
Bovine IFN-ε has moderate antiviral activity on MDBK, BT, EBK and PK-15cells, but not on
Bovine IFN-ε gene can be constitutively expressed in liver, thymus, kidney, small intestine
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and testis, but not in the heart.
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BHK-21 and MDCK cells.
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