Identification of Epitope in EAV N Protein

Identification of Epitope in EAV N Protein Identification of a novel conserved B cell epitope in the N protein of EAV (Bucyrus strain) Running title: Identification of epitope in EAV N protein. Highlights: One EAV N-specific mAb 1C11 was developed. A minimal linear peptide epitope within the N protein was identified. The identified epitope was conserved among different regional EAV strains. The mAb and identified epitope may be useful diagnostic tools for EAV infection. Abstract Objective: To identify the minimal epitope of N protein of the equine arteritis virus (EAV). Methods: The full-length sequence of EAV N gene was cloned by RT-PCR and ligated into pET32a vector for expression. The recombinant pET-N protein was expressed in E. coli and purified by Ni affinity chromatography. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay (IFA). The peptides were identified using the prepared mAb by indirect ELISA and Western blot. The homology analysis was performed using DNAMAN software. Results: Recombinant EAV N protein was successfully expressed in the procaryon expression system. An EAV N-reactive mAb was selected and designated as 1C11. Indirect ELISA results showed that overlapping domain of MBP-N10 and MBP-N11 was recognized by the mAb 1C11. Further, the indirect ELISA and Western blot showed that 101QRKVAP106 was the minimal linear epitope of the EAV N protein. The homology analysis showed that the identified epitope is conserved among all EAV isolated strains, with the exception of the ARVAC which is a modified live virus vaccine strain. Conclusion: One EAV N-specific mAb was developed and a minimal linear peptide epitope within the N protein was identified. The EAV N-specific mAb and the defined linear peptide epitope of EAV N protein may be useful for the development of specific diagnostic tools and design of vaccine. Keywords: Epitope; N protein; Equine arteritis virus; Monoclonal antibody Introduction Equine arteritis virus (EAV) is the etiologic agent of equine viral arteritis (EVA) which is a respiratory and reproductive disease of horses [1-3]. EAV was à ¯Ã‚ ¬Ã‚ rst isolated from horses in Ohio in 1953. It is the prototype virus of the family Arteriviridae (genus Arterivirus, order Nidovirales) [4, 5]. EAV infection of horses has been reported in many countries including New Zealand, Australia, and South Africa [6-10]. EAV is a positive-sense, enveloped and single-stranded RNA molecule with a length of 12.7kb [11]. It contains two large open reading frames (ORFs, 1a and 1b) and seven smaller ORFs (2a, 2b, and 3 to 7). ORFs 1a and 1b encode two replicase polyproteins (pp1a and pp1ab), whereas the ORFs 2a, 2b, 5, 6, and 7 encode the known EAV structural proteins E, GS, GL, M, and N, respectively [12]. Moreover, ORFs 3 and 4 encode glycosylated membrane-associated proteins whose functional role is still under debate [13, 14]. EAV N can be used as an alternative protein candidate of diagnostic antigens and accounts for 35-40% of the total virion protein [15]. B cell epitopes involved in the immune response against EAV [16]. In the present study, we aimed to identify the precise B cell epitope using a monoclonal antibody (mAb) against EAV N protein. Our result will provide important information for developing serological diagnosis of EAV infection and understanding the antigenic structure of EAV N protein and vaccine design. Materials and methods Ethics statement Care and use of laboratory animals and all animal experiments were in accordance with animal ethics guidelines established by the Institutional Animal Ethics Committee in China. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032). Cell lines and virus SP2/0 myeloma and Rabbit kidney 13 (RK-13) cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) in a humidified 5% CO2 atmosphere at 37 °C. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Invitrogen) and antibiotics (0.1mg/ml streptomycin and 100 IU/ml penicillin).The Bucyrus strain of EAV (GenBank accession No. NC-002532.2, a highly cell culture-adapted strain provided by the key laboratory of Tropical and Subtropical Animal Viral Diseases in Yunnan province, China) was propagated in RK-13 cells and stored at -80à ¢Ã¢â‚¬Å¾Ã†â€™. Expression and characterization of recombinant EAV N protein The full-length sequence of EAV N gene was cloned by RT-PCR using the following primers: 5†²-CCCGGATCCATGGCGTCAAGACGATC-3†² (upstream) and 5†²-TTTGTCGACTTACGGCCCTGCTGGAGGCGCAAC-3†² (downstream). The primers contained BamH I and Sal I restriction sites (italicized). The purified and digested PCR product was ligated into an expression vector pET32a (Novagen, Germany). The pET-N recombinant plasmid was transformed into E. coli BL21 (DE3) and 1mM isopropyl-ÃŽ ²-D-1-thiogalactopyranoside (IPTG, Invitrogen, USA) was used for inducing expression of N protein. The recombinant proteins were obtained from the bacterial lysates. The insoluble inclusion bodies were washed and solubilized with phosphate buffered saline (PBS, pH 7.4). Then, the recombinant N protein fused with 6 His-tags was evaluated by SDS-PAGE and purified by Ni affinity chromatography according to manufacturer’s instruction (Invitrogen). Preparation and characterization of mAbs against N protein EAV N-reactive mAb was generated as previously described [17]. Briefly, 6-week-old female BALB/c mice were immunized with the purified recombinant N protein (100ÃŽ ¼g per mouse) mixed with an equal volume of Freund’s complete adjuvant (FCA, Sigma, USA). Two booster injections containing the same amount of purified N protein in an equal volume of Freund’s incomplete adjuvant (FICA) were given at 2-week intervals. The purified N protein without adjuvant was injected intraperitoneally as the final immunization. After three days of the final injection, the mice were euthanized and their splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol (PEG4000, Sigma). The hybridoma cells were seeded into 96-well plates and selected in hypoxanthine-aminopterin-thymidine (HAT) selection medium (DMEM containing 20% fetal bovine serum, 100g/ml streptomycin, 100IU/ml penicillin, 100mM hypoxanthine, 16mM thymidine, and 400 mM aminopterin). After 5 days, the medium was re moved and replaced with hypoxanthine-thymidine (HT)-DMEM medium (DMEM containing 20% fetal bovine serum, 100g/ml streptomycin, 100IU/ml penicillin, 100mM hypoxanthine, and 16mM thymidine). After selection in HAT and HT medium, hybridoma supernatants were screened for evaluating reactivity and specificity of mAb by Western blot and immunofluorescence assay (IFA). The class and subclass of the mAb was determined using a SBA ClonotypingTM System/HRP (Southern Biotechnology Associates, Inc., Birmingham, AL35260, USA). Polypeptide design and expression Eleven overlapping peptides spanning the N protein were designed (Table 1,). For each peptide, a pair of oligonucleotide strands was synthesized. Each pair of oligonucleotide strands was annealed and cloned into the BamHà ¢Ã¢â‚¬ ¦Ã‚   and Sal I sites of pMALâ„ ¢-C4x vector and expressed as MBP-N fusion proteins. These MBP-fused proteins were named consecutively MBP-N1 to MBP-N11. The recombinant plasmids were transformed into E.coli Rosetta (DE3) (Novagen). Each MBP-fused polypeptide was induced by IPTG and screened by indirect ELISA. Briefly, MBP tags and purified N protein were used as negative and positive controls, respectively. Ninety-six-well microtiter plates were coated with expressed MBP-N fusion proteins at 4à ¢Ã¢â‚¬Å¾Ã†â€™ overnight and blocked with 5% skim milk for 1 h at 37à ¢Ã¢â‚¬Å¾Ã†â€™. After washing three times by PBST (PBS plus 0.5% Tween-20), 100 ÃŽ ¼l of mAb was added to wells and incubated at 37à ¢Ã¢â‚¬Å¾Ã†â€™ for 1 h. Then, the plates were washed three ti mes by PBST and incubated with diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam, UK) at 37à ¢Ã¢â‚¬Å¾Ã†â€™ for 1 h. The color was developed and the reaction was stopped with 2M H2SO4. The absorbance at 450 nm was measured. All assays repeated three times and the average of the three values was calculated. Identification of the epitopes The MBP-N-fusion proteins were identified by indirect ELISA and Western blot using the mAb. Indirect ELISA was performed as mentioned above. For Western blot, the purified MBP-N recombinant proteins were electrophoresed on SDS-PAGE, and then transferred to a nitrocellulose membrane. Nonspecific antibody binding sites on the membrane were blocked with 5% skim milk in PBS overnight at 4à ¢Ã¢â‚¬Å¾Ã†â€™. The membrane was washed and incubated with mAbs for 1h at 37à ¢Ã¢â‚¬Å¾Ã†â€™. The membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody after five times washing with PBST. Following another five times washing, the color was developed using 3,3-diaminobenzidine (DAB) and terminated by rinsing the membrane with deionized water. Homology analysis To evaluate the conservation of the identified linear epitope among EAV from different geographic areas, the identified epitope and the corresponding regions of other regional EAV virus strains were aligned using DNAMAN software (Lynnon BioSoft Inc., USA). Results Production of recombinant EAV N protein and mAb As shown in Fig.1a, Recombinant EAV N protein was successfully expressed in the procaryon expression system. A clear single target band with expected molecular weight was displayed. Accordingly, the recombinant EAV N protein was suitable as an antigen for immunization and hybridoma screening. Purified proteins were utilized to immunize BALB/c mice. After cell fusion and selection, an EAV N-reactive mAb generated from one hybridoma cell line was selected for its strong reactivity against N protein. This mAb was designated as 1C11. As shown in Fig.1b, c, mAb 1C11 reacted with recombinant N protein and total protein of EAV (Fig.1b, c). The reactivity of mAb was also assessed using RK-13 cells by IFA (Fig.1d). The mAb only reacted with EAV infected cells and not reacted with uninfected control RK-13 cells. Identification of EAV N epitope To localize linear antigenic epitopes within the N protein, 11 16-amino acid long MBP fused peptides (MBP-N1 MBP-N11) were expressed and probed by mAb 1C11 by indirect ELISA. The results showed that MBP-N10 (91TVSWVPTKQIQRKVAP106) and MBP-N11 (95VPTKQIQRKVAPPAGP110) epitopes were recognized by the mAb 1C11 (OD450 > 1) (Fig. 2a). All the left fragments (MBP-N1-9) failed to react with the mAb. Because adjacent epitopes have 12 overlaps, we deduced that the linear epitope located in the overlapping domain of MBP-N10 and MBP-N11 (95VPTKQIQRKVAP106). To identify the minimal linear peptide epitope within this overlapping domain, a series of truncated polypeptides were expressed as MBP-fusion proteins. Ultimately, the indirect ELISA and Western blot showed that 101QRKVAP106 was the minimal linear epitope for the reactivity of the EAV N protein recognized by mAb 1C11 (Fig. 2c, d). Homology analysis Sequence alignment was performed to evaluate the conservation of the identified epitope among different regional EAV viruses (Fig. 3). The identified epitope is conserved among all EAV isolated strains, with the exception of the ARVAC which is a modified live virus vaccine strain. Discussion Mapping location of viral protein epitopes and defining the degree of their conservation may play an important role for understanding of the antigenic structure, virus-antibody interactions. It may be very useful for vaccine design and clinical applications. In this study, the B cell epitopes of EAV N protein were identified using a mAb. Epitope mapping using mAbs has become a powerful tool to study protein structure and provides new tools to diagnose diseases and design vaccines [18]. Here, we defined one peptide epitope of EAV N protein in by using an EAV N-specific mAb. To our knowledge, epitope on the N protein of EAV has been published, but no previous studies about 101QRKVAP106 have been reported. Starick et al. [19] produced a mAb against the N protein to detect EAV. Weiland et al. [20] used the same method to produce a mAb against the N protein of EAV and to distinguish different virus isolates from semen and tissue samples after passaging through RK-13, Vero and fetal equine kidney cells. However, the minimal epitope of these mAb was not defined precisely. Similar to the work of Starick et al. and Weiland et al. [19, 20], a mAb named 1C11 against EAV N protein was prepared by using recombinant N protein expressed in E. coli and used for identifying B-cell epitopes on EAV N protein. mAb 1C11 reacted well with EAV by WB and IFA, thus this antibody may be a useful detection tool in EAV diagnosis. mAbs are useful and effective for mapping antigenic epitopes of viral proteins. In this study, for epitope mapping, 11 overlapping peptides from EAV N protein were expressed with MBP tags and identified by ELISA to screen linear epitopes. The ELISA results showed that the epitope located in the sharing region of MBP-N10 (91TVSWVPTKQIQRKVAP106) and MBP-N11 (95VPTKQIQRKVAPPAGP110). Then this region (95VPTKQIQRKVAP106) was expressed, and 7 peptides with deletions were obtained to identify the precise epitope. According to the results of ELISA and Western blot, 101QRKVAP106 was considered as the minimal linear epitope of EAV protein. This result is different from the previous studies [15, 21] which stated that the precise epitope of N protein located in amino acids 1-69. This may be due to the difference of the specificity and reactivity of the mAbs. Sometimes, a mAb can react with different locations of a viral protein. Sequence alignment showed that the identified epitope is very conservative among distinct regional EAV strains, but with a mutation of one amino acids on the ARVAC N protein epitope. This result suggests a slight regional difference emerged in this epitope. Therefore, it is possible to distinguish anti-Bucyrus EAV antibody from anti- ARVAC EAV antibody by using the epitope as antigen. This will be helpful in distinguishing the distinct regional EAV infection. This finding indicates that the N epitope of EAV identified in our study have a potential use in serological monitoring and differential diagnosis. In conclusion, one EAV N-specific mAb was developed and a minimal linear peptide epitope within the N protein was defined. The EAV N-specific mAb and the defined linear peptide epitope of EAV N protein may be useful for the development of specific diagnostic tools and design of vaccine.