Identification of Epitope in EAV N Protein

Identification of Epitope in EAV N ProteinIdentification of a young hold B carrell epitope in the N protein of EAV (Bucyrus strain)Running claim Identification of epitope in EAV N protein.HighlightsOne EAV N-specific mAb 1C11 was create.A nominal elongated peptide epitope indoors the N protein was see.The identify epitope was conserved among divergent regional EAV strains.The mAb and identified epitope whitethorn be utilizable symptomatic tools for EAV infection.AbstractObjective To invest the minimal epitope of N protein of the equine arteritis computer virus (EAV).Methods The full-length sequence of EAV N ingredient was cl geniusd by RT-PCR and ligated into pET32a vector for expression. The recombinant pET-N protein was verbalised in E. coli and purified by Ni affinity chromatography. The purified N protein was utilise to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by westerly pip and immunofluorescence assay (IFA). The peptides were identified using the prepargond mAb by verifying ELISA and Western blot. The homology outline was performed using DNAMAN bundle.Results Recombinant EAV N protein was successfully verbalized in the procaryon expression system. An EAV N-reactive mAb was selected and designated as 1C11. Indirect ELISA results showed that co-occur 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 elongate epitope of the EAV N protein. The homology analysis showed that the identified epitope is conserved among all(a) 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 bilinear 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 intentionful for the development of specific diagnostic tool s and design of vaccine.Keywords Epitope N protein Equine arteritis virus Monoclonal antibodyIntroductionEquine 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 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 blood cell with a length of 12.7kb 11. It contains two large open reading frames (ORFs, 1a and 1b) and septet 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 know EAV structural proteins E, GS, GL, M, and N, respectively 12. Moreover, ORFs 3 and 4 encode glycosylated membrane-associated proteins whose functional th eatrical role is still under debate 13, 14.EAV N can be employ 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 domain, we aimed to identify the precise B cell epitope using a monoclonal antibody (mAb) against EAV N protein. Our result allow provide important information for developing serological diagnosis of EAV infection and understanding the antigenic structure of EAV N protein and vaccine design.Materials and methodsEthics statementCare and use of research lab physicals and all animal experiments were in accordance with animal ethics guidelines established by the institutional Animal Ethics Committee in China. All animal studies were authorise by the Animal Ethics Committee of Harbin Veterinary Research set of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032).Cell lines and virusSP2/0 myeloma and rock rabbit kidn ey 13 (RK-13) cells were cultured and maintained in Dulbeccos modified eagles medium (DMEM Invitrogen, Carlsbad, CA, USA) in a humidified 5% CO2 zephyr at 37C. All culture media were supplemented with 10% heat-inactivated foetal bovid serum (GIBCO, Invitrogen) and antibiotics (0.1mg/ml streptomycin and 100 IU/ml penicillin).The Bucyrus strain of EAV (GenBank accession zero(prenominal) 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 -80.Expression and characterization of recombinant EAV N proteinThe full-length sequence of EAV N gene was cloned by RT-PCR using the following primers 5-CCCGGATCCATGGCGTCAAGACGATC-3 (upstream) and 5-TTTGTCGACTTACGGCCCTGCTGGAGGCGCAAC-3 (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--D-1-thiogalactopyranoside (IPTG, Invitrogen, USA) was utilise for inducing expression of N protein. The recombinant proteins were obtained from the bacterial lysates. The insoluble inclusion bodies were swear out and solubilized with phosphate buffered saline (PBS, pH 7.4). because, the recombinant N protein f employ with 6 His-tags was evaluated by SDS-PAGE and purified by Ni affinity chromatography according to manufacturers command (Invitrogen).Preparation and characterization of mAbs against N proteinEAV N-reactive mAb was generated as previously described 17. Briefly, 6-week-old womanly BALB/c mice were immunized with the purified recombinant N protein (100g per mouse) mixed with an equal lot of Freunds complete assistant (FCA, Sigma, USA). Two booster injections containing the same summation of purified N protein in an equal volume of Freunds incomplete adjuvant (FICA) were given at 2-week intervals. The purified N protein without adjuvant was injected intraperitoneally as the final immunisation. later on 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-amino grouppterin-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 removed 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 covered 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 expressionEleven overlapping peptides spanning the N protein were designed (Table 1,). For each peptide, a pair of oligonucleotide strands was synthesized. apiece pair of oligonucleotide strands was annealed and cloned into the BamH and Sal I sites of pMAL-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 4 overnight and blocked with 5% skim milk for 1 h at 37. After washing three time by PBST (PBS plus 0.5% Tween -20), 100 l of mAb was added to wells and incubated at 37 for 1 h. Then, the plates were washed three times by PBST and incubated with diluted horseradish peroxidase (HRP)-conjugated buttocks anti-mouse IgG (Abcam, UK) at 37 for 1 h. The polish 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 epitopesThe 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 rachis sites on the membrane were blocked with 5% skim milk in PBS overnight at 4. The membrane was washed and incubated with mAbs for 1h at 37. The membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody af ter flipper 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 analysisTo evaluate the conservation of the identified linear epitope among EAV from unalike geographic areas, the identified epitope and the corresponding regions of other regional EAV virus strains were aligned using DNAMAN software (Lynnon BioSoft Inc., USA).ResultsProduction of recombinant EAV N protein and mAbAs shown in Fig.1a, Recombinant EAV N protein was successfully expressed in the procaryon expression system. A clear single target reverberate 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 stron g 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 epitopeTo localize linear antigenic epitopes within the N protein, 11 16-amino superman 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 overlapp ing 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 analysisSequence coalescency 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.DiscussionMapping location of viral protein epitopes and defining the phase 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.m Abs 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 say that the precise epitope of N protein located in amino acids 1-69. This may be due to the distinction 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 unprogr essive 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.