Identification of the B-cell epitopes on N protein of type 2 porcine reproductive and respiratory syndrome virus, using monoclonal antibodies

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Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (N) is the immunodominant region of PRRSV viral proteins. However, B cell epitopes present on N protein have not been well characterized.

In this study, The ORF7 gene was amplified by RT-PCR and inserted into the expression vector pET-28a, the constructed pET-28a-N was transformed into Escherichia coli BL21. After expression, purification and identification, the recombined N protein was used as the target to generate monoclonal antibody (mAb). Strains of hybridoma cells secreting anti-N mAbs were obtained by the hybridoma technique. Three of them (named 1G4, 1C6, 3D11) were specifically reacted with PRRSV by IPMA and IFA. To identify the B-cell epitopes within PRRSV N protein, six serial overlapping synthesized peptides (P1-P6) covering the whole region of N were used to define the epitopes recognized by 1G4, 1C6, 3D11. Importantly, after the identification of dot-ELISA and indirect ELISA, we found that 1-15aa was a new epitope which had never been reported before and that it was highly conserved among some HP-PRRSV isolates of type 2 PRRSV.

The results of this study might open new perspectives on the detection of PRRSV and have important implications for studing the structure of the N protein.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide for almost 30 years, causing annual economic losses of about US $664 million to the US swine industry [1]. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped, single-stranded RNA (+) virus belonging to the order Nidovirales, family Arteriviridae [2]. PRRSV is categorized into two genotypes based on the genetic diversity. Genotype 1 (European) and genotype 2 (North American) share only 65% nucleotide identity at the genomic level [3]. The North American and European PRRSVs, represented by VR2332 and Lelystad virus (LV), respectively [4].

The PRRSV genome contains at least nine open reading frames (ORFs)-ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5b and ORF7, as well as the 5′ and 3′ untranslated regions [5,6]. ORF1a and ORF1b encode viral replicase polyproteins, which are translated immediately upon viral entry and are then proteolytically processed by virally encoded proteinases into 14 mature nonstructural proteins (NSP1a, NSP1b, NSP2-NSP6, NSP7a, NSP7b, and NSP8-NSP12) [7,8]. The structural-protein coding region of PRRV encodes eight structural proteins: GP2, E, GP3, GP4, GP5, M, GP5a and N [9].

The nucleotide protein (N), 123–128 amino acid, is considered to be one of the most conserved PRRSV proteins [10]. The N protein is divided into an N-terminal RNA-binding domain and a C-terminal dimerization domain. The aminoterminal half of the N sequence (residues 1–57) is presumed to be mostly disordered and contains a large number of positive charges, consistent with a role in RNA binding [11]. The greatest sequence difference between PRRSV strains and with other arteriviruses is in this domain [12], probably due to relaxed structural constraints. The crystal structure of the C-terminal (residues 58–123) showed a dimer comprised of a four-stranded antiparallel-sheet floor that is capped and flanked by-helices [13,14].

In addition, N protein has rich antigenic determinants, which induce production of long-time antibodies after the PRRSV infecting cells [15]. It has good immunogenicity and reaction activity [16,17]. Initial work indicated that the antibodies are directed mainly to the N-protein and are non-neutralizing [18].

The localization of viral protein epitopes is very important for determining antigenic structure and virus-antibody interactions at the molecular level. In this study, we successfully expressed and purified PRRSV N protein, and produced mAbs against PRRSV N, which could be used to recognize PRRS, as well as identify the B cell epitopes on N protein of the North American genotype PRRSV strain. Importantly, we found that 1-15aa was the new epitope which had never been reported before and that it was highly conserved among some HP-PRRSV isolates of type 2 PRRSV. The results of this study might open new perspectives on the antibody–antigen reaction and have important implications for studying the structure of the N protein.

Section snippets

Cell lines, viruses, and reagents

MARC-145 cells and SP2/0 myeloma cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL, Paisley, UK) with 15% fetal bovine serum (FBS) (Gibco-BRL, USA). HP-PRRSV Strain JX-A1 (GenBank accession no. EF112445) was kept in our laboratory. All chemical reagents were from commercial sources and of analytical grade; Ni Sepharose™ 6 Fast Flow(GE)；Taq DNA polymerase, DNA restriction enzymes, T4 DNA ligase were purchased from TaKaRa; IPTG, imidazole, Triton X-100 were from Sigma (USA);

Expression and purification of recombinant His-tagged N

The ORF7 gene was amplified by RT-PCR (Fig. 1, lane 7) and inserted into the expression vector pET-28a, resulting in the construct pET-28a-N. The plasmid pET-28a-N was transformed into Escherichia coli BL21 (DE3) for expression of the histidine (His)-tagged N protein. The recombinant His-fused N protein was successfully expressed in E. coli BL21 cells (Fig. 1, lane2). His-N was found almost completely in soluble form of the IPTG-induced E. coli after ultrasonication (Fig. 1, lane 3 and 4).

Discussion

In this study, the pET-28a was used for cloning and high-level expression of His-tagged N protein, with molecular weight of about 19 KDa, which was found almost completely in Soluble form of the IPTG-induced E. coli after ultrasonication (Fig. 1, lane 3 and 4). His-N was subsequently purified by Ni-NTA affinity chromatography columns according to the manufacturer's instruction. (Fig. 1, lane 6). Interestingly, the protein dissolved in 400 mM imidazole solution had a better purification effect

Conflict of interests

The authors declare that they have no competing interests.

Acknowledgments

This work was funded by grants from the National Key Research and Development Program of China (2016YFD0500704) and National Natural Science Foundation of China (31472177) PRRSV structural protein T cell epitope mapping.

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    Authors Gaiping Zhang and Ning Li equally contributed to this paper, and Ning Li is the same role as the first author in contribution to this paper.

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