Identification and characterization of linear B cell epitopes on the nucleocapsid protein of porcine epidemic diarrhea virus using monoclonal antibodies

Abstract

The nucleocapsid (N) protein of porcine epidemic diarrhea virus (PEDV), the most important pathogen causing severe diarrhea in piglets, is a highly conserved structural protein. In this study, 5 monoclonal antibodies (McAbs) against the PEDV N-protein were prepared and identified. Three new epitopes, ⁵⁶QIRWRMRRGERI⁶⁷, ³¹⁸GYAQIASLAPNVAALLFGGNVA VRE³⁴² and ³⁹⁸HEEAIYDDV⁴⁰⁶, were firstly identified in the viral N-protein, by using McAbs 3F10, 6A11, and 1C9. The epitope ³⁹⁸HEEAIYDDV⁴⁰⁶ was deleted in SH strain (isolated by our lab) and different between CV777 and YZ strain (isolated by our lab). To study the characters of this epitope, four peptides were synthesized according to the sequence of SH and CV777 and used in the study. The result showed that the 398^th amino acid maybe an important amino acid of the epitope. Biological information analysis showed that the three B cell linear epitopes are highly conserved among different PEDV isolates. In addition, McAb 1C9, which attached to the epitope ³⁹⁸HEEAIYDDV⁴⁰⁶, showed variant reactivity with PEDV CV777, SH, YZ and MS strains. McAb 1C9 reacted with PEDV strains CV777 and YZ, but not with SH which had a deletion from 399 to 410 amino acids in N-protein (No. MK841494). Among the three McAbs, 6A11, 3F10 and 1C9, only 6A11 reacted with porcine transmissible gastroenteritis virus (TGEV) in immunofluorescence assay, therefore the other two could be used to distinguish TGEV and PEDV. These mAbs and their defined epitopes may provide useful tool for the study of the PEDV N-protein structure and function, and facilitate the development of diagnostic methods for PEDV.

Keywords: Epitopes; McAbs; Nucleocapsid protein; Porcine epidemic diarrhea virus (PEDV).

Fig. 1

Schematic diagram of the truncated N proteins To identify the epitopes of PEDV to the McAb, the N gene was divided into 17 truncated genes (A, B, C, A1–A6, C1–C8). A: 1-190Aa, B:101-318Aa, C:241-441Aa, A1:28-190Aa, A2:61-190Aa, A3:80-190Aa, A4:36-190Aa, A5:44-190Aa, A6:52-190Aa, C1:255-372Aa, C2:255-409Aa, C3:241-342Aa, C4:241-350Aa, C5:241-360Aa, C6:241-381Aa, C7:241-389Aa, C8:241-399Aa.

Fig. 2

Identification of monoclonal antibodies by IFA(A) and Western-blot(B). A: After inoculated with PEDV CV777 strain, the Vero cells were fixed and analyzed by IFA with the five McAb. Non-inoculated cells were used as control. To confirm the specificity of McAb, positive serum and the RPMI-1640 containing 20 % FCS (N) were used as antibody to incubated the cells as control. The Vero cells inoculated with CV777 could reacted with the five McAb and positive serum, and showed obvious green fluorescence on the surface. The non-inoculated cells and N control showed no any fluorescence. B: The purified PEDV and N protein expressed in Baculovirus were separated by 10 % agarose gel and transferred onto a nitrocellulose (NC) membranes. After blocking, the membranes were incubated with McAb overnight at 4 °C. After 3 times washes with PBST, the membranes were incubated with HRP-labeled Goat Anti-Mouse IgG (H + L) antibody. The reaction was visualized by using ECL Western blot substrate. Vero cells and Sf9 cells were used as control. Line 1: Purified PEDV; Line 2: Vero cell control; Line 3: N protein expressed in Baculovirus; Line 4: Sf9 cell control.

Fig. 3

Epitope mapping of McAbs by Western blot. The truncated N proteins and the oligopeptides were separated by 12 % agarose gel and transferred onto a nitrocellulose (NC) membranes. After blocking, the membranes were incubated five monoclonal antibody and HRP-labeled Goat Anti-Mouse IgG antibody successively. The line 6P-1 means that the expressed protein of the pGEX-6P-1 vector reacted with the McAb. (A) The McAb 3F10 could reacted with the truncated proteins A including A1, A4, A5, A6, A6-1, A6-2. (B) The McAb 6A11 could reacted with proteins C, C1, C2, C3, C4 and C5. (C) The McAb 1C9 could reacted with the truncated proteins C2, C2-2 and C2-3.

Fig. 4

Reactivity of McAb with the epidemic PEDV strains by IFA and Western blot. Vero cells inoculated with PEDV strain YZ, SH and MS were incubated with 1C9, 6A11 and 3F10, and then stained with FITC labeled goat anti-mouse IgG. Vero cells inoculated with YZ and MS showed specific fluorescence, but Vero cells inoculated with SH 2016 only reacted with the McAb 3F10 and 6A11, not with 1C9. To further verify reactivity of McAb with the epidemic PEDV strains, Vero cells inoculated with PEDV strain YZ, SH2016 and MS were separated by 12 % agarose gel and transferred onto a nitrocellulose (NC) membranes. Non-specific antibody binding sites were blocked with PBST containing 10 % skim milk at room temperature for 2 h. The membranes were successively incubated with McAbs and HRP-labeled Goat Anti-Mouse IgG (H + L) antibody (Beyotime, China). Then the reaction was visualized by using ECL Western blot substrate (Thermo). The result of Western blot was consistent with that of IFA. Vero cells inoculated with PEDV strain CV777 were used as positive control.

Fig. 5

Reactivity of McAb with TGEV by IFA. ST cells were inoculated with TGEV and then successively incubated with the McAb and FITC-labeled Goat Anti-Mouse IgG(H + L) antibody. The specific fluorescence was observed in the cells incubated with McAb 6A11, but not with 3F10 and 1C9.

Fig. 6

Multiple alignments of the epitopes among PEDV isolates and different coronavirus. The epitope ⁵⁶QIRWRMRRGERI⁶⁷ and ³⁹⁸HEEAIYDDV⁴⁰⁶ has little gene differences in all of the PEDV strains, and the epitope ³¹⁸SGYAQIASLAPNVAALLFGGNVA VRE³⁴² was completely conservative in all of the PEDV strains (A). While the three epitopes had much differences between different coronaviruses (B).

Fig. 7

Reactivity of McAb and mice sera against PEDV YZ and SH strain by iELISA. Four synthesized polypeptides were coated in the ELISA plate, respectively. McAb and mice sera were added into the wells and then and HRP-labeled Goat Anti-Mouse IgG (H + L) (Beyotime, China) was added into each well. After developing colour with TMB substrate (Beyotime, China) and stoping with 2 M H₂SO_4, absorbance was read on Multiscan Spectrum (Epoch, Biotek) at 450 nm. PEDV SH: mice sera against PEDV SH strain; PEDV YZ: mice sera against PEDV YZ strain; 1C9: McAb 1C9.

Fig. 8

Localization of the identified epitopes. The relative localization of the identified epitopes of 3F10 marked in red, 6A11 marked in green, 1C9 marked in orange, in a partially predicted 3D structure of N protein is highlighted in spheres (A) and a cartoon representation (B). (C) Structural features of N protein was predicted using PROTEAN software. All four epitopes are shown in boxes.