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. 2016 Dec 10;8(12):327.
doi: 10.3390/v8120327.

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

Affiliations

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

Hui Dong et al. Viruses. .

Abstract

The coronavirus membrane (M) protein acts as a dominant immunogen and is a major player in virus assembly. In this study, we prepared two monoclonal antibodies (mAbs; 1C3 and 4C7) directed against the transmissible gastroenteritis virus (TGEV) M protein. The 1C3 and 4C7 mAbs both reacted with the native TGEV M protein in western blotting and immunofluorescence (IFA) assays. Two linear epitopes, 243YSTEART249 (1C3) and 243YSTEARTDNLSEQEKLLHMV262 (4C7), were identified in the endodomain of the TGEV M protein. The 1C3 mAb can be used for the detection of the TGEV M protein in different assays. An IFA method for the detection of TGEV M protein was optimized using mAb 1C3. Furthermore, the ability of the epitope identified in this study to stimulate antibody production was also evaluated. An immunodominant epitope in the TGEV membrane protein endodomain was identified. The results of this study have implications for further research on TGEV replication.

Keywords: coronavirus; endodomain; immunodominant epitope; membrane protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Preparation of monoclonal antibodies (mAbs) against the M protein of TGEV. (a) Expression and purification of GST-M protein. The proteins were detected after western blotting with a GST mAb; (b) Reactivity of the 1C3 and 4C7 mAbs with the GST-M protein and the TGEV M protein. PM represents protein marker. T+ represents the cell lysates of TGEV-infected porcine kidney 15 (PK-15) cells. T− represents the cell lysates of mock infected PK-15 cells.
Figure 2
Figure 2
Identification of the epitopes of the 1C3 and 4C7 mAbs. (a) Scheme of the M protein and M fragments; (b) Western blotting analysis of the GST-M1, GST-M2, GST-M3, GST-M4, and GST-M5 proteins using the 1C3 and 4C7 mAbs; (c) Western blotting analysis of the GST-M6 and GST-M7 proteins using the 1C3 and 4C7 mAbs; (d) Five peptides were reacted with the mAb 1C3 and nine peptides with 4C7 by peptide ELISA. aa represents amino acids. PM represents protein marker.
Figure 3
Figure 3
Location of the identified epitope in the predicted structure of the TGEV M protein. (a) The location of the epitope (shown in red) for mAb 1C3 (YSTEART) in the TGEV M protein is highlighted; (b) Conservation of the M epitopes (YSTEART) in TGEV, porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). Dots indicate identical residues.
Figure 4
Figure 4
Application of the generated mAbs 1C3 and 4C7 in immunofluorescence assay (IFA) and immunoperoxidase monolayer assay (IPMA). (a) IFA analysis of the M protein in TGEV-infected PK-15 cells using 1C3 and 4C7 mAbs; (b) IPMA assay of the M protein in TGEV-infected PK-15 cells using 1C3 and 4C7 mAbs. The mAb against the N protein of TGEV was used as a positive control.
Figure 5
Figure 5
Application of the generated mAbs 1C3 and 4C7 in IP and immunohistochemistry (IHC). (a) Immunoprecipitation analysis of the M protein in TGEV-infected PK-15 cells using 1C3 and 4C7 mAbs. T+ represents the cell lysates of TGEV-infected PK-15 cells. T− represents the cell lysates of mock-infected PK-15 cells. The mIgG represents mouse control IgG; (b) IHC analysis of the M protein in the small intestines of TGEV-inoculated animals using 1C3 (1) and 4C7 (2) mAbs and an N-protein mAb (3) as a positive control. Staining of the small intestines of mock-inoculated animals with 1C3 mAb is shown as a negative control (4).
Figure 6
Figure 6
Optimization of the IFA method using mAb 1C3 for the M protein. (a) Purification of mAb 1C3 IgG. Lanes 1–7: purified IgG; (b) Optimization of the IFA method for M protein detection using the purified mAb 1C3 IgG in PK-15 cells. PM represents protein marker.
Figure 6
Figure 6
Optimization of the IFA method using mAb 1C3 for the M protein. (a) Purification of mAb 1C3 IgG. Lanes 1–7: purified IgG; (b) Optimization of the IFA method for M protein detection using the purified mAb 1C3 IgG in PK-15 cells. PM represents protein marker.
Figure 7
Figure 7
Antibody responses to the identified epitopes. (a) Humoral responses elicited by the aa 243-249 and aa 243–262 epitopes; (b) Reaction of antibodies elicited by epitopes with the TGEV virus in PK-15 cells.

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