Identification Of Antigenic Epitopes On The Membrane Protein And Nucleocapsid Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an enteric disease of swine mainly characterized by enteritis, diarrhea, vomiting, and even dehydration. The pathogen of PED is porcine epidemic diarrhea virus (PEDV). The prevalence of PED all over the world has resulted severe economic losses to the swine industry.In the present study, Membrane (M) protein gene and Nucleocapsid (N) protein gene of PEDV CH/SHH/06 strain were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified genes were cloned into the pGEM-T vector for sequencing, respectively. After validation of the correction of the inserted genes, the truncated M gene (tM, 295bp～678bp ) was amplified by polymerase chain reaction (PCR) with the verified plasmid pGEM-T-M as template and the tM gene was inserted into the prokaryotic expression plasmids pET-30a and pGEX-6P-1, respectively. After digestion of N protein gene from verified plasmid pGEM-T-N by restriction enzymes, N gene was inserted into the prokaryotic expression vector pET-30a to generate recombinant plasmid pET-30a-N. Recombinant proteins tM-Hi_s6 and GST-tM and N-Hi_s6 were expressed by inducing pET-30a-tM transformed E.coli and pGEX-6P-1-tM transformed E.coli and pET-30a-N transformed E.coli with IPTG. E.coli srains transformed by pET-30a and pGEX-6P-1 were operated in parallel as negative controls. The molecular weitht of the recombinant proteins were 15Ku (tM-Hi_s6), 40Ku (GST-tM) and 49Ku (N-Hi_s6), respectively, shown by SDS-PAGE analysis. And it was revealed by Western blot assays that all the recombinant proteins could react with rabbit anti-PEDV serum specifically, indicating that the recombinant proteins have good reactivity.To prepare MAbs against M and N protein of PEDV, BALB/c mice were immunized with purified recombinant proteins tM-Hi_s6 or N-Hi_s6. One strain of hybridoma secreting antibodies against M protein and three hybridomas against N protein, designed as D4, 4G1, 4H7 and 2G3, respectively, were obtained. The MAb D4 was IgG1 subtype withκchain and all of the three MAbs of N protein were IgG2b subtype withκchain. Vero E6 cells infected with PEDV showed immunofluorescence with the MAbs D4, 4G1 or 4H7 as primary antibody proved by Indirect immunofluorescence assay (IFA). MAb D4 could react with authentic M protein revealed by Western blot analysis.Through a series of expression of truncated M proteins and N proteins, the antigenic epitope against MAbs D4 , 4G1,4H7 and 2G3 were preliminary established, which were"¹⁹³TGWAFYVR²⁰⁰","⁶⁰RMRRGERIEQPS⁷¹","¹⁴³ERDLKDIPEWRRIPKG¹⁵⁸"and"337aa～396aa"of N protein, respectively.In order to finely identify the antigenic epitopes of PEDV M protein and N protein, the terminal amino acid residues both from carboxy and amino terminals of the epitopes were deleted one by one to determine the core anmino acids of the epitopes. The results indicated that"¹⁹⁵WAFYVR²⁰⁰", "⁶⁰RMRRGERIEQPS⁷¹", and"¹⁴⁸DIPEWRR¹⁵⁴"were the core epitope recognized specifically by MAbs D4, 4G1 and 4H7. Homologous analysis showed that"¹⁹⁵WAFYVR²⁰⁰"and"⁶⁰RMRRGERIEQPS⁷¹", the corn epitops of M or N protein, were highly conserved."¹⁴⁸DIPEWRR¹⁵⁴", another corn epitope of N protein, share a relative conservation among M proteins of different PEDV strains. The epitope"¹⁹⁵WAFYVR²⁰⁰"could be recognized by porcine anti-PEDV positive serum, but not porcine anti-TGEV positive serum proved by Western blot assay.We identified the antigenic epitopes on M protein and N protein of PEDV, which will provide useful informations for understanding the antigenic structure of M protein and N protein and designing vaccine and diagnosis methods based on epitopes.