Identification Of A Novel Epitope In The S1 Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). The disease has frequently broken out in Europe and Asia pig industries over the last 40 years, with the virus first appearing in England and Belgium in the early 1970s, resulting in significant losses to the global pig industry. The S glycoprotein which is localized on the vision surface, plays a key role in the interaction with the cellular receptor to regulate viral entry and induction of the neutralizing antibodies. The S protein can be divided into S1 (the 1-789 amino acid) and S2 (the 790-1383 amino acid) domains. In addition, the S1 domain contains multiple neutralizing epitopes and receptor binding domain. Therefore, screening and identification of the antigenic epitopes on S1 domain were carried out in this study. The arm is to provide the valuable information for development of antibody testing and immunologic specificity of S protein and the research of novel vaccine. The results are as follows:1. Antigenic analysis of S1 proteinFive overlapping fragments, which covered the S1 gene, were separately amplified by PCR and designated as SA, SB, SC, SD and SE respectively. These fragments were cloned into prokaryotic expression vector pCold TF. The confirmed plasmids were transformed into DE3 and induced by IPTG. The fusion proteins were purified according to the manufacturer’s protocols, and then prepared for polyclonal antibody. Ninety six well microtiter plates were coated with the purified SA-SE recombinant protein. The results of ELISA indicated that SE protein was recognized by the polyclonal antibody and showed strong binding activity, furthermore the results of Western blot and IFA suggested that the immune serum against SE showed that SE protein had good immunogenicity.2. Establishment of indirect ELISA for SE proteinThe indirect enzyme-linked immunosorbent assay (ELISA) has established based on the immundominant region protein SE and the paramenters of the ELISA were optimized. The cutoff value determined is 0.155 by analyzing optical density OD450 values of 32 PEDV negative sera. Coincidence rate of PEDV positive and negative sera tests revealed that the coincidence rate was 93.7%. Repeatability tests showed that the coefficients of variation of the intra and inter batch were both less than 10%.3. Preparation of monoclonal antibody to SE proteinTo identify the antigenic epitope of the S1 domain, the SE protein-specific monoclonal antibody was prepared. The spleen cells from immunized mice were fused with SP2/0 myeloma cells. After four times of the clone, one hybridoma clone that secreted McAb specific for the PEDV SE protein was isolated and designated 2E10. The titer of purified monoclonal antibody by established ELISA was 1×105. As shown by Western blot analysis, the monoclonal antibody 2E10 shown a strong reaction with PEDV, but not with Porcine epidemic diarrhea disease Virus, Japanese encephalitis virus, Classical swine fever virus and Porcine pseudorabies virus interactions. In conclusion, these results indicated that the McAb 2E10 has a high specificity.4. Epitope identification of SE proteinFirst, four overlapping fragments (SE1～SE4), covering the SE protein, were expressed and subjected to Western blot with McAb 2E10. As demonstrated by Western blot, SE2 and SE3 showed reactivity with the McAb 2E10. Subsequently, SE2 and SE3 were divided into two overlapping fragments (SE5～SE6) and were probed by McAb 2E10. The results showed that SE6 harboted the antigenic epitope. In order to identify core amino acides on epitopes SE6, pepscan often synthesized peptides (SE7～SE16) was carried out with McAb 2E10. In ELISA, the SE16 showed a strong reaction with McAb 2E10. The results of the ELISA were confirmed further by Western blot analysis and the results of Western blot were in accordance with the results of the ELISA. These data demonstrate that SE16 (721SSTFNSTREL730) are core amino acides of SE.All together, we determined an important immunodominant region of S1 protein and developed ELISA methods for antibody detection. A monoclonal antibody to SE protein and a linear antigenic epitope SE16 (721SSTFNSTREL730) were identified on the SI protein of PEDV. These datas could provide the basis for the structure and function of S1 protein and the development of neutralizing epitope.