The Clone Expression And Immunogenicity Analysis Of The Specific Epitope Protein In The COE Region Of PEDV S Protein

Up to now,Porcine Epidemic Diarrhea Virus PEDV has become popular among countries in the world,and it has brought an economic loss to the pig industry.The high tolerance of PEDV to the environment and the wide spread of its transmission make it a difficult to prevent infectious disease in pig industry.At present,in-depth studies have been carried out at home and abroad,mainly on the specific diagnosis and related vaccine research.COE region is a part of protective antigen gene in PEDV s protein,This study aims to obtain the strong specific antigenic protein in COE region of PEDV s protein,and lay the foundation for the subsequent establishment of specific ELISA detection method.According to the reported antigen epitope sequence of COE region of PEDV s protein,primers were designed to extract virus RNA from intestinal tissue and lymph node tissue of piglets suffering from PEDV death,extract virus target RNA and reverse transcription to obtain cDNA,and obtain target gene sequence by PCR.The target gene was connected to pMD-18T vector.The target gene was identified by sequencing,and the sequencing results showed that the gene sequence was correct.According to bioinformatics analysis,the protein encoded by the gene contains six potential T-lymphocyte antigen determinants?specific motifs?,10 immune dominant T-lymphocyte antigen sites,8 amino acid sequence regions in the fragment with an epitope analysis index of 1.7,and 50%hydrophobic analysis.It is suitable for water-soluble and fat soluble adjuvants.The protein can form three The domain is located on the surface of S protein,indicating that the protein fragment is suitable to be used as antigen protein.The target gene was digested by EcoR I and Hind III,and the target gene fragment was connected to prokaryotic expression vector PET-32a.The target gene was digested by BamH I and Hind III,and the target gene fragment was connected to the prokaryotic expression vector pMal c2x.The prokaryotic expression vectors PET-PSC and pMal-PSC were successfully constructed.After IPTG induction,E.coli expressed the target protein.PET-PSC inclusion body protein was extracted by Ni agarose resin affinity chromatography.The protein of pMal-PSC was obtained by the purification of amlose resin.Kunming mice were divided into four groups with SPF.They were immunized with 206 adjuvant and PET-PSC,AL?OH?₃ and CPG,PET--PSC,PET--PSC and PBS respectively.After three times of immunization,spleen were extracted and the levels of cytokines inf and IL-2 and IL-4in serum were measured.Western blot showed that the target protein was successfully obtained.The results showed that the increase level of INF-?in the protein group with Freund's adjuvant was significantly higher than that control group?P<0.05?,and the increase level of IL-2 and IL-4 in the protein group with Freund's adjuvant were significantly higher than that control group?P<0.05?,The SI value of CpG immunoadjuvant group was about 3.5,which was significantly higher than that of antigen protein and PBS group?P<0.05?.The SI value of 206 protein group was 3.0,which was significantly higher than that of antigen protein and PBS group?P<0.05?.The results showed that the target protein had good immunogenicity.PET--psc polyclonal antibody was used as positive sample to optimize the coating concentration.The optimal coating concentration of pMal-PSC protein was 2?g/mL.The sensitivity of ELISA method was tested by using pmal Recombinant Prokaryotic Expression antigen as positive sample.The results showed that the A₄₅₀ value of the antibody was still above 0.5after diluting to 25600 times.The results showed that when the antibody was diluted to 25600,it could be used to detect antigen protein.The PSC protein was obtained successfully.It had good immunogenicity by IL-2,IL-4,IFN-?and lymphocyte proliferation test.PSC protein is highly sensitive by ELISA and can be used in ELISA.