| Porcine epidemic diarrhea(PED),which is caused by the porcine epidemic diarrhea virus(PEDV),is a highly contactable enteric infectious disease.The disease is characterized by acute watery diarrhea,dehydration,and vomiting,with high morbidity and mortality in suckling piglets.Since the winter of 2010,variant strains of PEDV associated with high mortality emerged in China and cause huge economic losses in China's swine industry.This disease has seriously hindered the healthy development of China's pig industry.There is currently no effective vaccine against PEDV.The S protein(spike protein)of PEDV consists of two domains,S1 and S2.Among them,S1 protein is the major immunogenic protein of PEDV,contains multiple epitopes and neutralizing epitopes,and is enriched in multiple receptor binding domains.It is closely related to virus antigenicity and adsorption invasion,therefore,it can be used as a target protein for the development of PEDV genetic engineering vaccines.In this study,recombinant baculovirus rB-S1 containing PEDV s1 gene was constructed,recombinant baculovirus rB-S1 was shown to efficiently express S1 protein in Sf9 insect cells by IFA,SDS-PAGE and Western blot,and It can react specifically with PEDV positive serum and has good immunogenicity.In this study,a 75-day-old sow was immunized with a subunit vaccine prepared with recombinant S1 protein,and an inactivated vaccine and a blank control group were established.Serum and colostrum were collected before the sows were delivered PEDV-specific IgG antibodies in serum,IgA antibodies in milk and serum neutralizing antibodies were detected.The results showed that the levels of IgG and IgA antibodies produced in the subunit vaccine group were higher than those in the inactivated vaccine group and the control group,indicating that the genetic engineering subunit vaccine could stimulate the sows to produce immunity.Piglets were challenged with a PEDV virulentstrain(CT strain)at 5 days of age.The piglets in the subunit vaccine group were in good condition in the first three days after the challenge,and a few piglets developed diarrhea three days later.The protection rate was 75.32%,which was higher than the inactivated vaccine and the blank control group.The results of the challenge protection experiment confirmed that the PEDV S1 subunit vaccine has a good protective effect against the PEDV CT strain.To screen for adjuvant suitable for PEDV subunit vaccines,four different adjuvants were used in combination with S1 protein to prepare subunit vaccines.The 70-day-old pigs were immunized and selected the best adjuvant by serum ELISA,neutralization assay,flow cytometry method,etc.The results showed that the ISA 660 VG adjuvant vaccine can induce higher levels of IgG antibodies and neutralizing antibodies,and can more effectively stimulate the activation of CD8 T lymphocytes,which is superior to ISA 201 VG,IMS 1313 VG N and GEL 01.To further determine the optimum immunization dose for subunit vaccines,this experiment was conducted to immunize 70-day-old pigs with immunization doses of 50,100,200,and 400 ?g of S1 protein respectively,and screened out the best Immunization dose by serum ELISA,neutralization assay,flow cytometry method,etc.The results showed that 400 ?g of S1 protein can induce higher levels of IgG antibodies and neutralizing antibodies,and can more effectively stimulate the activation of CD8 T lymphocytes.Therefore,it was determined that the optimal immunization dose for the subunit vaccine in this study was 400 ?g/head/time,and the best adjuvant was ISA 660 VG.In summary,the S1 protein of the PEDV CT strain was successfully expressed by using the insect baculovirus expression system in this study.The PEDV subunit vaccine prepared from this protein has a good immune-protective effect and is expected to be able to provide reference for the development of PEDV subunit vaccine. |
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