| Porcine epidemic diarrhea virus(PEDV) belongs to Coronaviridae family and causes severe enteritis, vomiting, watery diarrhea and dehydration in swine, especially is characterized by significant morbidity and mortality in neonatal piglets. The economic impacts of the PED are substantial. Now the vaccine of PED had been commercialized and widely used in China, but did not control the prevalence of the disease, and the immune efficiency needs to be improved. So it is necessary to develop a more effective and safer vaccine to prevent the PEDV infection.Spike protein is on the viral surface of PEDV, and the major antigen determinant is on it. It performs a very crucial function in the recognition of target cells and attachment of the viral particles to the receptors of host cells, with subsequent penetration into the cells via membrane fusion. It also had been identified as a stimulating factor for the production of neutralizing antibodies within the host. So it is the target protein for researching the vaccine of PED.To develop a safer and more effective vaccine of PED, the two DNA fragments of spike gene was amplified by RT-PCR and cloned into the transfer vector pUSG11/P28to get the recombinant transfer vector pUSG11/P28-S1and pUSG11/P28-S2. After homologous recombination with wild swinepox virus(SPV) genome, the target gene was inserted into SPV with Lipofectamine2000. By eGFP selection we get recombinant SPV named rSPV-S1and rSPV-S2. Amount of neutralization antibodies was observed in the serum of mice immunized with rSPV-S1. By PCRã€Western blotting and indirect immunofluorescence assay, the S2gene was identified in the recombinant SPV and was correctly expressed in PK-15cells infected by rSPV-S2. The results suggested that the recombinant SPV-S could be a promising candidate vaccine for the preventive of PEDV.In this research, the DNA fragment of spike gene(250bp-831bp, S2) was amplified by RT-PCR and cloned into the transfer plasmid pFastBacHTA to get the recombinant transfer plasmid pFastBacHTA-S2containning the S2gene. Then the plasmid pFastBacHTA-S2 was transformed into the E. coli DH10Bac cells. By blue/white selection and PCR analysis, recombinant transfer Bacmid plasmid DNA(rBacmid-S2DNA) was obtained, and then used to transfect into Sf9insect cells with Lipofectamine2000to produce the recombinant baculovirus that named vBacmid-S2. After the third generation of vBacmid-S2in Sf9cells, the expressed recombinant S2protein band of approximate24kD was identified by SDS-PAGE and Western blotting. The recombinant Baculovirus vBacmid-S2antigen was visualized in infected Sf9cells by indirect immunofluorescence assay. The results indicated that the partial spike gene was well and correctly expressed in insect cells, and could be the diagnosis antigen for the development of subunit vaccine of PED. |
