| Porcine epidemic diarrhea is an intestinal disease.Mucosal immune plays an important role.Listeria monocytogenes is a food-brone intracellular bacterium.To develop and evaluate attenuated Listeria monocytogenes-based oral vaccine for porcine epidemic diarrhea,the main results in this study are as followed:(1)Whole genome sequence of porcine epidemic diarrhea virus(PEDV)strain PEDV/CHN/SHANGHAI 5/2012 was analyzed,this strain belongs to Non-S INDEL clade with high virulence.Phylogenetic analysis showed that this strain is far away from the classical vaccine strain CV777 which is genetically closer to emerging S INDEL strains.(2)With N gene as target,a TaqMan probe-based RT-qPCR was developed for PEDV quantitative detection,sensitivity reached at 1.4×10~2copies.Meanwhile,a conventional duplex RT-PCR for PEDV qualitative detection was also developed for based on both N gene and S gene.RT-qPCR and conventional duplex RT-PCR demonstrated high specificities.(3)To screen appropriate E.coli expression vectors for expressing antigenic COE and S1D,a series of recombinant vectors were constructed and transformed into E.coli BL21,IPTG induction and SDS-PAGE electrophoresis demonstrated that both COE and S1D were successfully expressed.Western-bolt results showed that anti-PEDV positive sera could strongly react with the Ni column purified polypeptides COE and S1D,these two polypeptides can be used as coating antigens to develop an indirect ELISA for detecting antibodies of vaccinated animals in our next work.(4)InlB mediates LM to infect hepatocytes,so InlB encoding inlB gene was knocked out from chromosome of ?actA/plcB strain to further attenuate virulence.Expression vectors pKSV7-phly-COE-EGFP and pKSV7-phly-S1D-EGFP were constructed and electroporated into?actA/plcB/inlB strain,respectively.Confocal results showed both COE and S1D have been expressed.The animal experiment is in progress. |
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