| Porcine epidemic diarrhea(PED)is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus(PEDV).The affected pigs mainly show clinical signs such as vomiting,diarrhea and severe dehydration.Pigs of all ages can be infected,but the mortality rate of newborn piglets within 7 days of age is as high as 95%.It is difficult to establish active immunity in a short period of time by vaccination of newborn piglets,so it is of scientific importance to use antibody preparations for prevention or emergency treatment since active immunity cannot be established by vaccination of newborn piglets.In this experiment,the recombinant Lactobacillus p PG-T7g10-sp-Fab expression vector was constructed using the Lactobacillus constitutive expression vector p PG-T7g10-PPT,which secretes the neutralizing antibody Fab fragment expressing PEDV.Western Blot assays showed that the expression of the PEDV-neutralizing antibody Fab fragment was detected in both the bacterium and medium of recombinant Lactobacillus royale p PG-T7g10-sp-Fab/J31.Enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence assay(IFA)demonstrated that the PEDV-neutralizing antibody Fab protein expressed by recombinant Lactobacillus royale p PGT7g10-sp-Fab/J31 specifically binds to PEDV.The phage-spot reduction assay demonstrated that the PEDV-neutralizing antibody Fab protein expressed by recombinant Lactobacillus royale p PGT7g10-sp-Fab/J31 had a neutralizing effect on PEDV.To determine the passive immune protection of recombinant Lactobacillus p PG-T7g10-sp-Fab/J31 against PEDV-infected piglets,6 newborn piglets were divided equally into 2 groups of 3 piglets each,one group was orally administered recombinant Lactobacillus p PG-T7g10-sp-Fab/J31,and the other group was a control group,orally administered with the transformed unexpressed antibody empty vector Lactobacillus royi p PG-PPT/J31,and after 3 d of continuous infusion,the above six piglets were reared in the same cage with the PEDV-infected piglets.The weight change and clinical signs of each piglet were recorded daily,and nasal and anal swabs were collected from the piglets every 6 h for detection of piglet detoxification using RT-PCR and q PCR methods.After 7 d of cohabitation,the non-dead piglets were executed and dissected to observe the intestinal pathological changes and perform intestinal pathological histological examination.The results showed that all six piglets could be infected with PEDV after cohabitation with PEDV-infected piglets,and the survival rate of p PG-T7g10-sp-Fab/ J31 piglets orally administered with recombinant Lactobacillus was 100% after cohabitation,while two control piglets died at the 6th day after cohabitation with PEDV-infected piglets.In the control group,PEDV was detected in nasal and anal swabs 48 h after cohabitation,and the detoxification time of p PG-T7g10-sp-Fab/ J31 piglets was later than that of the control group,and the amount of detoxification was significantly lower than that of the control group.In conclusion,this study successfully constructed recombinant Lactobacillus royi p PG-T7g10-sp-Fab/J31,which can secretly express PEDV-neutralizing antibody Fab fragment.p PG-T7g10-spFab/J31 expressed Fab protein with PEDV-neutralizing activity,and oral administration of recombinant Lactobacillus royi p PG-T7g10-sp-Fab/J31 to newborn piglets reduced clinical symptoms caused by PEDV infection,decreased mortality,and reduced detoxification time and amount.Fab/J31 alleviated the clinical signs caused by PEDV infection,reduced piglet mortality,and decreased the duration and amount of detoxification.The results of this study provide a new idea for the prevention and emergency treatment of PEDV infection. |
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