| Porcine epidemic diarrhea(PED)is a highly contagious disease which severely threatens the pig industry worldwide.PED is a disease caused by porcine epidemic diarrhea virus(PEDV)infect and cause disease in all age of pigs characterized by water diarrhea,vomiting,dehydration and death,especially the neonatal piglets with a high mortality reaching 100%.Due to the development of immune system of neonatal piglets is not complete,it is crucial to obtain passive protection via maternal antibody.At present,the high level of Ig G antibody produced by the current PEDV commercial vaccine cannot provide effective protection for neonatal piglets.It’s reported that SIgA produced by mucosal immune response play a major role in preventing and controlling PED.SIgA comprises two monomers Ig A,a connecting chain(J chain)and a secretion component(SC).SC is the extracellular portion of the polymeric immunoglobulin receptor(pIgR),a unique component of SIgA and a sign that can be used to distinguish Ig A and SIgA.In the current,there is no compelling commercial kit to detect SIgA against PEDV in sow milk.In this study,we successfully developed the ELISA method to detect SIgA against PEDV using the purified PEDV virions as antigen and prepared monoclonal antibodies against SC as the secondary antibody.The developed kit facilitates to evaluate the mucosal immune response induced by the PEDV vaccine and provides a power tool to prevent and control PED.(1)For the preparation of the monoclonal antibodies against porcine pIgR protein,primers were designed on the basis of according to the extracellular domain of the porcine pIgR gene sequence.The amplified fragments were cloned into the prokaryotic expression vector p ET-30a(+)via PCR,enzyme digestion and ligation.The successfully constructed recombinant plasmid was transformed into E.coli BL21(DE3)competent cells to express the protein induced by IPTG,and purified pIgR protein was obtained using NiNTA affinity chromatography.The results showed that the recombinant plasmid was correctly constructed,and pIgR protein was successfully expressed.(2)BABL/c mice were immunized with purified pIgR protein mixed with an adjuvant.PEG was employed to fuse the splenocytes from immunized mouse and mouse myeloma cells(SP2/0).Hybridoma cells secreting specific monoclonal antibodies were screened by enzyme-linked immunosorbent assay(ELISA)with three rounds of subcloning.A total of four hybridoma cell lines stably secreting monoclonal antibodies against pIgR protein were eventually obtained.The eukaryotic expression vector pc DNA3.1-pIgR was constructed to express pIgR in 293 T cells to evaluate the reactivity of the prepared monoclonal antibodies via IFA and Western blotting.(3)The reaction conditions of the ELISA for the detection of SIgA against PEDV were optimized: the concentration of coating antigen(purified PEDV virion)is 4 μg/m L,the filed sample dilution is 1 : 2,the dilution of prepared monoclonal antibodies against pIgR is 1 : 1 000,the blocking buffer is 2.5% BSA,the TMB incubation time is 5 min.The threshold value(P ≥ 0.538,N < 0.474)of the method was determined by detecting 30 negative sow milk samples.The coefficients of variation of the repeatability tests were all less than 10%,indicating that the method has good reproducibility.In order to evaluate the sensitivity of the ELISA,20 positive milk samples were 2-fold serially diluted.The results showed that the sensitivity of the developed ELISA was superior to the commercial PED Ig A detection kit produced in Korea.The coincidence rate of the ELISA was 88.9% compared with PED Ig A detection kit. |
