Prokaryotic Expression Of The Truncated S1Gene Of Porcine Epidemic Diarrhea Virus And The Establishment Of Indirect ELISA

Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigscharacterized with vomiting, severe diarrhea and death from dehydration. Porcine epidemicdiarrhea virus (PEDV) is the causative agent of PED. All ages of pigs, especially theneonatal piglets, are susceptible to the disease. Piglets within1-week old infected withPEDV usually die after2to4days when disease onsets, with a mortality up to90%. PEDhas caused significant economic losses to the pig industry.The S1gene of the S protein of PEDV harbors critical determinants which can inducestrong neutralizing antibodies, and thus a set of primers specific to S1gene of PEDVCV777strain was designed. Utilizing designed primers, a truncated S1gene was amplifiedby RT-PCR. Afterwards, the truncated S1gene was cloned into the PMD-18-T vector,which was subsequently confirmed by PCR and sequencing analysis, and then subclonedthe truncated S1into pET-32a (+) vector to construct a recombinant expression plasmidreferred as pET-S1. pET-S1was then transformed into E. coli BL21(DE3), followed by S1protein expression induced with IPTG. The expressed S1protien was identified as the formof inclusion bodies. Via a seria of tests, optimized conditions for protein expression weredetermined, which ware as follows: the IPTG concentration was0.6mmo1/L, theinduction time was4hrs. The S1protein, an expression product, was purified by Nicolumns, and highly pure recombinant S1protein was refolded by dialysis to restore theactivity of the protein. Western-blot and ELISA analyses showed that the recombinant S1protein could specifically react with PEDV positive sera and induce high titers of antibodyagainst PEDV when immunized rabbits, indicating that the recombinant protein had a goodreactogenicity.The purified recombinant S1protein was used as a coating antigen, and thus an indirectELISA for the detection of PEDV antibodies was established. The optimum workingconditions were determined through checkerboard methods: the concentration of coatingantigen was10ug/ml, and coated at4℃overnight: blocking time was2h at37C with5%Skim milk; a10240-fold dilution of sera tested was incubated at37℃for45min;1:2000diluted HRP-antibody conjugate was incubated at37℃for60min; and OPD substratesolution was incubated at37℃for15min in dark room. The cut off value of the positiveresults was0.125. The evalution and validation results demonstrated that indirectantibody-capture ELISA established in this study for the detection antibodies againstPEDV was highly specific and reproducible. The results from the ELISA establishedbased on the tests of189pig sera showed that the positive rate for PEDV was48.68%(92/189). The indirect ELISA established in this study with the truncated S1protein generated bya prokaryotic expression system will provide an alternative methodology for the suevey ofPEDV antisera and a potential subunit vaccine candidate as well. The results are valuablefor the prevention and control of PED.