Establishment Of ELISA Protocol For Pseudorabies Virus Antibody Test In Porcine And Prokaryotic Expression Of Japanese Encephalitis Virus EIII Protein

Pseudorabies?PR?is an economically important viral disease of pigs in many countries.The disease is caused by pseudorabies virus?PRV?.PRV is a member of the genus Varicellovirus of the subfamily Alphaherpesvirinae within the family Herpesviridae.Pigs are the natural host for PRV and the only animals to become latent carriers,although the virus can infect numerous other species of mammals.Japanese encephalitis?JE?is an zoonotic infectious disease caused by Japanese encephalitis virus?JEV?,a zoonotic mosquito-borne flavivirus.JEV can causepregnant sows abortion,producing stillbirth or mummified tires,and also cause the boar's acute testicular or infertility.It has seriously affecting the development of the pig industry.PRV gE gene amplified by PCR was cloned into pET-32a to construct prokaryotic expression plasmid pET-gE.Transformed the plasmid pET-gE and the plasmid pET-EIII saved by the lab into E.coli BL21?DE3?.By using the SDS-PAGE analysis,as well as ImageJ and Graphpad prism 5.0 software,the factors closely related to the expression of the recombinant gE protein and the recombinant EIII protein were optimized which were culture growth temperature,induction OD₆₀₀,and induction IPTG concentration.The optimized expression conditions of the recombinant gE protein were that when the OD₆₀₀reached about 1.2,adding IPTG to final concentration of 1.5 mM,and shaking 175 rpm for 16 h at 16?.The optimized expression conditions of the recombinant EIII protein were that when the OD₆₀₀ reached about 1.6,adding IPTG to final concentration of 1 mM,and shaking 175 rpm for 16 h at 16?.To obtain highly purified recombinant gE protein and recombinant EIII protein,the purification conditions were also optimized to determine the concentration of imidazole.The research showed that the concentration of imidazole in the washing buffer was 100 mM and in the eluate was 500 mM for recombinant gE protein.However it was the concentration of imidazole in the washing buffer 100 mM and in the eluate was 300 m M for recombinant EIII protein.Western blot showed that the purified recombinant gE protein could react with PRV-positive sera,and the purified recombinant EIII protein could react with JEV-positive sera,but they all couldn't with negative serum.These results confirmed that the purified gE protein and the purified EIII protein had good immunogenicity and specificity.PRV gE-ELISA method was established with the purified recombinant gE protein as the coating antigen through optimizing the reaction conditions.The coating concentration protein was 0.5?g/m L,incubated at 37?2 h.The optimal blocking buffer was consisted with 1%gelatin and 2.5%BD skim milk.The plates were blocked for 2.5 h at 37?.Serum sample was diluted with 10×and incubated for 45 min at 37?.Rabbit anti-pig IgG-HRP was diluted with 3?g/mL and incubated for 60 min at 37?.The color developing time was incubated for 15 min at 37?.The ELISA cut-off value was 0.721which was determined by using TG-ROC method.Thus,samples with S/P ratios?0.721were considered negative against PRV,and those with ratios>0.721 were considered positive against PRV.The gE-ELISA was high specificity for detection of the antibody against PRV,but no cross-reactions to the positive sera of classical swine fever virus,transmissible gastroenteritis virus,porcine reproductive and respiratory syndrome virus,porcine circovirus type 2 virus,porcine epidemic diarrhea virus,actinobacillus pleuropneumoniae,streptococcus suis type 2.The coefficient of variation of intra-and inter batch repeatability test was lower than 5.95%and 9.62%,repectively.Moreover,comparison of the established gE-ELISA with four commercial kits for detection of 250clinical samples to PRV,the results showed that the highest concordance rate was 83.6%.It would provide a rapid assay for detection of PRV infection and serosurvey of the disease.