The Study On Differential Diagnosis Between Vaccine TJ And Wild-type Strain Of Porcine Reproductive And Respiratory Syndrome

Porcine reproductive and respiratory syndrome(PRRS),commonly known as swine blue aural disease,is a viral disease that not only causes respiratory symptoms in piglets or even swine at all ages,but also abortion and stillbirth in pregnant sows.PRRS has a high mortality rate,transmission rate,and can cause immune suppression.Its outbreak has brought serious losses to the pig industry all over the world.Given that different strains of porcine reproductive and respiratory syndrome virus have differences in virulence,antigenicity and biological characteristics,and strain variations,recombination occur frequently,a single stains PRRSV vaccine cannot protect pigs from heterologous strains infection.The extensive abuse of the disease vaccines in quantity and variety can reduce clinical symptoms,but can not prevent infection of virulent strains and persistent infection,and that makes it difficult to eliminate the threat of PRRS fundamentally,so the eradication of PRRS is becoming the choices of many hog farms.Due to the lack of a stable and accurate method for the detection of the wild strains and vaccine strains,it is urgent to establish a method for the identification of antibodies between PRRSV wild virus infection and vaccine immune antibody.In this study,Nsp2 gene of the PRRSV TJ strain was found to have a deletion of 307 nucleotides in PRRSV strain sequence and specific primers were designed.The viral antigen RNA of PRRSV JX strain containing complete Nsp2 gene was extracted and the PRRSV-Nsp2-N1N2 gene(fragment gene deleted)was amplified by RT-PCR.The expression plasmid pET-32a-N1N2 was constructed and the fusion protein was expessed by E.coli BL21 prokaryotic expression system.It's confirmed through SDS-PAGE that the fusion protein was of soluble expression and existed in the supernatant.By means of purification of the fusion protein by His affinity chromatography,fusion protein with high purity and specificity was obtained.Choosing the purified fusion protein pET-32a-NlN2 as the solid phase coated antigen,an indirect ELISA method was established.The reaction conditions were optimized and negative and positive values were determined.The chosen optimal concentration of coating antigen was 75ng/well detected serum dilution was 1:200,enzyme labeled antibody concentration was 1:5000,reaction time for sealing liquid,tested serum and TMB was 60min,60min and 15min,respectively.Criterion for positive is P>0.4401 and P/N>2.5.In the compliance test,138 positive serum samples and 50 negative serum samples were compared to commercial PRRSV indirect ELISA detection kit,and the resulted compliance rate was 100%.The results of this trial showed that the established ELISA detection method has high sensitivity,repeatability and specificity,can distinguish the TJ stain vaccine antibody and the wild virus infection antibody of PRRS,and provides technical support for the purification of PRRS.