Establishment Of ELISA And Colloidal Gold Immunochromatography Method For Identify Antibodies Of PRRSV Wild Strain And TJM-F92 Vaccine Strain

Porcine reproductive and respiratory syndrome is an infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is mainly caused reproductive disorders in sows and dyspnea in piglets,which has a significant impact on the global pig industry.Vaccine immunization is an important method to prevent and control the disease.At present,there are classic strains and highly pathogenic strains in the approved vaccines in China,in which TJM-F92 vaccine strain is based on highly pathogenic TJ strain and there is a natural deletion of 120 amino acids in Nsp2.In this study,PRRSV conserved nucleocapsid protein(N protein)and TJM-F92 strain deletion fragment TJ120 were prokaryotic expressed and purified,and indirect ELISA and colloidal gold test strips were established to identify antibodies against PRRSV wild strain and TJM-F92 vaccine strain.In this study,ORF7 and the deletion fragment TJ120 of TJM-F92 strain were amplified by PCR technique and prokaryotic expression vector p ET28a(+)-PRRSVN and p GEX-6P-1-Nsp2-TJ120 were constructed and expressed and purified.Using purified p ET28a(+)-PRRSV-N protein as coating antigen,indirect ELISA and colloidal gold strip for detection of PRRSV antibody were established by optimizing reaction conditions.In the optimized indirect ELISA,the best coating concentration of p ET28a(+)-PRRSV-N is 2μg/m L,the critical value of negative and positive is0.3172,the best dilution of serum is 1:320.Compared with that of the IDEXX commercial kit,the overall coincidence rate of this method is 88.8%.In the optimized p ET28a(+)-PRRSV-N colloidal gold strip,the colloidal gold-SPA optimal dilution is 1:1,the optimal coating concentration of rabbit anti-porcine Ig G is1.0mg/ml,the best coating concentration of p ET28a(+)-PRRSV-N is 0.6mg/ml,the sensitivity is 1:160.Compared with that of the IDEXX commercial kit,the overall coincidence rate of this method is 81%.Using purified p GEX-6P-1-Nsp2-TJ120 protein as coating antigen,indirect ELISA and colloidal gold test strips for differential diagnosis of TJM-F92 vaccine antibodies were established by optimizing reaction conditions.In the optimized indirect ELISA the best coating concentration of p GEX-6P-1-Nsp2-TJ120 is 4ug/m L,the critical value of negative and positive is 0.3097,and the best dilution of serum is1:320.Compared with that of the IDEXX commercial kit,the overall coincidence rate of serum antibody detection of unimmunized TJM-F92 vaccine and inactivated vaccine of this method was 85%.In the optimized p GEX-6P-1-Nsp2-TJ120 colloidal gold strip,the colloidal gold-SPA optimal dilution is 1:1,the optimal coating concentration of rabbit anti-porcine Ig G is 1.0mg/ml,the best coating concentration of p GEX-6P-1-Nsp2-TJ120 is 0.8mg/ml,the sensitivity is 1:80.Compared with that of the IDEXX commercial kit,the overall coincidence rate of serum antibody detection of unimmunized TJM-F92 vaccine and inactivated vaccine of this method was 79%,which can identify the field of wild antibodies and TJM-F92 vaccine antibodies.In summary,indirect ELISA and colloidal gold immunochromatographic methods were established to identify PRRSV wild virus antibody and TJM-F92 vaccine strain antibody in this study.In the pig farms with TJM-F92 vaccine strain,p ET28a(+)-PRRSV-N was used to detect the negative and positive of PRRSV antibodies,and p GEX-6P-1-Nsp2-TJ120 was used to identify the vaccine antibodies and wild virus antibodies in the above positive antibodies.Comprehensive analysis of the two results can accurately identify the immune effect and provide technical support for the prevention and control of the disease.