Prokaryotic Expression Of African Swine Fever Virus VP72 Gene And Establishment Of Two Antibody Detection Methods

African swine fever,acute high fever and accompanied by bleeding throughout the body and internal organs,is an acute infectious disease of pigs caused by the African swine fever virus.Once infected,the morbidity and fatality can run 100%.It is classified as a notifiable disease in China and easy to cause misdiagnosis because clinical manifestations are very similar to classical swine fever.The outbreak of african swine fever in Shenyang on August 3,2018 that has spread across the country after three months.This study performed prokaryotic expression and protein purification of ASFV VP72 gene,preparation of rabbit anti-recombinant P72 protein polyclonal antibody,and established an indirect ELISA method for detecting ASFV antibody and a method for detecting ASFV antibody immunochromatographic test strips.1.Prokaryotic expression of ASFV VP72 geneIn this study,uses the prokaryotic expression system to express the protein of ASFV VP72 gene.The expression conditions were optimized,and recombinant proteins were purified and identified for antigenicity.The results showed that the expression protein molecular weight was 125 k D as inclusion bodies.The optimal conditions for inducing temperature was 15?,IPTG induction concentration was 1.0 mmol/L and 5 h.According to the Ni-Agarose His Tag Protein Purification Kit to purify recombinant protein that was1.46 mg/m L.Western Blotting identification results showed that the expressed recombinant protein is the target protein.The results of the double agar diffusion test showed that when the rabbit polyclonal antibody against the ASFV VP72 recombinant protein was 1:16,the precipitation line was still clearly observed.It shows that the recombinant protein expressed in this study has good immunogenicity and antigenicity,and provides a scientific basis for subsequent experiments.2.Establishment of indirect ELISA antibody test methods for ASFVWith the purified P72 protein as a coating antigen was coated,optimize coating conditions,determine the best working conditions of ELISA and standard threshold.The established indirect ELISA antibody detection method will be studied for specificity,reproducibility and compliance tests.Results showed that optimal antigen coating concentration was 1.46 mg/m L and the serum dilution concentration was 1:50.The optimal dilution of enzyme-labeled secondary antibody was 1:4000 and the best color development time of the TMB is 10 min.The standard cut-off value of indirect ELISA:when OD₄₅₀nm?0.3385,it was judged as positive;when OD₄₅₀nm<0.3385,it was judged as negative.Specific test results showed positive reactions with standard ASFV positive sera and other virus standard sera is negative,with indicating good specificity.The results of the repeatability test showed that the coefficients of variation within the batch and between batches were 3.15%?5.741%and 2.753%?5.523%,respectively,indicating that the established indirect ELISA method has good repeatability.The results of the compliance test showed that the comparison of the clinical sample test results with the IDVET commercial kit showed that the coincidence rate of the two was 86.67%(13/15),which was met the expected goal of the test.3.Development and application of colloidal gold immunochromatographic strips for detecting antibody against ASFVIn this study,trisodium citrate reduction method was used to label the purified P72protein on gold-labeled particles(30 nm in diameter)as the capture antigen and the optimal amount of labeled protein and the optimized colloidal gold p H were screened.The rabbit anti-recombinant protein polyclonal antibody-labeled quality control line(C line)and the HRP rabbit anti-pig Ig G labeling detection line(T line)were fixed on the NC membrane,optimization of antibody dosage by T-line and C-lines and specificity and sensitivity tests.The results showed that the color of the prepared colloidal gold solution was burgundy;The optimal labeling p H was 7.5 and the optimal labeling amount was 30?L;The maximum dilution concentration of the T line and the C line was 1:8;The performance test results show that the GICA test strip has good specificity and repeatability;When the ASFV standard positive serum is diluted to 32 times,a weak red line appears on the T line,which proves that the GICA test strip has good sensitivity.The coincidence rates of clinical sample test results with the IDVET commercial kit positive is 80%(12/15).It is great significance for clinical ASFV antibody detection and disease prevention.