Prokaryctic Expression Of P54 And PI215L Proteins Of African Swine Fever Virus And Establishment Of Indirect ELISA For Detecting Antibodies

African swine fever(ASF)is a viral infectious disease caused by the African swine fever virus(ASFV),which can infect pigs of all ages and types.It is characterized by fever,respiratory disorders and systemic hemorrhage as significant clinical characteristics,and is the only arbo-DNA virus.After the outbreak,the fatality rate can reach 100%,causing huge economic losses to pig industry.The E183 L gene is about 555 bp in length and encodes the p54 protein which is one of the main structural proteins of the ASFV.It has strong antigenicity and can induce the production of neutralizing antibody.It participates in the process of virus invasion into host cells and the transformation of virus inner envelope,and induces cell apoptosis.The I215 L gene is about 639 bp in length,the p I215 L protein encoded has similar functions to E2 ubiquitin conjugating enzyme.It plays a key role in viral DNA replication and genome transcription.In this study,the p54 and p I215 L proteins were expressed and purified,and their specific polyclonal antibodies were prepared.Using the purified p54 and p I215 L recombinant proteins as the coating antigen,two indirect ELISA methods for the detection of ASFV antibody was established.The specific contents are as follows:1.The prokaryotic expression and identification of ASFV p54 and p I215 L proteinsASFV E183 L and I215 L genes were amplified by PCR and cloned into E.coli expression plasmid p ET-28a(+).Two recombinant plasmids of p ET-28a-p54 and p ET-28a-I215 L were constructed.The recombinant plasmids were transformed into E.coli Rosetta and induced by IPTG.The results showed that the recombinant p54 and p I215 L proteins were soluble.The proteins were purified by affinity chromatography after centrifugation.The purified proteins were identified by SDS-PAGE and western blot.The results showed that the target proteins had good antigenicity.The purified recombinant proteins were used to prepare mouse polyclonal antibodies.The results of ELISA and IFA showed that the anti-p54 and anti-p I215 L serum had good specificity,which provided material support for the study of biological function of the proteins and the establishment of ELISA antibody detection methods.2.Establishment of indirect ELISA antibodies detection methods by ASFV p54 and p I215 L proteinsUsing the above-mentioned purified recombinant proteins of p54 and p I215 L as coating antigens,two indirect ELISA for detection the antibodies against p54 and p I215 L proteins were estalished.The optimized reaction conditions were: the optimal coating concentration of the antigen was 2.0 μg/m L,and the coating time was 37°C for two hours then 4°C overnight,the blocking solution was 0.5% BSA solution,the blocking time was37°C for 3 hours,the best dilution of the serum to be tested was 1:100,the best time for serum to be incubated at 37°C for 30 mins of p54 protein and 1 hour of p I215 L protein,the best dilution ratio of enzyme-labeled SPA was 1:5000 and the best action time was15 mins,the best action time of TMB was 37°C for 10 mins of p54 protein and 5mins of p I215 L protein.Maximum value of P/N were calculated according to the OD450 nm value read by the microplate reader as the criterion after stopping coloring.The critical value of p54-ELISA was determined as follows: the sample was positive when the S/P value tested was ≥ 0.227,and negative when the S/P value was < 0.172,and suspicious when it was between 0.172 and 0.227.The sensitivity of the method was 95%,the coefficient of variation of intra batch and inter batch repeatability was 0.66%~7.34%.The critical value of p I215L-ELISA was determined as follows: the sample was positive when the S/P value was ≥ 0.208,negative when the S/P value was < 0.159,and suspicious when it was between 0.159 and 0.208;the sensitivity of the method was 100%,the coefficient of variation of intra batch and inter batch repeatability was 1.31%~6.57%.There were no cross reactions between the two methods and the positive sera of PCV2,SVA,PRRSV,FMDV,PRV and CSFV.Two methods were used to compare with IDvet commercial kit from France,the coincidence rates of p54-ELISA and p I215L-ELISA were 91.3% and 88.2%.The results showed that p54-ELISA estalished was better than p I215L-ELISA in detecting ASFV serum antibodies.