Expression Of P30 Protein Of African Swine Fever Virus Mediated By Potato Virus Y In Nicotiana Benthamiana

With the continuous innovation of genetic engineering technology,the transient expression system of foreign genes mediated by plant virus vector has developed rapidly in recent years,which has broad prospects for research and development due to its fast expression speed,high expression quantity,good safety,and can be produced on a large scale.African swine fever(ASF)is a serious swine infectious disease caused by African swine fever virus(ASFV)infection,and the fatality rate can reach 100%.Due to the complexity of the virus and the existence of multiple genotypes,no effective vaccines and drugs have been developed,which has exerted a significant impact on the development of animal husbandry and animal food safety.Therefore,the establishment of rapid and accurate diagnostic methods for ASFV can effectively prevent the spread of the disease.The ASFV p30 protein is an early membrane protein,which is highly expressed in the early stage of virusl infection,and can stimulate the body to produce a high level of antibody response.It can be used as an ideal antigen for serological diagnosis.In this study,the p30 recombinant protein was expressed in Nicotiana benthamiana by using Potato virus Y(PVY)mediated plant transient expression system.An indirect ELISA method for the detection of ASFV antibody was established using the expressed recombinant protein p30 as the antigen.The results are analyzed as follows:1.The PVY vector plasmid was connected with the optimized p30 gene by the In-fusion kit,and the PVY-p30 recombinant virus plasmid was successfully constructed.The plasmid was inoculated into Nicotiana benthamiana by friction.2.Observed regularly the inoculated Nicotiana benthamiana.It was found that the inoculated plants showed the same symptoms as wild type PVY infection.RT-PCR analysis showed that p30 gene could be correctly transcribed in plant virus genome;Indirect ELISA analysis showed that the p30 recombinant protein began to express at the first week of virus inoculation,and the expression level of the recombinant protein was highest at the fifth week of inoculation;Western-blot analysis showed that p30 recombinant protein reacted specifically with ASFV pig positive serum,it has good reactogenicity.The p30 recombinant protein expressed in Nicotiana benthamiana was purified by Ni-NTA-agarose affinity chromatography column.Western-blot analysis showed that the purified p30 recombinant protein could be specifically recognized by the p30 protein monoclonal antibody.3.An indirect ELISA method was established for the detection of ASFV antibody by using purified p30 recombinant protein as the coated antigen.The optimal reaction conditions are: The antigen concentration was 0.5 μg/m L,coated at 37℃ for 1 h;5% skim milk closed at 37℃ for1h;The dilution ratio of serum to be tested was 1:400,and incubate at 37℃ for 0.5 h;HRP-conjugated secondary antibody was incubated at 37℃ for 0.5 h;TMB color developed at room temperature and protected from light for 10 min.50 ASFV negative serum samples were tested,and the critical value of negative and positive was 0.461.Evaluate the performance of the detection method established in this experiment.When the dilution ratio of ASFV positive serum is 1:2560,it can still be determined as positive;The method established in this experiment has no reaction with the positive sera of other common diseases of pigs,and can specifically detect ASFV antibodies;The intra-assay and inter-assay variability coefficients were less than 10%.The method established was used to detect 214 clinical pig serum samples in parallel with the ASFV detection kit,and the overall coincidence rate was 85.0%.In this experiment,the indirect ELISA method for the detection of ASFV antibody based on the p30 recombinant protein expressed in Nicotiana benthamiana as antigen has high clinical application value.