Preparation Of Monoclonal Antibody To P30 Protein Of ASFV And Establishment Of Indirect ELISA

African swine fever(ASF)is a highly contagious disease of pigs,caused by African swine fever virus(ASFV).In the case of infection with virulent strains,the mortality rate of infected pigs is extremely high,even up to 100%.ASF is listed in a legally reported animal diseases by OIE,while ASF belongs to a class I of animal diseases in China.Since the first diagnosis of ASF in China in August 2018,the ASF epidemic outbroke in many places,which made a big economic losses in pig industry.At present,there is still a lack of safe and effective vaccines for the prevention and control of ASFV,so the establishment of a rapid and accurately diagnostic method is very important for the prevention and control of the disease.ASFV p30 protein is a structural protein encoded by the open reading frame CP204L in the genome and appears in large amount in the early stage of viral infection.p30 protein plays an important role in the mechanism of virus infection and invasion of host cells.At the same time,p30 protein has good antigenicity,so it is an ideal serological diagnosis and immunological detection antigen.In this study,by constructing eukaryotic and prokaryotic expression plasmids of ASFV p30,monoclonal antibodies specific to p30 protein were developed using prokaryotic expression of recombinant proteins for immunization.It provides basic biological materials for rapid diagnosis methods and immune prevention of ASF.The main research contents results include the following three parts:1.Clone and expression of p30 gene of African swine fever virus.In this study,a pair of specific primers were designed and synthesized according to the p30 gene sequence of ASFV published on GenBank.The target gene was amplified by polymerase chain reaction(PCR)and cloned into the eukaryotic expression vector pcDNA3.1 and the prokaryotic expression vector pET-32a respectively to construct the eukaryotic recombinant plasmid pcDNA3.1-p30 and the prokaryotic recombinant plasmid p30-his.The eukaryotic recombinant plasmid pcDNA3.1-p30 was transfected into 293T cells,and analyzed by indirect immunofluorescence(IFA)and Western Blot.The results showed that the recombinant p30 could be expressed on 293T cells and reacted well with the positive ASFV serum.The prokaryotic expression plasmid p30-his was transformed into BL21(DE3)competent cells,and the expression was induced by isopropyl-?-D-thiogalactoside(IPTG).The results of SDS-PAGE and Western Blot showed that the recombinant protein could be expressed and mainly existed in the form of inclusion bodies,and the molecular weight was about 42 kDa.2.Preparation of monoclonal antibodies against ASFV p30.In this study,BALB/c mice at age of 6-8 weeks were immunized with p30 prokaryotic recombinant protein every 10 days.After three times immunization,the sera titers of mice were tested by IFA.SP2/0 cells were fused with the spleen cells from the immunized mice.The supernatant of hybridoma cells was screened by IFA with the 293T cells transfected with the eukaryotic recombinant plasmid pcDNA3.1-p30.Finally,two cell lines secreting monoclonal antibodies were obtained,named ASFV-p30-2F9 and ASFV-p30-3F9.Western Blot showed that the two monoclonal antibodies obtained can specifically react with p30 prokaryotic and eukaryotic expressed proteins.Both monoclonal antibodies were IgG1 subclass/Kappa chain.IFA titer of ASFV-p30-2F9 monoclonal antibody ascites was 1:6400 in IFA,and ASFV-p30-3F9 was 1:3200.3.Establishment of indirect ELISA for detecting antibody against African swine fever virusUsing the purified p30 recombinant protein as coating antigen,an indirect ELISA for detecting antibody against African swine fever virus was established.All factors related to the indirect ELISA were optimized.The optimal concentration of antigen coating was 0.25 ?g/mL,the dilution of serum was 1:400,the blocking solution and incubation time were 5%skim milk for 2 h.The incubation times for primary antibody and conjugated antibody was 1 h respectively.The color development time was 10 min.This method could detected antibody to ASFV but not antibodies against PRV,PEDV,CSFV.It indicated that this method has good specificity.The cutoff value of ELISA was determined by detecting 12 ASFV negative sera.It was negative when OD450nm?0.284,and positive when OD450nm?0.341.The OD450nm value of the serum to be tested was suspicious between 0.284 and 0.341.The ELISA method had good intra-and inter-batch repeatability,the coefficient of variation was less than 10%.This method will be very useful in surveillance of ASFV infection in pigs.