Establishment Of A Competitive ELISA Detection Method Based On African Swine Fever Virus P72 Protein Nanobodies

African swine fever(ASF)is a contagious swine infectious disease with high lethality.The World Organization for Animal Health(OIE)lists it as one of the legally reported severe infectious diseases with a mortality rate of up to 100% It poses a huge threat to the global pig industry.It poses a huge threat to the global pig industry.In 2018,African swine fever was introduced to China.Due to the lack of effective vaccines,it has caused serious economic losses to China's pig industry.African swine fever virus(ASFV),the causative agent of the disease,belongs to the African swine fever virus genus.ASFV is a large double-stranded DNA virus with a complex structure.Its entire genome is between 170-193 kb,and different strains contain about 151-167 genes.Nanobody(Nb)is a single-chain antibody that exists in camelids and naturally lacks the first constant regions of light and heavy chains.The antibody has the advantages of small molecular weight,stable structure,good solubility,strong specificity,easy genetic engineering modification and low production cost.In recent years,it has been widely used in the diagnosis and treatment of human and animal diseases.Compared with traditional antibodies,it has some irreplaceable advantages.However,the application of Nanobodies in the diagnosis and prevention of ASFV has not been reported.In this study,the prokaryotic expression system was used to express the p72 protein of ASFV,and then it was immunized with Bactrian camel,and the anti-p72 protein nanobody was obtained by phage display technology.Subsequently,the fusion protein of anti-p72 protein nanobody and horseradish peroxidase(HRP)was prepared,and the fusion protein was used to establish a competitive ELISA method for rapid detection of anti-ASFV antibody in pig serum.The main results obtained in this study are briefly described as follows:1.Prokaryotic expression and purification of African swine fever virus p72 proteinWith reference to the p72 gene sequence of ASFV Chinese isolates on Gen Bank,the prokaryotic expression vector p ET-28a-ASFV-p72 of ASFV-p72 protein was successfully constructed by gene synthesis,PCR amplification,enzyme digestion,ligation and transformation.Then,IPTG induced expression,ultrasonic lysis and nickel column affinity chromatography purification,SDS-PAGE showed that the expected recombinant recombinant p72 protein of 75 k Da was obtained,and the protein existed in the form of inclusion bodies.Western blot analysis showed that the expressed recombinant p72 protein It can react with ASFV positive pig serum and has good antigenicity.2.Construction of anti-African swine fever virus p72 protein nanobody library and screening of specific nanobodiesThe expressed p72 protein was immunized into Bactrian camel.After 5 immunizations,indirect ELISA was used to detect anti-p72 protein-specific antibody titer in the serum of immunized camel of 1: 256000.After collecting peripheral blood from camels,isolating lymphocytes,extracting total RNA,and transforming E.coli TG1 cells by nested RT-PCR,enzyme digestion,ligation and electroporation,we successfully constructed a 3.8 × 107 and rich diversity Nanobody Phage display library.Subsequently,using the purified p72 protein as the coating antigen,using phage display technology,after 3 rounds of panning,9 strains of specific anti-ASFV-p72 recombinant protein specific Nanobodies were successfully screened and named Nb1-Nb9.Indirect ELISA analysis showed that Nanobody Nb4 had the highest affinity for recombinant p72 protein.3.Competitive ELISA for detection of anti-ASFV antibodies in pig serum based on Nanobody-HRP fusion proteinBased on the rapid expression platform of Nanobody and HRP fusion protein established in the early stage,p EGFP-N1-p72-Nb4-HRP eukaryotic expression vector was constructed by enzyme digestion,ligation and transformation.After transfection into HEK293 T cells,immunofluorescence and direct ELISA The results showed that the fusion protein of Nanobody Nb4 and HRP was successfully secreted and expressed in HEK293 T cells.The affinity verification of the fusion protein revealed that its affinity titer with p72 protein reached 104.The optimal antigen coating amount of the established competitive ELISA was 80 ng / well,the optimal dilution of fusion protein was 1: 100,and the optimal dilution of pig serum to be tested was 1:20.The Cut-off value is 30.8%.Sensitivity,characteristic and repeatability analysis found that the established competitive ELISA method has high sensitivity,strong specificity,good repeatability and simple operation.It has a 93.33% overall coincidence rate with commercial kits in clinical testing.In summary,based on the advantages of Nanobodies,this study successfully screened and obtained 9 specific Nanobodies against ASFV-p72 protein,and subsequently successfully prepared anti-ASFV Nanobodies and HRP using Nanobody-HRP fusion protein expression platform Fusion protein,and use the fusion protein to quickly establish a competitive ELISA method for detecting anti-ASFV antibodies in swine serum.This method has the advantages of simple operation,strong sensitivity,high specificity and low production cost.Surveillance and prevention and control of the disease laid the foundation.