Development Of The CELISA And Identification Of The Linear Epitopes Based On The Nb-HRP Of African Swine Fever Virus Structural Protein P54

African swine fever(ASF),which is caused by the ASF virus(ASFV),a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and brings huge economic losses to world swine industry.ASF has been classified as a notifiable disease by the World Organization for Animal Health(WOAH).ASFV is an enveloped icosahedral virus containing a double-stranded DNA genome of 170-194 kb.In the absence of an effective vaccine,control of ASF must rely on early,effective and costeffective detection and rigorous control and elimination strategies.The p54 protein encoded by E183L is an attractive candidate antigen for ASF detection and vaccine development.However,traditional serological detection methods are generally costly,complex and technically demanding.Nanobodies(Nb)are a promising alternative to traditional antibodies as a diagnostic tool.It has the advantages of small molecular weight,simple preparation,rapidity,good stability and solubility,high affinity and specificity,and the ability to recognize hidden epitopes that are not recognized by ordinary antibodies.Currently,the Nbs in ASFV antibody assay has not been reported.Based on the above research background,this study used phage display technology to screen for specific nanobodies against In this study,we used phage display technology to screen specific nanobodies against ASFV p54,and established a competitive enzyme-linked immunosorbent assay(cELISA)for the rapid detection of ASFV-specific antibodies in pig serum.In this study,a cELISA was developed for the rapid detection of ASFVspecific antibodies in porcine sera using nanobodies.The discovery of a highly conserved natural B-cell epitope in ASFV gene type Ⅱ provides a necessary basis for future vaccine design and p54 as an effective diagnostic tool.The results of this study are as follows The main contents and results of this study are as follows:1.The prokaryotic recombinant plasmid pET-30a-p54 was successfully constructed and expressed in the E.coli(Transetta DE3)by IPTG.SDS-PAGE showed that it was successfully expressed(16 kDa)and soluble.Then,the p54 recombinant protein was purified by affinity chromatography on a nickel column with a high purity of more than 90%and a yield of about 25 mg/L.Western blot showed that the purified p54 recombinant protein reacted with inactivated ASFV antibody-positive pig serum and had good antigenic reactivity.2.Four-year-old male Bactrian camel was immunized with soluble p54 recombinant protein combined with Fredrin adjuvant.After five immunizations,the antibody titer reached 1:256000,indicating that soluble p54 recombinant protein had good immunogenicity.The anticoagulant blood of Bactrian camels was collected,PBMC was isolated,total RNA was extracted and cDNA was reverse-transcribed.The heavy chain antibody variable region(VHH)gene was amplified by nested PCR and cloned into phage vector pCANTAB 5E.The VHH library was successfully constructed by electrotransformation into TG1 E.coli.cells.The storage capacity is 4.25×108 CFU/mL.A total of 13 strains of p54 specific nanobodies were obtained by three rounds of bio-panning using phage display technology.Indirect ELISA analysis showed that Nb8 and Nb83 showed high affinity and specificity for the p54 recombinant protein.3.Overlap PCR was used to fuse the human Ig kappa(Hm Ig κ)chain and codonoptimized horseradish peroxidase(HRP),and cloned them into pCAGGS-HA vector to construct pCAGGS-Nb8-HRP/Nb83-HRP recombinant plasmid successfully.The Nb8HRP and Nb83-HRP fusion protein was transfected into HEK 293T cells,and the protein(65 kDa)was successfully expressed by Western blot and IFA analysis.Indirect ELISA results showed that the affinity and specificity of Nb8 to p54 recombinant protein were much higher than Nb83.Subsequently,HEK 293T cell line expressing Nb8-HRP fusion protein was established by lentivirus expression system.By optimizing the reaction conditions,a rapid and low-cost cELISA for the detection of ASFV-specific antibodies in pig serum was established using Nb8-HRP fusion protein as a probe for the first time.According to ROC analysis,the optimal critical value of cELISA was 52.5%.There was no cross reaction with positive sera of porcine pseudorabies virus(PRV),porcine epidemic diarrhea virus(PEDV),swine fever virus(CSFV)and porcine reproductive and classical swine fever virus(CSFV)and porcine reproductive and respiratory syndrome respiratory syndrome virus(PRRSV).A total of 209 serum samples were tested using the developed cELISA and the commercial ELISA kits.The results showed that the relative specificity and sensitivity of cELISA were 98.97%and 93.3%,respectively,and the consistency rate of the two ELISA methods was 98.56%.4.13 pCAGGS-Nbs-HRP recombinant plasmids were successfully constructed by using Overlap PCR with human Igκ strand and codon-optimized HRP gene fusion.After transfection into HEK 293T cells,13 Nbs-HRP fusion proteins(65 kDa)were successfully expressed as identified by Western blot and IFA.Direct ELISA showed that Nbs-HRP fusion protein was successfully secreted into the supernatant.Western blot and indirect ELISA showed that Nb8 had the best reaction with p54 recombinant protein expressed in E.coli.IPMA and IFA results showed that Nb8-HRP fusion protein could specifically bind to ASFV Pig/HLJ/18 infected cells.The epitope of p54 was then identified using Nb8.Dot blot,ELISA,Western blot and IFA showed that Nb8 could recognize the truncated p54-T1 protein expressed in eukaryotic cells.Six overlapping short peptides spanning p54-T1 were subsequently synthesized.Dot blot and peptidebased ELISA revealed a previously unreported minimal linear B cell epitope 76QQWVEV81.ELISA results showed that it could react with the positive serum of inactivated ASFV antibody from naturally infected pigs,indicating that it was a natural linear B cell epitope.Alanine scanning mutagenesis results indicated that 76QQWV79 was the core binding site of Nb8.Bioinformatics analysis of the 76QQWVEV81 epitope showed that it was highly conserved in the genotype Ⅱ ASFV strains.The structural prediction results showed that the identified epitope was located inside p54 and had a high antigenic index and hydrophobicity.It may be a component of the α helix and β fold,indicating that 76QQWVEV81 May be a key B-cell epitope on p54 protein of the genotypeⅡ ASFV strain.In conclusion,we obtained ASFV p54 specific nanobodies for the first time and provided useful tools for further study of the structure and function of p54.Using Nb8HRP fusion protein as a probe,we established cELISA with low cost,good specificity,sensitivity and repeatability for the detection of anti-ASFV antibodies in pig serum,which provided a new method for the detection of anti-ASFV antibodies in pig serum and laid a foundation for the prevention and control of the disease.The discovery of 76QQWVEV81,a previously unreported minimal linear epitope of natural B cells,provides a theoretical basis for the further development of Nbs into effective anti-ASFV therapeutic drugs or diagnostic reagents and provides valuable information for the design of new vaccines.