Establishment Of Indirect ELISA Antibody Detection Method Based On African Swine Fever Virus C-type Lectin Protein

African swine fever virus(ASFV)can infect domestic pigs and wild boars,causing acute,hot and highly lethal hemorrhagic infectious diseases,namely African swine fever(ASF),which causes huge economic losses to pig breeding industry in China and other countries in the epidemic area.At present,the detection of ASFV pathogenic gene or its antigen(antibody)in pigs is an important means to determine whether swines are infected,and serological detection methods can be used for ASF surveillance screening and auxiliary diagnosis in swines,The C-type lectin protein encoded by ASFV is a multifunctional protein expressed in the early and late stages of viral infection,and has good immunogenicity.In this study,the sequence analysis and antigenic epitopes prediction of C-type lectin were performed by bioinformatics software.BALB / c mice were immunized with C-type lectin expressed in mammalian system(HEK-293 T cells)to prepare polyclonal antibody against C-type lectin and evaluate its antigenicity.Finally,the indirect ELISA method for detecting ASFV Ig G antibody was established by optimizing the reaction conditions with C-type lectin as coating antigen.The results are as follows:1.Bioinformatics analysis of C-type lectin encoded by EP153 R gene.Homology analysis and antigen epitope prediction were performed on the nucleotide sequences of C-type lectin from 10 representative ASFV isolates in China.The results showed that the nucleotide sequence similarity of C-type lectin was 95%-100%,and the C-type lectin antigen linear epitopes B(KKYIGLINKKEGLKKK)and D(NEEKNYNN)were highly conserved(100% similarity).The extracellular amino acid sequences of C-type lectin of 79 ASFV strains(10 domestic sequences and 69 foreign sequences)and the corresponding extracellular amino acid sequences of CD2 v strains were analyzed.The phylogenetic tree was constructed by neighbor-joining method using MEGA11.0 software.The phylogenetic tree based on the extracellular domain of C-type lectin showed that the strains of different serum groups were located in different branches,and the amino acid homology in the same branch was 88% –100%.The phylogenetic tree was constructed based on CD2 v and C-type lectin extracellular regions.The ASFV epidemic strains in China were all located in the same branch with the known serum group SG8 strain,and the amino acid similarity within the branch reached 99%.2.Immunogenicity of ASFV C-type lectin protein.In this study,293 T cells were used to express the extracellular domain protein of ASFV C-type lectin,and the C-type lectin protein was mixed with the adjuvant in proportion.BALB/c mice at the age of 7 weeks were immunized once every two weeks,and a total of three immunizations were carried out to explore its immunogenicity.The C-type lectin protein was used as the coating antigen,and the antibody level of mouse serum was detected by ELISA.The results showed that the Ig G antibody level in the protein-immune group was significantly higher than that in the non-immune group,and the antibody titers of the five mice reached 1:64000.The PK-15 cells transfected with the C-type lectin plasmid were detected by IFA.The specific fluorescence of the mouse C-type lectin antibody group showed that C-type lectin had good immunogenicity.The C-type lectin polyclonal antibody prepared in the experiment can lay an experimental foundation for subsequent related studies.3.Establishment and preliminary application of indirect ELISA for C-type lectin protein.The ELISA Ig G antibody detection system was established and optimized with Ctype lectin protein as the coating antigen.The optimal coating concentration of antigen protein was 4 ng/μL,and 1% BSA was the optimal concentration of blocking solution.The optimal dilution multiple of serum(primary antibody)was 1:100,and the optimal reaction time was 15 min.The dilution multiple of enzyme-labeled antibody(secondary antibody)was 1:6000.The optimal reaction time of serum antibody was 30 min,and the optimal reaction time of substrate was 5 min.The critical value of 26 serum samples of ASFV-negative pigs was 0.2982.The further experimental results showed that the ELISA method had good specificity and repeatability,and the coefficients of variation within and between batches were less than 10%.The results of clinical samples showed that the coincidence rate with French ID-Vet kit was 98.18%(54/55),the positive detection rate was 97.92 %(47/48),and the negative coincidence rate was 100%(7/7),which provided a new method for serological diagnosis of ASFV infection.