Preparation Of Monoclonal Antibodies Against P72 And P30 Proteins Of African Swine Fever Virus And Establishment Of Blocking ELISA

ASFV is an important pathogen of swine industry,which can cause acute death in domestic pigs.The mortality of ASF can reach 90-100%.In August 2018,a case of African swine fever was reported for the first time in Shenyang,Liaoning Province.Since then,the disease spreads rapidly throughout the country,causing huge economic losses to the swine industry.At present,there is no effective commercial vaccine and treatment for ASF,and its prevention and control mainly depends on early diagnosis.In order to effectively prevent and control the disease,it is urgent to study on the biological function of the virus protein,and develop a sensitive,specific,simple and rapid detection methods.In this study,recombinant p30 and p72 proteins of ASFV were expressed by prokaryotic system,and monoclonal antibodies against p30 and p72 proteins were prepared by immunizing mice with the recombinant proteins.And 6 epitopes of p72 protein were identified by using the12 monoclonal antibodies.And an anti-p72 monoclonal antibody 6E5 was choiced to establish a blocking ELISA for ASFV antibody detection.The blocking ELISA method established in this study has high sensitivity and specificity,therefore it is suitable for ASFV diagnosis and epidemiological investigation.The main contents of this study are as following:1.Preparation and identification of monoclonal antibodies against p30 and p72 proteins of African swine fever virusAccording to the CP204L（p30）and B646L（p72）genes of ASFV pig/HLJ/2018（Gene Bank MK333180）,PCR primers were designed to amplify the p30 and p72 genes fragments from ASFV positive clinical samples,and then cloned into prokaryotic expression vectors p ET-32a（+）and p ET-28a（+）,respectively.The recombinant plasmids were transformed into E.coli BL21,and the recombinant proteins were expressed,identified and purified.The 6-8 weeks old female BALB/c mice were immunized with the purified proteins by three times,and the spleen cells were isolated and fused with SP2/0cells.Fourteen hybridoma cells secreting monoclonal antibody（Mc Ab）against p30 and p72 of ASFV were obtained by indirect ELISA and subcloning.The hybridoma cells of passage 5,10 and 20 could stably secrete the antibodies,and the antibody titers of the cell supernatant arrange from 1:1,600 to 1:12,800.The subtype identification results showed that the heavy chain of 14 Mc Abs belonged to Ig G1 and the light chain belonged to kappa type.Western blot results showed that 12 anti-p72 Mc Abs can reacted with the ASFV and have a specific band at 72 KDa;2 anti-p30 Mc Abs can reacted with the ASFV and have a specific band at 32 KDa.IFA results showed that anti-p72 Mc Abs 6C6 and 6G5 and anti-p30Mc Abs 2B9 and 8b9 could specifically react with ASFV infected PAMs.This study laid an important material support for biological research and diagnostic methods of p30 and p72proteins.2.Identification of linear B cell epitopes of ASFV p72 protein by using the monoclonal antibodiesIn this study,26 truncated p72 recombinant proteins were expressed by prokaryotic expression system.The epitopes of p72 protein were identified by Western blot with 12anti-p72 Mc Abs.A total of 6 new epitopes were identified.The epitope recognized by 6C6and 6G5 Mc Abs was ⁵¹QIEETHL⁵⁷;the epitope recognized by 6F7 and 8G4 Mc Abs was⁶³HFKPYVPV⁷⁰;the epitope recognized by 8B8 and 9H11 Mc Abs was ⁸³TPTLGNKL⁹⁰;the epitope recognized by 6E1 and 10B7 Mc Abs was ¹⁴⁷QTPLEGAVYTL¹⁵⁷;the epitope recognized by 6C1,6E5 and 10D2 Mc Abs was ²⁰⁸TTLVRKFCI²¹⁶;the epitope recognized by 5H11 Mc Abs was ²⁴³CNIHDLHK²⁵⁰.The p72 protein amino acid analysis of the different ASFV genotypes strains showed that the epitopes of ⁵¹QIEETHL⁵⁷,⁶³HFKPYVPV⁷⁰,⁸³TPTLGNKL⁹⁰,¹⁴⁷QTPLEGAVYTL¹⁵⁷and ²⁰⁸TTLVRKFCI²¹⁶ were highly conserved in different ASFV strains.However,²⁴³CNIHDLHK²⁵⁰ epitope showed multiple amino acid variation in Xb and XII genotypes.The variate amino acids were I²⁴⁵V,H²⁴⁶Q,L²⁴⁸M and K²⁵⁰M.The recombinant p72 protein of Xb and XII genotypes was constructed by base point mutation technique.It was found that 5H11 Mc Ab only reacted with genotype II p72 protein.Furthermore,the structural informatics results exproled high antigenic and hydrophilic properties of the ²⁰⁸TTLVRKFCI²¹⁶ epitope,which exposes to the surface of p72 protein and maybe an important linear B cell epitope in p72 protein.It lays a foundation for the study of biological function of the protein.3.Establishment and application of blocking ELISA for detection of the antibody to African swine feverIn this study,the recombinant p72 protein of ASFV was used as detection antigen,and the horseradish peroxidase（HRP）-labeled monoclonal antibody 6E5 was used as blocking antibody.Through the optimization of reaction conditions,a blocking ELISA for the detection of the anti-p72 protein antibody was established.By detecting the antibody in 119ASFV negative serum,the criteria of the blocking ELISA were examined as:the serum sample with the PI≥50%could be judged as positive,the serum sample with PI≤40%judged as negative,and the serum sample with PI between 40%and 50%judged as suspicious.The method has a sensitivity of 94.0%and no cross reaction with PRV,PRRSV,CSFV,PCV2,SVA and FMDV positive serum.This method was used to detect 447 pig serum samples that simultaneously detected by ID Screen（?）African Swine Fever Competition ELISA kit.The coincidence rate of this two methods was 94.9%.In summary,the blocking ELISA method established in this study has high sensitivity and specificity,it is suitable for ASFV diagnosis and epidemiological investigation...