Establishment Of An ELISA Based On The MGF110-5L-6L Prokaryotic Protein For Detecting Antibody Against African Swine Fever Virus

African swine fever（ASF）is a highly lethal hemorrhagic disease caused by African swine fever virus（ASFV）.Since 2018,the outbreak and prevalence of ASFV in China has caused serious economic losses to the entire industry.ASFV is a DNA virus with 170 to 193 thousand base pairs（kbps）,containing 150 to 167 open reading frames,encoding 150 to 200 different proteins.Currently,there is no effective vaccine or diagnostic method to prevent and control African swine fever,which seriously hinders the development of the pig industry.The detection volume of African swine fever is large,and the market requires more sensitive and accurate detection techniques,The serological detection of African swine fever plays a key role in the diagnosis process.Therefore,this study established an indirect ELISA method for detecting ASFV antibodies using ASFV MGF110-5L-6L protein as the coating antigen.The MGF110-5L-6L protein of African swine fever virus has high immunogenicity.According to the MGF110-5L-6L gene sequence of Anhui strain of ASFV（Gen Bank:MK128995.1）published by NCBI,this study conducted bioinformatics analysis on its gene sequence by using transmembrane region,signal peptide,DNASTAR Protein and other software,Intercept the amino acids at positions 105～205 of the dominant gene segment with no signal peptide,good hydrophilicity and Antigenicity,And add 6 downstream×His tag was cloned into the prokaryotic expression vector p ET-32a（+）and a fusion protein expression plasmid p ET32a-MGF110-5L-6L was constructed.The recombinant plasmid p ET32a-MGF110-5L-6L was transformed into the competent state of Escherichia coli BL21（DE3）,and the inclusion body protein MGF110-5L-6L was induced to express by IPTG.The highest protein expression was induced at 37°C for 6 h.The protein concentration was 1.50 mg/m L after purification by Ni-Sepharose affinity chromatography.The Western blot analysis showed that the protein had good reactivity.New Zealand rabbits were immunized with a mixture of refolded MGF110-5L-6L truncated protein and an immune adjuvant in equal amounts.Three immunizations were conducted to prepare rabbit anti p MGF110-5L-6L polyclonal antibodies with a titer of1:512000.293T cells were transfected with p CAGGS-EGFP-MGF110-5L-6L full-length gene recombinant plasmid.Western blot detection and indirect immunofluorescence test showed that p MGF110-5L-6L polyclonal antibody could specifically recognize MGF110-5L-6L protein,which proved that the prokaryotic expression protein had good Antigenicity,laying a foundation for further research on the function of MGF110-5L-6L protein and establishing an ELISA antibody detection method for ASFV.In this study,an indirect ELISA method was established using MGF110-5L-6L protein as the coating antigen and optimized.The optimal antigen coating concentration and serum dilution were determined using chessboard titration.The results were determined based on positive（P）/negative（N）≥2.1.Finally,the optimal antigen coating concentration was determined to be 4μg/m L,and the serum dilution was 1:400;The coating buffer is Na₂CO₃-Na HCO₃ buffer,and the coating conditions are incubation at 37℃for 1 hour in a incubator,and coating at 4℃overnight;The blocking solution was 3%BSA and incubated at 37℃for0.5 h;The dilution of the enzyme labeled second antibody is 1:2500,and the optimal incubation time for both serum and second antibody is 1 hour;The optimal reaction time for TMB color developing solution is 15 minutes.Calculate the critical value based on the S/P value.When the sample S/P value is≥0.3450,the sample is positive;When the S/P value is<0.3450,the sample is negative.This method does not cross react with the positive serum of PRRSV,PCV2,CSFV,and PRV,and can detect positive serum with a dilution of 1:6400.The coefficient of variation（CV）within and between batches is<10%,indicating that this detection method has good specificity,sensitivity,and repeatability.The indirect ELISA method established in this test was used to detect 149 clinical samples,and compared with the commercial African swine fever virus antibody detection kit,the result showed that the coincidence rate was 97.99%,indicating that the indirect ELISA method established in this paper has good application value.In summary,this study established an indirect ELISA method for detecting antibodies to African swine fever virus based on MGF110-5L-6L protein.This method has good specificity,sensitivity and repeatability,and has a good coincidence rate with commercial kits.The indirect ELISA antibody detection method established in this study provides technical support for the prevention and control of African swine fever.