Preparation And Application Of Tag-free Recombinant K205R Antigens For African Swine Fever Antibody Detection

Currently,control of African swine fever(ASF)relies solely on the stamping out policy and biosafety measures due to lack of safe and effective vaccine.Therefore,the quick and accurate diagnosis is very important.Serological detection is important for ASF diagnosis and epidemiological investigation.OIE recommended tests for ASFV antibody detection include enzyme-linked immunosorbent assay(ELISA),indirect immunofluorescent assay(IFA)and Western blotting.The OIE-recommended ELISA uses the purified antigen from virally infected cells and thus can be performed in the reference laboratories only.There are at least three ELISA kits are available in the international market,the supply of which is limited and expensive with lower detection sensitivities.Although the expression of ASFV recombinant antigens has been reported in China,most of them have the His-tag after purification,which can cause the cross reaction with the immune serum of His-tagged subunit vaccine-immunized pigs.In this study,we constructed two vectors for fusion expression of ASFV K205R protein with elastin-like polypeptide(ELP),purified them by inverse transition cycling(ITC)and cleaved ELP tag with active inclusion bodies of tobacco etch virus(TEV)protease,aiming at simplifying the purification process and avoiding cross reaction of purification tag.By using two different directions,two vectors,namely pELP-K205R and K205R-ELP,were constructed for expression of ELP-K205R fusion proteins.After transforming into BL21(DE3)E.coli,the expression of fusion proteins was induced with 0.1mM IPTG at 20?.SDS-PAGE analysis showed that both ELP-K205R and K205R-ELP fusion proteins were expressed correctly as soluble proteins.The optimization experiment showed that the transition temperature of ELP-K205R protein was 28? in the presence of 3M NaCl and 0.5%Triton X-100.After one cycle of ITC,ELP-K205R protein was purified to more than 80%purity with a yield of 35mg/mL.The ELP tag could be cleaved completely from ELP-K205R fusion protein,but not from K205R-ELP fusion protein.After additional cycle of ITC,the recovered K305R protein had more than 90%purity,which could be recognized by ASFV antibody in LISA and Western blotting assays.By systemic optimization,recombinant K205R antigen ELISA was established for ASFV antibody detection.The optimal conditions for antigen coating was 5 ?g/mL K205R protein for 2h at 37? or overnight at 4?.The blocking condition included incubation for 2h a 37? with 5%defatted milk powder and 0.05%Tween 20 in PBS.The optimal condition for detection serum was 1:200 dilution in blocking buffer for 30min at 37?.The optimal condition for HRP-conjugated goat anti-pig IgG was 1:10000 for 30 min at 37?.Detection of 24 ASFV antibody-negative sera showed that recombinant K205R antigen ELISA had a cutoff value of 0.066±0.09 according to OD450 detection.By parallel diction with 116 pig serum samples,the K205R antigen ELISA had 100%detection agreement with ASFV multi-antigen ELISA kit.By mixing the K205R antigen with recombinant p54 and B602L antigens,an immune blotting strip was established.Among 116 serum samples detected by ELISA,48 were detected to be ASFV antibody positive by the immune blotting strip with an overall detection agreement of 98.3%.These data suggest the tag-free recombinant K205R antigen can be used for ASFV antibody detection.