Preparation And Application Of Tag-free Recombinant P54 And B602L Antigens For African Swine Fever Antibody Detection

African swine fever(ASF)serological diagnosis methods include immunofluorescent test,enzyme-linked immunosorbent assay(ELISA)and Western blotting.Among them,ELISA antibody detection method includes World Animal Health Organization(OIE)recommended ELISA(OIE-ELISA)and commercial ELISA kits.OIE-ELISA uses the purified antigen from virally infected cells and thus can be performed in ASF reference laboratories only.Both supplication and detection sensitivity of commercial ELISA kits can not meet the need of practical use.Both p54 and B602L are the immunodominant antigens of ASFV.Although their expression has been reported in China,these recombinant p54 and B602L antigens are expressed His-tagged proteins which require expensive nickel affinity column for purification with cross-reaction with serum samples from His-tagged subunit vaccine-immunized pigs.In this study,both p54 and B602L proteins were expressed as elastin-like polypeptide(ELP)fusion proteins and ELP tag was removed using active inclusion bodies of tobacco etch virus(TEV)protease,aiming at simplification of recombinant protein purification and avoiding cross reaction of fusion tag.First,p54 and B602L genes of African swine fever virus(ASFV)were cloned individually into ELP fusion expression vector in two different orientations,and the recombinant vectors pELP-P54,pP54-ELP,pELP-B602L and pB602L-ELP were transformed into E.coli.The expression of fusion proteins was induced with IPTG.SDS-PAGE analysis showed that ELP-P54 and ELP-B602L fusion proteins were efficiently expressed,but not P54-ELP and B602L-ELP fusion proteins.The transition temperatures of ELP-P54 and ELP-B602L fusion proteins were 28? and 26?,respectively.In the presence of 0.5%Triton X-100 and 2 M NaCl,the two fusion proteins were purified to 90%or 85%purity by inverse transition cycling(ITC).After incubation with TEV protease inclusion body,the cleavage efficiencies of the two fusion proteins were up to 95%.After an additional cycle of ITC,the recovered tag-free p54 and B602L proteins had more than 90%purities,which could be recognized by ASFV antibody-positive serum.The chess-board titration assay showed that the two recombinant proteins reacted positively with the antibody-positive serum and negatively with the negative serum with a good linear relationship between OD450 values and serum dilutions.Then,mixed antigen ELISA was established by mixing tag-free recombinant p54 and B602L antigens with tag-free recombinant K205R antigen prepared in our research group.The detect result of 47 serum samples from pigs survived from ASFV infection showed that 45 samples were ASFV antibody positive with a detection sensitivity of 96%,which was consistent with that of recombinant p54 antigen dipstick.In contrast,only 23 serum samples were detected to be ASFV antibody positive by Ingenasa ELISA kit with a detection sensitivity of 49%and 4 suspicious samples.These data demonstrated that tag-free recombinant p54 and B602L antigens can be used to detect ASFV antibodies.