Preparation And Application Of Antibodies Against ASFV Immune Protection Related Antigens

African swine fever（ASF）is a highly contagious swine viral disease with a mortality rate close to100%in domestic pigs.ASF is caused by a large double-stranded DNA virus ASF（African swine fever virus,ASFV）.The natural hosts of this virus include domestic pigs,wild boars and arthropod（ticks from the genus Ornithodoros）.ASFV infection is occasionally asymptomatic and develops into a persistent infection.In contrast,infecting domestic pigs usually develop fatal hemorrhagic fever.ASF was first diagnosed in Shenyang,China in August 2018,and then rapidly spread to more than 30provinces and cities,causing the most severe economic losses to the pig industry.As no effective vaccine available so far,detection of ASFV infection and biosecurity measures were taken into account for prevention and control of the disease.In this study,the prokaryotic expression system was used to express ASFV p17,p30,p54,and p72proteins,and using the purified recombinant proteins as immunogens,polyclonal antibodies to each of the four proteins and a monoclonal antibody against p54 protein were developed.Using purified p54protein and anti-ASFV p54 protein monoclonal antibody,an indirect ELISA and a competitive ELISA for detection of antibodies to ASFV were established.1.Prokaryotic expression of ASFV genes encoding p17,p30,p54 and p72 protein and preparation of polyclonal antibodiesRefer to sequence of the ASFV strain SY18（Gen Bank:MH713612.1）,the D117L gene（coding for p17）,CP204L gene（coding for p30）,E183L gene（coding for p54）,and B646L gene（coding for p72）were synthesized and ligated into the p UC57-simple and p IRES vector.Then,the four genes were amplified by polymerase chain reaction,and sub-cloned into prokaryotic expression plasmids p Cold-I,p ET-30a,or p Cold-TF,respectively,generating recombinant plasmids p Cold-I-p30,p Cold-I-p72,p ET-30a-p54-JD,and p Cold-TF-p17-JD.BALB/C mice were immunized with the four purified proteins p30,p72,p54（54-185aa）,p17（61-118aa）,respectively,and the polyclonal antibodies to each of the four proteins were tested by Western blot and IFA with good reactivity and specificity.2.Preparation of anti-ASFV p54 protein monoclonal antibody and identification of its target epitopeThe mice were immunized with purified p54 protein three times,the spleen cells from mouse with higher titer of antibodies were selected and fused with SP2/0 cells.The monoclonal antibody against p54 protein was screened by indirect ELISA.The results of Western blot and IFA showed that monoclonal antibody 1H9 could specifically recognize p54 protein.The target epitope recognized by1H9 was further identified by Western blot and ELISA with a series of fragmentated p54 protein as¹⁷⁵YTHKDLENSL¹⁸⁴.3.Establishment of indirect and competitive ELISA methods for for detection of ASFV p54 protein antibodyIn the study,an indirect ELISA method was established with purified p54 protein as coating antigen.A competitive ELISA method was also established with purified p54 protein as coating antigen and monoclonal antibody 1H9 as competitive antibody.Both methods were used for detecting ASFV antibodies and showed good specificity and reproducibility.The above results provide important diagnostic tools for monitoring of ASFV infection.