Production And Epitope Mapping Of Monoclonal Antibodies Against HP-PRRSV Nsp2 And Nsp12

Porcine reproductive and respiratory syndrome（PRRS）is an economically important infectious disease of swine and is characterized by reproductive failure and respiratory disease both in sows and in pigs of all ages.The disease was first reported in the USA in the late 1980s and it has become one of the most important diseases in the swine industry.The causative agent of this disease,PRRS virus（PRRSV）,was first identified in 1991 in the Netherlands,and then in the USA in 1992.PRRSV is a positive-sense RNA virus,which is classified into the genus Arterivirus.ORF 1a and ORF1b encode replicase polyproteins pp1a and pp1ab,which are autoproteolytically cleaved into fourteen non-structural proteins（Nsps）:Nsp1α,Nsp1β,Nsp2/3/4/5/6,Nsp7α,Nsp7βand Nsp8/9/10/11/12.Nsp2 is unique due to its largest size,genetic heterogeneity,its participation in diverse roles in supporting the viral replication cycle,and its packaging within the PRRSV virion.Nsp12 is essential in virus replication.However its function is still unknown.The cDNAscorresponding to Nsp2（1-233aa）and Nsp12 of HP-PRRSV HuN4 were amplified by Polymerase Chain Reaction（PCR）and cloned into the pET-28a vector and then named as pET-28a-Nsp2（1-699bp）and pET-28a-Nsp12.Then two recombinant proteins,named His-Nsp2（1-233aa）and His-Nsp12were expressed and purified as antigens to immune the BALB/C mice.Three Monclonal antibodies（MAbs）against PRRSV Nsp2,named 4A12,4G8,8H11 and one MAb against PRRSV Nsp12,named 1E5 were then produced and screened out.Their antigenicities were tested by Western blot and IFA analysis.Our findings showed that 4A12,4G8,8H11 can recognize Flag-Nsp2（1-233aa）expressed in HEK293 cells and HP-PRRSV HuN4 Nsp2 and 1E5 can recognize Flag-Nsp12 expressed in HEK293 cells and HP-PRRSV HuN4 Nsp12.The B cell epitopes recognized by 4A12 and 1E5 were then defined by Western blot and ELISA.The Results showed that the B cell epitope of Nsp2 recognized by 4A12 is ¹⁸⁸ELSDDSNRPV¹⁹⁷ and the B cell epitope of Nsp12 recognized by 1E5 is ¹³⁰KANATSMRFH¹³⁹.An analysis was used to assess the conservation of epitopes among different strains published in NCBI.Our data suggest that¹⁸⁸ELSDDSNRPV¹⁹⁷ is relatively conserved among some HP-PRRSV isolates of type 2 PRRSV.And¹³⁰KANATSMRFH¹³⁹ is highly conserved among some HP-PRRSV strains of type 2 PRRSV,but not the classical PRRSV strains of type 2 PRRSV or the isolates of type 1 PRRSV.Taken together,4 MAbs against HP-PRRSV HuN4 Nsp2 and Nsp12 were prepared by cell fusion,which provides the foundation to further study the biological functions of Nsp2 and Nsp12.And a precisely method was used to map the B epitopes of on the HP-PRRSV HuN4 Nsp2 and Nsp12.We found that 1E5will be used to establish a new method to test HP-PRRSV infection,and has important significance for PRRSV phylogenetic analysis and epidemiological investigation.