| Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure of pregnant sows and respiratory distress of piglets and growing pigs. The disease is caused by the PRRS virus. Since the main permissive targets for PRRSV in vivo are cells of the monocytic lineage and the nature of its highly variation, persistent infection and antibody dependent enhancement (ADE), therefore, safe and effective vaccine against the disease is not available. Antigenic epitope information of the virus is not only useful for investigating the relationship between antigenic structure and function of the PRRSV but also useful for diagnosis of PRRSV infection and developing safe and effective multi-epitope vaccine against the disease.In this study, six recombinant plasmids harboring different structural protein of PRRSV CH-1a (designated as pET30a-N, pGEX6p-rtM, pGEX6p-rtGP5, pET30a-GP4, pGEX6p-rtGP3 and pGEX6p-rtGP2) were constructed and expressed in E.coli. The expressed fusion proteins were detected with sera of PRRSV infected pigs by Western-blotting. BALB/c mice were immunized with expressed fusion proteins and monoclonal antibodies (MAbs) were developed. 16 anti-N MAbs, 15 anti-GP5 MAbs, 5 anti-GP3 MAbs, one anti-M MAb, one anti-GP4 MAb and one anti-GP2 MAb were identified, respectively.The ORF7 gene were divided into four overlapping fragments and expressed in E. coli respectively. The reactivity of different fragments expressed in E. coli were probed with 16 anti-N MAbs by ELISA. Only seven out of 16 MAbs could react with ORF7-N2 and ORF7-N3 fusion proteins, others only reacted with complete N protein. The overlapping region (49-70aa) of fragments ORF7-N2 and ORF7-N3 were then divided into seven fragments (NEP1-NEP7). six out of the seven MAbs reacted with fusion peptide NEP3(49-58aa) while MAb N1H11 only reacted weakly with fragment in region 49-70aa, suggesting that the segment 49-70aa is a part of the epitope recognized by N1H11. The NEP3 gene was truncated from both ends and the smallest function unit that recognized by each mAb was identified. The function epitopes recognized by MAbs N2H7 and N2F7 are H54FPLA58 and K52PHFPLA58, respectively. The function epitope recognized by MAbs N1A2, N1E3, N1G4 and N2E5 is E51KPHFP56. Sequence analysis revealed that the overlapping region of three antigenic epitopes are well conserved among 56 isolates of PRRSV (including both North American genotype and European genotype). The Western blot analysis indicated that only NEP3 could be recognized by PRRS positive pig sera, suggesting that the epitope is probably more immunodominant.M protein was expressed in two overlaping fragments and tested for reactivity with MAbs M2B3. The results showed that both fragments could react with MAbs M2B3, suggesting that theepitope recognized by M2B3 located in the overlaping region (112-134aa) of the two fragments. A series of oligoes coding for region 112-134aa were synthesized with truncating from both ends and expressed in a fusion form of peptide, only MEP1(112-134aa), MEP2(117-134aa) and MEP3(112-129aa) could react with MAb M2B3, suggesting that amino acid segment 117-129 is antigenic region recognized by MAb M2B3. Western-blotting analysis indicated that all three fusion peptides could be recognized by PRRS positive pig sera, suggesting that this epitope is also a immunodominant epitope of the PRRSV. Sequence comparison found that this region is relatively conservative among North American PRRSVs.To map epitopes of the PRRSV GP5 protein, the ORF5 gene coding for GP5 was divided into four overlaping fragments. The reactivity of different fragments expressed in E. coli was probed with MAbs by indirect ELISA. The results showed that two MAbs could react with GP5-P4 and GP5-P3, other 13 MAbs only reacted with GP5-P4. In Western blotting, only GP5-P4 could react with PRRS positive pig sera. To map the precise antigenic sites of the GP5-P4, seven fragments based on GP5-P4 were synthesized and expressed in E. coli. Based on reactivity of MAbs to the expressed fusion peptides, 15 MAbs were divided into three groups which recognized peptides GP5EP3(146-156aa), GP5EP5(164-180aa), and GP5EP7(192-200aa), respectively. Further analysis of GP5EP3, GP5EP5 and GP5EP7 with end truncating found that the function epitopes within GP5EP3, GP5EP5, GP5EP7 were R151LYRWR156, E169GHLIDLKRV178 and Q196WGRL200, respectively. In comparison with other North American PRRSVs, we found that L152YRWR156 is highly conservative, while only one amino acid substitution of L200 or P200 in Q196WGRL200. Substitution analysis showed that 5 MAbs could only recognize the peptide with L20 and 4 MAbs could recognize both L200 or P200. In Western blot with sera from pigs infected by PRRSV BJ-4 or CH-la, we found that serum from CH-la infected pig was reactive to GP5EP5 and GP5EP7(L200), while serum from BJ-4 infected pigs could recognize GP5P5 and mutant GP5EP7(P200).With the same procedure, antigenic epitopes of GP3 were investigated. The ORF3 gene coding for was divided into two overlaping fragments. The reactivity of different fragments expressed in E. coli was probed with five MAbs by indirect ELISA. The results showed that all five MAbs reacted with ORF3-P1 but not with ORF3-P2. To map the precise antigenic sites of the ORF3-P1, nine fragments with different length were synthesized and expressed in E. coli. From the results obtained, two MAbs could recognize epitope GP3EP3(58-72aa)and other three MAbs could recognize epitope GP3EP7(66-81aa). Further analysis with truncated GP3EP3 and GP3EP7 demonstrated that epitopes within GP3EP3 and GP3EP7 were W74CRIGHDRCGED85 and Y67EPGRSLW74, respectively. Within all North American isolates, there are two mutations in region 74-85aa(H79/Y79 and/or G83/E83) and two mutations in region 66-81 aa(R72S73/K72V73 or R72S73/K72A73). Mutation analysis indicated that these amino acids are essential as epitope. Western blot analysis with sera from BJ-4 and CH-1a infected pigs found that peptides within GP3(50-65aa,58-72aa, 66-81aa, 73-87aa, 80-95aa, 88-101aa and 94-109aa) were all immunodominant epitopes.In addition, antigenic epitopes of GP2 and GP4 recognized by MAbs 26D8 and 42H12 were located in region 38-148aa and 61-178aa, respectively.Taken together, this study was based on the analysis of structural proteins of PRRSV and epitope mapping with monoclonal antibodies, and the epitope or immunodominant information regarding a prototype PRRSV(CH-1a) circulating in China may be helpful in understanding molecular properties of structural proteins of PRRSV, as well as relationship between immunogenicity or pathogenesis and gene variations of the virus. Therefore, the results from the study may also be useful for diagnosis of PRRSV infection based on antigenic epitope or developing a new strategy for vaccine design. |
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