Identification Of A B-cell Epitope In GP3of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus HuN4Strain

Highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS） is one of the mosteconomically significant viral diseases, causing great losses in China and continuing to be a threat forthe swine industry. The glycosylated protein3（GP3） is considered to be associated with protectiveimmunity, but its function is poorly known.In this study, BALB/c mice were immunized with HP-PRRSV HuN4to generate monoclonalantibodies （MAb） against GP3. Indirect immunofluorescence assay （IFA） based on HuN4andeukaryotic expressed HuN4GP3was used to screen the positive hybridoma cell clones. A hybridomacell named as4G5that secreted MAb against HP-PRRSV GP3was obtained, the titer of4G5MAbascetic detected by IFA is1:5,120. Western blot showed that4G5could react with GP3expressed inE.coli. In order to identify the minimal epitope recognized by4G5, the ORF3gene was truncated intoseveral overlapping fragments, and fusion expressed with GST-tag in E.coli BL21（DE3）, then theproteins were probed with4G5by Western blot. Based on the analysis above, we determined theminimal epitope was localized to W⁷⁴CRIGHDRCS⁸³. Sequence alignments of23PRRSV isolatesrevealed that the epitope sequence was relatively conserved except for H⁷⁹â†'Y⁷⁹and S⁸³â†'G⁸³/E⁸³. Inaddition, the IFA showed that4G5had reactivity to PRRSV attenuated live vaccine strains HuN4-F112(H⁷⁹…S⁸³), CH-1R (H⁷⁹…S⁸³) and JXA1-R (N⁷⁹…S⁸³), but not to RespPRRS MLV (Y⁷⁹…E⁸³), whichindicated that the mutation (H⁷⁹/N⁷⁹…S⁸³â†'Y⁷⁹…E⁸³) may change the antigenicity of the epitope. Inorder to determine the effect of the mutated amino acid (S⁸³â†'G⁸³) on epitope antigenicity, the mutatedepitope (W⁷⁴CRIGHDRCG⁸³) was expressed and examined. This suggested that the mutated amino acidcouldn’t cause the antigenic differences of the epitope. So the antigenicity of the epitope is coincidentamong domestic classic and high pathogenic PRRSV strains. The GST-epitope4G5fusion protein wasexpressed, purifed, and employed as coating antigen to detect the pig sera by ELISA. The resultsshowed that antibodies against the epitope could experience an increasing process in the pigs postHuN4-F112vaccine immunization.4G5could be useful for further study of the structure and functionof HP-PRRSV GP3.