Production of virus-specific antiserum corresponding to sequences in the lactate dehydrogenase-elevating virus (LDV) ORF6 protein

Abstract

The elucidation of the antigenic structure of the envelope proteins of Arteriviridae which includes lactate dehydrogenase-elevating virus (LDV) will provide further understanding of a mechanism of strict host cell specificity. To analyze the linkage between LDV envelope proteins, M/VP-2 and VP-3, which may play an important role in viral infectivity, we generated specific antibody against M/VP-2 that has not been reported in previous studies. A synthetic polypeptide corresponding to the C-terminal region of LDV strain C (LDV-C) ORF6, which encodes M/VP-2, was chemically synthesized and coupled to keyhole limpet hemocyanin (KLH). The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages showed that the antibody was able to react specifically with authentic virion protein. The immunoreactive antibody against LDV M/VP-2 described in this study will be useful for further studies of the specific roles of the envelope proteins in arterivirus assembly and infectivity.

Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins

“…Immunofluorescence analysis revealed that the localization pattern of expressed ORF 5 and ORF 6 proteins was similar to that of LDV VP-3 and M/VP-2 in infected macrophages [20,21]. In addition, ORF 5 and ORF 6 proteins were colocalized in most cells.…”

“…Because M/VP-2 appears to be associated with VP-3 in virions [14,27], colocalization of ORF 5 and ORF 6 proteins was expected. Therefore, an association of ORF 5 and ORF 6 proteins expressed in our established cell lines is necessary to be verified, however, we failed to detect the association by a co-immunoprecipitation assay using antibodies against LDV ORF 5 [22] and ORF 6 proteins [21]. Since the antibodies were unable to immunoprecipitate expressed ORF 5 and ORF 6 proteins solubilized in the lysis buffer described in Section 2, detailed experiments are required to select the suitable lysis buffer and condition for immunoprecipitation assay.…”

“…Purified LDV type C (LDV-C) was prepared with 4 and 8-week-old SJL/J mice (Jackson Laboratory, Maine, USA) as described previously [20]. A polyclonal antibody (#36) against LDV M/VP-2 was obtained after immunizing rabbits with a synthetic polypeptide corresponding to the LDV-C ORF 6 C-terminal region coupled with the keyhole limpet hemocyanin (KLH) [21]. A monoclonal antibody against LDV VP-3 (MAb no.…”

“…Purified LDV virion protein prepared as previously described [24,25] and expressed protein purified as described above were separated by SDS-PAGE on 12.5% polyacrylamide gels and electroblotted onto a PVDF membrane. Blots were reacted with rabbit antibody #36 (diluted 1:100) against LDV-C ORF 6 as reported previously [21] or monoclonal antibody no. 6 against LDV VP-3 [22].…”

“…In contrast, ORF 6 protein cell surface expression was unverified because there are no antibodies that react with cell surface M/VP-2. In our previous study [21], a rabbit antibody that reacted consistently with virion M/VP-2 in infected macrophages was generated, however, the antibody reacts only with the M/VP-2 cytoplasmic domain.…”