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. 2004 Jan;27(1):47-55.
doi: 10.1016/S0147-9571(03)00035-3.

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

H Takahashi-Omoe  1 , K Omoe, M Sakaguchi, Y Kameoka, S Matsushita, T Inada
Affiliations

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

H Takahashi-Omoe et al. Comp Immunol Microbiol Infect Dis. 2004 Jan.

Abstract
in English, French

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.

L'explication de la structure antigénique des protéines d'enveloppe des Arteriviridae, dont le virus de la lacticodéhydrogénase (LDV) fait partie, devrait permettre de mieux comprendre le mécanisme de la stricte spécificité des cellules hôtes. Pour analyser les liens entre les protéines d'enveloppe du LDV, à savoir M/VP-2 et VP-3, qui jouent certainement un rôle important dans le pouvoir infectant du virus, nous avons créé un anti-corps spécifique de la M/VP-2 dont il n'a pas encore été fait mention dans les études précédentes. Pour ce faire, nous avons chimiquement synthétisé un polypeptide correspondant à la zone de l'extrémité C d'un virus LDV de souche C (LDV-C), ORF6, codant M/VP-2, et nous l'avons couplé à l'hémocyanine de patelle (KLH). Ce peptide, immunogène chez le lapin, induit un anticorps spécifique à la protéine virale. Le transfert de type western et l'analyse en immunoflorescence de M/VP-2 du virion dans des macrophages infectés ont montré que l'anticorps pouvait réagir de manière spécifique avec une protéine authentique de virion. L'anticorps immunoréactif avec la M/VP-2 du LDV décrit dans cette étude va s'avérer utile dans les études qui vont être conduites sur le rôle spécifique des protéines d'enveloppe dans l'Arteriviridae et dans son pouvoir infectant.

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Figures

Fig. 1
Fig. 1
Titration curves of rabbit pre-immune sera and Ab#36 and #37 in ELISA using LDV-C ORF6 peptide as an antigen are shown in (A). Corresponding data using peptide 15-9 against major allergen of Japanese cedar pollen as an antigen are depicted in (B). (A) Pre-immune sera #36 (▵), Pre-immune sera #37 (♢), Ab#36 (▴), Ab#37 (♦). (B) Pre-immune sera #36 probed with LDV peptide (▵), Pre-immune sera #36 against peptide 15-9 (♢), antibody # 36 against LDV-C ORF6 peptide (▴), antibody #36 against peptide 15-9 (♦).
Fig. 2
Fig. 2
Western blotting analysis with anti-peptide antibodies to the LDV-C ORF6 protein, Ab#36 (A) and Ab#37 (B). Samples were loaded from normal SJL/J mouse sera (lane 1), LDV virion protein (lane 2).
Fig. 3
Fig. 3
Immunofluorescence analysis with Ab#36. LDV-infected macrophages were probed with pre-immune #36 IgG (A) and Ab#36 (C). Normal macrophages were probed with Ab#36 (B). Magnification, ×640.
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