RESUMO
A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/isolamento & purificação , Bovinos , Vírus da Diarreia Viral Bovina/genética , Drosophila melanogaster , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
We generated a recombinant vesicular stomatitis virus (VSV-E2) encoding the bovine viral diarrhea virus (BVDV) E2 glycoprotein with the VSV-G protein signal peptide. Infection of BHK21 cells with VSV-E2 induced the synthesis of a recombinant E2 (rE2) that comigrated with authentic BVDV-E2 in PAGE-SDS gels. Non-reducing immunoblots showed that rE2 is a disulfide bond-linked homodimer with at least 10-fold higher avidity for conformation-dependent anti-BVDV-E2 antibodies than its reduced monomeric counterpart. Immunofluorescence microscopy also showed that rE2 was transported to the plasma membrane of infected cells and analysis of purified particles demonstrated that dimeric rE2 was incorporated into VSV-E2 virions in approximately 1:10 ratio with respect to the G glycoprotein. BALB/c mice inoculated intranasally with VSV-E2 doses of up to 10(7) plaque forming units (pfu) showed no symptoms of viral-induced disease and developed a specific BVDV neutralizing response that lasted for at least 180 days post inoculation.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Glicoproteínas de Membrana , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Bovinos , Linhagem Celular , Quimera/genética , Quimera/imunologia , Cricetinae , DNA Recombinante/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/genética , Vacinas Virais/genéticaRESUMO
Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Cabañas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.