Production and characterization of a polyclonal antibody against rabies virus phosphoprotein
Keywords:
rabies virus, immunochemistry, phosphoproteins, recombinant proteins, Escherichia coli
Abstract
Introduction. The expression of recombinant viral proteins has been a useful tool to study molecular biology and pathogenesis of virus infections. Because commercial specific antibodies to rabies virus phosphoprotein (P) are currently unavailable, these antibodies must be generated de novo in order to study the role of P protein during the infectious process.Objective. A polyclonal antibody was produced and characterized for use against the phosphoprotein (P) of rabies virus. The antibody was raised in rabbits with a recombinant viral phosphoprotein (P) produced in Escherichia coli.
Materials and methods. Gene P coding for the viral phosphoprotein (P) was amplified by RTPCR and cloned into the expression vector PinPointTM Xa-1 T. The recombinant protein P was expressed in Escherichia coli, purified by affinity chromatography and used to produce a polyclonal antibody anti-P. The antibody anti-P was purified and characterized by immunocytochemistry, immunofluorescence, fluorometric CELL-ELISA and Western blotting.
Results. The recombinant viral phosphoprotein was successfully expressed as a 50 kd biotinylated fusion protein which corresponds to the whole protein P of rabies virus. The polyclonal antibody raised against this recombinant protein P was able to detect with high specificity, protein P in cultures of sensorial neurons infected with rabies virus.
Conclusions. The P protein obtained from heterologous expression in Escherichia coli became a specific antigen that was used to produce a polyclonal antibody capable of detecting native P protein in rabies virus infected cells.
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References
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3. Gupta AK, Blondel D, Choudhary S, Banerjee AK. The phosphoprotein of rabies virus is phosphorylated by a unique cellular protein kinase and specific isomers of protein kinase C. J Virol. 2000;74:91-8.
4. Jacob Y, Badrane H, Ceccaldi PE, Tordo N. Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. J Virol. 2000;74:10217-22.
5. Castellanos JE, Martínez-Gutiérrez M, Hurtado H, Kassis R, Bourhy H, Acosta O, et al. Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglio cultures. J Neurovirol. 2005;11:403-10.
6. Rincón V, Corredor A, Martínez-Gutiérrez M, Castellanos JE. Fluorometric cell-Elisa for quantifying rabies infection and heparin inhibition. J Virol Methods. 2005;127:33-9.
7. Smith PK, Krohn RI, Hermanson G, Mallia A, Gartner FH, Provenzano M, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85.
8. Wiechelman KJ, Braun RD, Fitzpatrick JD. Investigation of the bicinchoninic acid protein assay identification of the groups responsible for color formation. Anal Biochem. 1988;175:231-7.
9. Chenik M, Chebli K, Blondel D. Translation initiation at alternative in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J Virol. 1995;69:707-12.
10. He Y, Gao D, Zhang M. Expression of the nucleoprotein gene of rabies virus for use as a diagnostic reagent. J Virol Methods. 2006;138:147-51.
11. Reyes del Valle J, Del Angel RM. Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand. J Virol Methods. 2004;116:95-102.
12. Tuffereau C, Bénéjean J, Blondel D, Kieffer B, Flamand A. Low-affinity nerve growth factor receptor (p75NTR) can serve as a receptor for rabies virus. EMBO J. 1998;17:7250-9.
13. Langevin C, Jaaro H, Bressanelli S, Fainzilber M, Tuffereau C. Rabies virus glycoprotein (RVG) is a trimeric ligand for the N-terminal cysteine-rich domain of the mammalian p75 neurotrophin receptor. J Biol Chem. 2002;277:37655-62.
14. Blondel D, Regad T, Poisson N, Pavie B, Harper F, Pandolfi PP et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies. Oncogene. 2002;21:7957-70.
15. Nadin-Davis SA, Sheen M, Andel-Malik M, Elmgren L, Armstrong J, Wandeler AI. A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination. J Clin Microbiol. 2000;38:1397-403.
16. Raux H, Iseni F, Lafay F, Blondel D. Mapping of monoclonal antibody epitopes of the rabies virus P protein. J Gen Virol. 1997;78:119-24.
17. Motoi Y, Inoue S, Hatta H, Sato K, Morimoto K, Yamada A. Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli. Jpn J Infect Dis. 2005;58:115-8.
2. Real E, Rain JC, Battaglia V, Jallet C, Perrin P, Tordo N, et al. Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides. J Virol. 2004;78:7410-7.
3. Gupta AK, Blondel D, Choudhary S, Banerjee AK. The phosphoprotein of rabies virus is phosphorylated by a unique cellular protein kinase and specific isomers of protein kinase C. J Virol. 2000;74:91-8.
4. Jacob Y, Badrane H, Ceccaldi PE, Tordo N. Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. J Virol. 2000;74:10217-22.
5. Castellanos JE, Martínez-Gutiérrez M, Hurtado H, Kassis R, Bourhy H, Acosta O, et al. Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglio cultures. J Neurovirol. 2005;11:403-10.
6. Rincón V, Corredor A, Martínez-Gutiérrez M, Castellanos JE. Fluorometric cell-Elisa for quantifying rabies infection and heparin inhibition. J Virol Methods. 2005;127:33-9.
7. Smith PK, Krohn RI, Hermanson G, Mallia A, Gartner FH, Provenzano M, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85.
8. Wiechelman KJ, Braun RD, Fitzpatrick JD. Investigation of the bicinchoninic acid protein assay identification of the groups responsible for color formation. Anal Biochem. 1988;175:231-7.
9. Chenik M, Chebli K, Blondel D. Translation initiation at alternative in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J Virol. 1995;69:707-12.
10. He Y, Gao D, Zhang M. Expression of the nucleoprotein gene of rabies virus for use as a diagnostic reagent. J Virol Methods. 2006;138:147-51.
11. Reyes del Valle J, Del Angel RM. Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand. J Virol Methods. 2004;116:95-102.
12. Tuffereau C, Bénéjean J, Blondel D, Kieffer B, Flamand A. Low-affinity nerve growth factor receptor (p75NTR) can serve as a receptor for rabies virus. EMBO J. 1998;17:7250-9.
13. Langevin C, Jaaro H, Bressanelli S, Fainzilber M, Tuffereau C. Rabies virus glycoprotein (RVG) is a trimeric ligand for the N-terminal cysteine-rich domain of the mammalian p75 neurotrophin receptor. J Biol Chem. 2002;277:37655-62.
14. Blondel D, Regad T, Poisson N, Pavie B, Harper F, Pandolfi PP et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies. Oncogene. 2002;21:7957-70.
15. Nadin-Davis SA, Sheen M, Andel-Malik M, Elmgren L, Armstrong J, Wandeler AI. A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination. J Clin Microbiol. 2000;38:1397-403.
16. Raux H, Iseni F, Lafay F, Blondel D. Mapping of monoclonal antibody epitopes of the rabies virus P protein. J Gen Virol. 1997;78:119-24.
17. Motoi Y, Inoue S, Hatta H, Sato K, Morimoto K, Yamada A. Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli. Jpn J Infect Dis. 2005;58:115-8.
How to Cite
1.
Castañeda NY, Chaparro-Olaya J, Castellanos JE. Production and characterization of a polyclonal antibody against rabies virus phosphoprotein. biomedica [Internet]. 2007 Jun. 1 [cited 2024 May 17];27(2):257-67. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/221
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