Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli

• Published in: Protein expression and purification Vol. 53; no. 1; pp. 209 - 215
• Main Authors: Simabuco, Fernando M., Carromeu, Cassiano, Farinha-Arcieri, Luis E., Tamura, Rodrigo E., Ventura, Armando M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-05-2007
• Subjects: Animals; Antibodies, Viral - biosynthesis; Antibodies, Viral - immunology; Antibody production; Antigens, Viral - immunology; Blotting, Western; Carcinoma, Hepatocellular - pathology; Carrier Proteins - metabolism; Cell Line, Tumor; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enteropeptidase - pharmacology; Escherichia coli; Escherichia coli - genetics; Female; Fluorescent Antibody Technique, Direct; Genetic Vectors; Human respiratory syncytial virus; Humans; Immunofluorescence; Liver Neoplasms - pathology; Maltose-binding protein; Maltose-Binding Proteins; Mice; Mice, Inbred BALB C; Nucleoprotein; Nucleoproteins - genetics; Nucleoproteins - isolation & purification; Nucleoproteins - metabolism; Phosphoprotein; Phosphoproteins - genetics; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Respiratory Syncytial Virus Infections - immunology; Respiratory Syncytial Virus, Human - chemistry; Respiratory Syncytial Virus, Human - genetics; Respiratory Syncytial Virus, Human - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Solubility; Transformation, Genetic; Vaccination; Human respiratory syncytial virus; Nucleoprotein; Phosphoprotein; Maltose-binding protein; Immunofluorescence; Antibody production
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.12.016

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS–PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.