Expression and characterization of a multivalent human respiratory syncytial virus protein

• Published in: Molecular biology (New York) 2010, Vol.44 (3), p.420-430
• Main Authors: Subbarayan, P, Qin, H, Pillai, S, Lee, J. J, Pfendt, A. P, Willing, G, Miller, M. E, Dennis, V. A, Singh, S. R
• Format: Article
• Language: English
• Subjects: Acrylamide; Adjuvants; Antibody response; Atomic force microscopy; Babies; Biochemistry; Biomedical and Life Sciences; Cell Molecular Biology; Enzyme-linked immunosorbent assay; Escherichia coli; Flagellin; Gel electrophoresis; Head and neck; Human Genetics; Human respiratory syncytial virus; Immune response; Immunity; Immunization; Immunoglobulin G; Infants; Infection; Life Sciences; Lymphocytes T; Microscopy; Mucosa; Protein structure; Proteins; Respiratory syncytial virus; Respiratory system; Respiratory tract diseases; Transmission electron microscopy; Vaccines; Western blotting
• ISSN: 0026-8933; 1608-3245
• DOI: 10.1134/S0026893310030106

Respiratory syncytial virus (RSV) has been recognized as one of the most common causes of severe respiratory tract infection in infants worldwide. As yet, a safe and effective vaccine has not been developed to protect humans from RSV. The F and G surface proteins have been widely investigated due to their potential to induce protective immunity. In addition, the M2 protein has been shown to be important in inducing a T-cell response. Our project involved the cl oning of the immunodominant regions of the RSV F, M2 and G proteins into a bacterial vector, pET-32a(+). The recombinant RFM2G protein was expressed in Escherichia coli and purified using His Bind columns. The purified rRFM2G protein was analyzed by poly-acrylamide gel electrophoresis and Western blotting. The predicted structure of the recombinant protein built by the Swiss PDB Viewer program suggested a rod shape with a distinct swollen head and neck which was confirmed by transmission electron microscopy and atomic force microscopy. BALB/c female mice were immunized with either RSV, rRFM2G alone, or rRFM2G in combination with flagellin as a mucosal adjuvant. Serum was collected on days 0, 14, 28 and 49 to assess the immune response by Enzyme-linked immunosorbent assay. Intranasal immunization of mice with the rRFM2G protein yielded significantly high serum IgG titers. Co-administration of the rRFM2G protein with flagellin did not augment the serum antibody response.