Purification And Quantification Of Human Respiratory Syncytial Virus F Protein

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory infection in infants and young children worldwide. A safe and effective RSV vaccine is not yet available, so it is important to pursue potential therapeutic measures against RSV infection. The RSV F protein is an attractive target for drug and vaccine development as it is essential for viral entry, is highly conserved, and is the major virus neutralization antigen. At present no satisfactory protein purification protocols are available for the F protein. The current method using preparative SDS-PAGE or immunoaffinity chromatography yields only small amounts of purified F protein and partially damage its antigenicity. In this study, we design a new two-step chromatographic process to purify F protein from RSV propagated in HEp-2 cells and set up a sandwich ELISA method to quantify the F protein. RSV infected HEp-2 cells and medium were collected and centrifuged. The resulting supernatant was precipitated by PEG6000, and then the precipitate was dissolved and further purified through sucrose gradient ultracentrifugation. The viral envelope proteins were subsequently solubilized with Triton X-100 detergent and the rough F protein was further purified by ion exchange chromatography and gel filtration chromatogry. Finally, RSV-F protein was obtained by this protein purification protocol. No loss of antigenicity could be observed during this procedure as the highly purified F protein remains detectable by a set of monoclonal antibodies. The purified F protein was recovered as a functional homodimer of 140 kD. Eighty-fiveμg purified F protein in total was obtained from 10 T-75 flasks of viral culture.