University thesis:
Zugl.: Hannover, Tierärztl. Hochsch., Inst. für Virologie, Diss., 2009
Footnote:
Description:
Phage Display, HRSV Fusions Protein, SARS-CoV Spike Protein. - For most viral diseases only symptomatic treatment is available. Highly pathogenic agents demand a search for compounds that can either be administered therapeutically or be employed in diagnostic assays in order to detect and confirm virus infection. Usually specific antibodies are used to provide evidence of viral antigen in ELISAs or immunofluorescence assays. This study evaluated the convenience of synthetic peptides as viral antigen detectors. To identify specific peptides for the targets of choice, the phage display technique was chosen due to the high variability of the phage library. In the first part of this work, general assay conditions were established by biopanning on the fusion protein of human respiratory syncytial virus presented on the surface of a recombinant Sendai virus. The peptides of the two isolated phages comprised the same twelve amino acids encoded by two different nucleotide sequences. The phage-displayed peptides recognised specifically the HRSV F protein in ELISA, immunofluorescence analysis, phage-overlay assay, co-immunoprecipitation and surface plasmon resonance analysis. The bovine respiratory syncytial virus is a close relative of HRSV. In ELISA, the binding of the phages to both viruses was equally strong. The phage-overlay assay and the co-immunoprecipitation experiments demonstrated a significantly weaker binding of the phages to bF than to hF. This might indicate that the structural domain that is recognised by the phages includes amino acids located in the F2 subunit which is different in both proteins. Based on the identified twelve amino acids, peptides were synthesised. These peptides recognised the HRSV F protein when they were coated in ELISA, but not when they were in solution while being incubated with the target. SPR analysis revealed that the immobilised peptides captured specifically the HRSV F protein; this suggests that a certain arrangement of several peptides is required for proper binding to the F protein. In the second part of this work, the spike protein of severe acute respiratory syndrome associated corona virus was selected to evaluate different ways of target presentation during the biopanning step. Neither S protein expression on BHK cells nor on the surface of VSV∆G yielded phages which displayed S protein specific peptides. In a third approach biopanning on concentrated cell culture supernatant containing soluble S protein was successful in isolating nine phages that recognised the target of interest. Although a consensus sequence failed to emerge, four phages bound to SARS-CoV in Sandwich-ELISA, immunofluorescence analysis and phage overlay assay. These clones might be considered specific for the S protein, although having a low affinity for their target; the five clones that lacked a positive binding signal in all assays other than the Sandwich-ELISA are most likely unspecific binders.