Bird¡¦s nest is the saliva from the Colloclia that have a lot of mucin and sialic acid. However the sialic acid is an important intermediate of influenza virus infection. In 2006, Guo et al was confirmed the Inhibitory effect of bird¡¦s nest extract on influenza virus infection. But the anti-virus component has not been isolated and purified. First, the bird's nest used in this study was proved to be a pure bird¡¦s nest by SDS-PAGE, sialic acid content and protein content. Qualification analysis by SDS-PAGE showed sample possessed three bands at about 50 kDa, 100 kDa and 150 kDa, by Warren assay showed sample possessed a sialic acid content about 10% of dry weight, by Kjeldahl method showed sample possessed a protein content about 50% of dry weight, and by fluorescence analysis in 365 nm showed sample emitted a blue-white light. According to the criteria on previous studies, the sample was considered to be a real and pure bird¡¦s nest. Subsequently, the sample was applied to pronase digestion and then separated by Bio-gel P2 for size exclusion chromatography. All fractions were performed carbohydrate analysis including sialic acid. In lectin blotting, Maackia amurensis agglutinin (MAA) lectin can interact with the fraction that suggest the structure of sialic acid is sialic acid-2,3 galactose in bird¡¦s nest. And carbohydrate membrane array to demonstrate the influenza virus of H5N1 and H1N1 can bind the fractions that suggest the antivirus effect of bird¡¦s nest. These fractions will be further analyzed by neutralization assay to study their antivirus effects in vitro.

<p>Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid <em>N</em>-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to <em>N</em>-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn²⁺. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH₂OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (<em>K</em>_i^*) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH₂OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuB^IS), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuB^RS), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-¹⁴C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuB^{RS<strong> </strong>}is required for complete inhibition.</p> / Master of Science (MSc)