1.Study On Purification, Characterization And Conformation Of Lycoris Radiata Agglutinin 2.Molecular Cloning And Analyzing Of Agglutinin From Amaryllis Vittata And Zephyranthes Grandiflora

The study on Lycoris radiata Agglutinin (IRA) main include the purification and characterization of LRA, the relationship between structure and bioactivity of LRA. LRA was isolated from the bulbs of Lycoris radiata by extraction, precipitation with (NHJiSO:,, ion-exchange chromatography on CM-Sepharose, DEAE- Sepharose and followed by gel filtration on Sephacryl S-100. The purified lectin showed a single protein band on SDS-PAGE, and the subunit molecular weight was 12KD. The molecular weight was 45KD determined by gel filtration on Sephacryl S-100. The result implied that there are four same subunits per LRA. LRA can agglutinate rabbit erythrocyte at 0.95ug/mL and E. coli cell at 1.9 ug/mL, and also can agglutinate Sacch. cerevisiae cells. The result of carbohydrate inhibition assay showed that Mannan and Thyroglobulin can inhibit the agglutinating activity of LRA. The LRA has strong mitogenetic activity toward T-lymphocyte. LRA showed antivirus activity to the Herpes simplex virus II (HSV-II), and the IC50=5~10 ug/mL, and it showed no cytotoxic activity toward vero cell proliferation at 500ug/mL; In the trials of inhibit HIV-1 and HIV-2, LRA could inhibit the infection of the HT-4 cell and CEM cell by the human immunodeficiency virus-1 (HIV-1) and HIV-2, theEC50=0. 43 ug/mL and 0. 79 ug/mL on inhibit the HIV-I, and the EC50=0. 60 ug/mL and 0. 59 ug/mL on inhibit the HIV-2. It showed no cytotoxic activity toward HT-4 and CEM cell proliferation at 71ug/mL. So LRA is a potent antivirus protein.The LRA exhibited a characteristic circular dichroic (CD) spectrum, a negative band centered at 222nm. It was estimated that LRA was a high - sheet protein. The fluorescence spectrum (FLS) of LRA excited at 280nm and 295nm showed a maximum peak at 338nm. The characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp. When LRA was excited at 295nm, the FLS showed a maximum peak at 338nm, the max of fluorescence emission spectrum blue-shifted more than 10nm compared with the max of free Tyr (348nm).The change of agglutinating activity , CD spectrum and FLS of LRA in different temperature, pH and different chemicals indicated that LRA had partial hemagglutinating activity at pH2. 0(50%), a temperature above 100 (60%) and after modified by N-bromosuccinimide (MBS), the activity lost completely , modified by DEPC, the LRA had a little activity, the other groups modified such as Arg, Tyr, Glu, Asp didn't effect the hemagglutinating activity of LRA. The result indicated that Trp residues were essential to the hemagglutinating activity and were involved in carbohydrate-binding site.The CD spectrum showed that the denaturation was a two-steps' progress. First, LRA didn't lose activity despite of its structure change because the change was out of the active center. Second, the active center was destroyed and LRA began to lose its agglutinate activity. So LRA has a comparatively stable structure. The FLS of LRA with different temperature and pH also showed there was little change. The study on fluorescence quenching showed that the fluorescence from Trpresidues were quenched by KI, acrylamide and CsCl, and 93. 6% of Trp were quenched by acrylamide. No sulfhydryl group has been detected when using N-ethymaleimide (MEM) as a modificatory reagent. There were 5. 3% Trp residues in a LRA molecular determined by colorimetry. Using NBS as the modificatory reagent, there were 12. 4 Trp residues in a LRA molecule. It means that every subunit contains three Trp residues.