New type microbicide for preventing human Aids virus and other pathogen propagation
A microbicide, HIV technology, applied in biocides, antiviral agents, animal repellents, etc., can solve problems such as unseen
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Embodiment 1
[0036] Example 1: Preparation method of β-D-(1-4)-glucan sulfate compound
[0037] Prepared according to the published patent 98118102.3 method, take 500 grams of Cynanchum otophyllum plant roots, pulverize into powder and extract with hot water (94℃) for 2-3 times, combine the extracts, cool and stand for 4 hours , Centrifugal precipitation (4000 times / min), pour out the upper layer, remove the sediment and discard it. The upper liquid was added with 2-3 times the volume of methanol, and white flocculent polysaccharides precipitated after shaking and stirring. After standing overnight, the upper liquid was clear and the lower sediment was tight. Aspirate the upper liquid with a pipette, centrifuge the lower sediment, discard the upper liquid, transfer the lower sediment to a suction filter funnel, and filter with oxygen. The filtered precipitate is washed with acetone, then washed with ether and filtered by pressure. In a vacuum desiccator (with phosphorus pentoxide temperature a...
Embodiment 2
[0038] Example 2: Experiments for the detection of anti-HIV effects of new microbicides in vitro
[0039] On a 96-well culture plate, the HIV-1 (H9 / HIV-1IIIB, gifted by MRC, UK, resuscitated, cultured, collected, aliquoted and frozen according to conventional methods) stock solution was diluted 5 times, 8 gradients in total, each Each gradient is set with 6 holes, and a control group is set at the same time. Add C8166 cells (T lymphocyte line, donated by the British Medical Research Council (MRC), AIDS Reagent Project) 50μl (2×10) to each well 4 / Well), the final volume of each well is 200μl. Incubate at 37°C with 5% CO2. On the third day, 100μl of fresh complete medium (RPMI-1640) was added. On the seventh day, the virus-induced cytopathic effect (CPE) in each well was observed under an inverted microscope to determine whether there was syncytium in each well. Form to determine; Calculate the virus TCID50 (50% Tissue culture infectious dose) according to the Reed & Muench method,...
Embodiment 3
[0041] Example 3: Inhibition of new microbicides on HSV-1 and toxicity on Vero cells
[0042] 1. PRA method to determine the inhibitory effect of new microbicides on HSV-1
[0043] 1. Add the Vero cell suspension to a 24-well plate (2-4×10 5 / well), 37°C, 5% CO2 incubator for 24 hours;
[0044] 2. After the cells grow into a dense monolayer and cover the bottom of the 24-well plate, quickly melt HSV-1, then add 200μl of virus supernatant or medium to each well, and shake the plate gently to make the virus dilution and cell monolayer sufficient Contact, incubate in a 37°C, 5% CO2 incubator for 1 hour;
[0045] 3. In another plate, use RPMI1640 to dilute the drug twice, and use acyclovir as a positive control;
[0046] 4. Carefully aspirate the medium (be careful not to hit the wall), add 1ml PBS to each well and gently wash the plate once;
[0047] 5. Add uncoagulated cover medium (mixture of A and B), 1ml per well. Add the diluted test solution in time, 0.5ml per hole, 6 holes (vi...
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