Materials
and
Methods
HIV-1 protease inhibition
studies.
- Expression system:E-coli strain BL-21(DE3),competitive vector pACYCT7pmt
- Enzyme solution:Cell lysates ,recombinant HIV-1 p55 Gag precursor
polypeptide,test compound dissolved in dimethysulfoxide(DMSO).
- Inhibition activity was determined by SDS-PAGE assay.
Measurement of the inhibition
constant(ki)
- HIV-1 protease was purified by ion exchange chromatography.
- Incubate with the synthetic
substrate,Ac-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2.
- Ki was determinend by Michaelis-Menten method.
Aspartic protease inhibition
studies.
- Pepsin ,Cathepsin D
- The amount of degraded substrate in the filtrate was measured by
ultraviolet absorption at a wavelength of 280 nm. IC50
Molecular modeling of inhibitor-protease
complex.
Anti-HIV-1IIIB activity
during chronic infection.
- HIV-1IIIB chronically infected Molt-4 cells.
- SDS-PAGE,Western blot analysis using anti-p17 monoclonal antibody.
Result