[HIV-1 integrase enzyme linked immunosorbent assay and inhibitors].
BACKGROUND
To establish an ELISA method for detecting the activity of HIV-1 integrase and screening of inhibitors.


METHODS
HIV recombinant plasmid F185K/C280S IN1-288 was transformed into the E.coli strain BL21(DE3) integrase with a histag was induced by adding isopropyl-?-D?thiogalactopyranoside (IPTG)and purified by nickel affinity chromatography. The synthesized donor substrate oligonucleotide representing the HIV-1 U5LTR was immobilized onto covalink polystyrene microtiter plates, and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate. The products were detected and quantified using a colorimetic avidin?linked alkaline phosphatase reporter system,and identified by 32? autoradiography. Some natural products and chemically synthesized compounds were screened for HIV-1 integrase inhibitors.


RESULTS
The purified integrase was identified by SDS?PAGE and showed integration activity by ELISA and?32P radioisotopic assay.?Coefficients of variation (CV)of ELISA in a lot and among the lots were 4.63% and 5.89% respectively, the mean of P/N was 2.836 0.161 under the optimal experimental condition. Some plant extracts were found as potent integrase inhibitors. The IC50s for CEH and CEHL were (20.41 5.68)?g/ml and (7.56 1.86)?g/ml respectively.


CONCLUSIONS
The authors have established a simple and rapid ELISA method with stable repeatability for detecting integrase activity, which can be used for screening and studying of specific inhibitors of HIV-1 integrase.
Department of Virology, Institute of medicinal Biotechnology, CAMS and PUMC, Beijing 100050, China.
Zhimin
Z
Guo
Hongshan
H
Chen
D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019427:HIV Integrase / Q000302:isolation & purification / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D000998:Antiviral Agents; D019428:HIV Integrase Inhibitors; D011993:Recombinant Fusion Proteins; D019427:HIV Integrase