Chinese Journal of Experimental and Clinical Virology 2002;16(2):119-123

HIV-1 integrase enzyme linked immunosorbent assay and inhibitors.

Zhimin GUO 1 ; Hongshan CHEN

Affiliations

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Country

China

Language

Chinese

Abstract

BACKGROUNDTo establish an ELISA method for detecting the activity of HIV-1 integrase and screening of inhibitors.

METHODSHIV 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.

RESULTSThe 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.

CONCLUSIONSThe 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.