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In vitro screening method of HIV-1 integrase chain transfer reaction inhibitor

A technology of HIV-1 and reaction inhibitors, applied in biochemical equipment and methods, material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of cumbersome steps, long detection time, cumbersome color reaction steps, etc., and achieve low cost , the effect of simple steps

Inactive Publication Date: 2013-01-02
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: long time-consuming, cumbersome steps, low throughput, high requirements for testing equipment and personnel, easy to produce radioactive pollution, and high cost
The disadvantages of this method are: it needs to coat and seal the microwell plate, which takes a long time; it needs to use a special microwell plate, and the cost is high
The disadvantages of this method are: the color reaction steps are cumbersome, the detection time is long, and the cost is high

Method used

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  • In vitro screening method of HIV-1 integrase chain transfer reaction inhibitor

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Embodiment 1

[0028] The HIV-1 integrase protein was expressed and purified by prokaryotic expression technology, and stored in aliquots; at room temperature, the samples of the compounds to be tested were prepared into solutions of different concentrations with dimethyl sulfoxide, fully shaken and dissolved for later use.

[0029] Dissolve single-stranded DNA1, single-stranded DNA2, single-stranded DNA3, and single-stranded DNA4 in sterile double-distilled water, mix single-stranded DNA1 and single-stranded DNA2 at equimolar concentrations in a dark environment, and place in a water bath at 95°C Boil for 3 minutes to denature, then slowly cool to room temperature, and then anneal to form donor DNA; mix single-stranded DNA3 and single-stranded DNA4 at equimolar concentrations in a dark environment, boil in a water bath at 95°C for 3 minutes to denature, and then slowly cool to room temperature, that is, annealing to form target DNA; the sequences of single-stranded DNA1, single-stranded DNA2...

Embodiment 2

[0036] The HIV-1 integrase protein was expressed and purified by prokaryotic expression technology, and stored in aliquots; at room temperature, the samples of the compounds to be tested were prepared into solutions of different concentrations with dimethyl sulfoxide, fully shaken and dissolved for later use.

[0037] Dissolve single-stranded DNA1, single-stranded DNA2, single-stranded DNA3, and single-stranded DNA4 in sterile double-distilled water, mix single-stranded DNA1 and single-stranded DNA2 at equimolar concentrations in a dark environment, and place in a water bath at 95°C Boil for 3 minutes to denature, then slowly cool to room temperature, and then anneal to form donor DNA; mix single-stranded DNA3 and single-stranded DNA4 at equimolar concentrations in a dark environment, boil in a water bath at 95°C for 3 minutes to denature, and then slowly cool to room temperature, that is, annealing to form target DNA; the sequences of single-stranded DNA1, single-stranded DNA2...

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Abstract

The invention relates to an in vitro screening method of HIV-1 integrase chain transfer reaction inhibitor and belongs to the field of biotechnology. The method includes using a FITC (fluorescein isothiocyanate) group to mark target DNA (deoxyribonucleic acid), detecting fluorescent signals of the FITC group to estimate chain transfer reaction activity of integrase or inhibitory activity of a sample to be tested. The process of the existing in vitro screening method of HIV-1 integrase chain transfer reaction inhibitor takes at least about 4h. The whole screening process of the in vitro screening method of HIV-1 integrase chain transfer reaction inhibitor is shortened to be within 2h, and the screening cost is reduced greatly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro screening method for inhibitors of HIV-1 integrase chain transfer reaction. Background technique [0002] HIV-1 inhibitors targeting integrase have found that inhibitors targeting integrase 3′ processing activity have poor anti-HIV-1 activity in vivo, while inhibitors targeting integrase strand transfer activity have better Anti-HIV-1 activity in vivo, so the inhibition of integrase chain transfer reaction activity is considered to be the key to the inhibitor's anti-HIV-1 activity in vivo (Hazuda DJ, Felock P, Witmer M, Wolfe A, Stillmock K, Grobler JA, Espeseth A, Gabryelski L, Schleif W, Blau C, Miller MD. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science, 2000, 287(5453):646-650). [0003] Currently, HIV-1 integrase chain transfer reaction inhibitors in vitro screening methods mainly fall into the follow...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/25G01N21/64
Inventor 刘昕刘斌李杉何红秋许先进谭建军张小轶李春华王存新
Owner BEIJING UNIV OF TECH
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