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In-vitro screening method for HIV-1 fusion inhibitor

A fusion inhibitor, HIV-1 technology, applied in the biological field, can solve the problems of high purity requirements of five-helix protein, long detection time, expensive detection equipment, etc., and achieve the effect of reducing screening cost, shortening time, and simplifying steps

Inactive Publication Date: 2013-10-16
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the purity of the five-helical protein is high, the detection time is long, and the required detection equipment is expensive.

Method used

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  • In-vitro screening method for HIV-1 fusion inhibitor
  • In-vitro screening method for HIV-1 fusion inhibitor
  • In-vitro screening method for HIV-1 fusion inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Incubate the histidine-tagged magnetic beads with the histidine-tagged HIV-1gp41 five-helix protein crude extract at 37°C for 20 minutes, shake off the liquid in the wells after precipitation on the magnetic plate, wash the plate and set aside ; Compound N-(4-carboxy-3-hydroxyl) benzene-2,5-dimethylpyrrole (NB-2 for short) known to have anti-HIV-1 fusion effect is formulated into 10mg / ml, 1mg / ml, 100 μg / ml, 10 μg / ml dimethyl sulfoxide solution, fully vortexed and dissolved, and then used for later use; set 3 wells of negative control in the microplate, add 2 μl dimethyl sulfoxide to each well, set 3 wells of positive control, do not add For the crude extract of five-helical protein, add 2 μl of different concentrations of NB-2 to each well of the other wells, and then add 98 μl of fluorescein isothiocyanate (FITC)-labeled C-helix solution to all wells, and mix thoroughly with a shaker , incubate at 15°C for 10 minutes, shake off the liquid in the well, wash the plate an...

Embodiment 2

[0025] Incubate the histidine-tagged magnetic beads with the histidine-tagged HIV-1gp41 five-helix protein crude extract at 37°C for 20 minutes, shake off the liquid in the wells after precipitation on the magnetic plate, wash the plate and set aside ; compound NB-2 known to have anti-HIV-1 fusion effect into 10 mg / ml, 1 mg / ml, 100 μg / ml, 10 μg / ml dimethyl sulfoxide solution, fully oscillate and dissolve for later use; For 3 wells of negative control, add 2 μl dimethyl sulfoxide to each well, set up 3 wells of positive control, do not add five-helical protein crude extract when coating, and add 2 μl NB-2 of different concentrations to each well of other wells, and then all Add 98 μl of FITC-labeled C-helix solution to each well, mix thoroughly with a shaker, incubate at 30°C for 10 minutes, shake off the liquid in the wells, and measure the relative fluorescence value (RFU) of each well with a fluorescent microplate reader after washing the plate , select the excitation wavele...

Embodiment 3

[0027] Incubate the histidine-tagged magnetic beads with the histidine-tagged HIV-1gp41 five-helix protein crude extract at 37°C for 20 minutes, shake off the liquid in the wells after precipitation on the magnetic plate, wash the plate and set aside ; compound NB-2 known to have anti-HIV-1 fusion effect into 10 mg / ml, 1 mg / ml, 100 μg / ml, 10 μg / ml dimethyl sulfoxide solution, fully oscillate and dissolve for later use; For 3 wells of negative control, 2 μl dimethyl sulfoxide was added to each well; for 3 wells of positive control, the crude extract of pentahelical protein was not added during coating, and 2 μl of NB-2 of different concentrations were added to each well of the other wells, and then all Add 98 μl of FITC-labeled C-helix solution to each well, mix thoroughly with a shaker, incubate at 40°C for 10 minutes, shake off the liquid in the wells, and measure the relative fluorescence value (RFU) of each well with a fluorescent microplate reader after washing the plate ,...

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Abstract

The invention discloses an HIV-1 fusion inhibitor screening method in vitro, which belongs to the biotechnology field. The method includes the steps that a magnetic bead which can absorb a histidine label is used for wrapping an HIV-1 gp41quintuple helix protein crude extract with a histidine label on a micro-perforated plate, wherein the micro-perforated plate is provided with three negative contrast holes and three positive contrast holes, each negative contrast hole is respectively added with 2 microlitres of dimethyl sulfoxide, when in wrapping, other holes are respectively added with 2 microlitres of compound samples which are to be detected and have different concentrations without adding the quintuple helix protein crude extract, and then all the holes are respectively added with 98 microlitres of reported radical label C-helix solution which is hatched for 10 min at a temperature of 15-40 DEG C, the liquid in the holes is flung, reported radical signals are detected by a fluorescence analyser after the plate is washed, and then the suppression ratios of the samples to be detected are calculated. The method is simple in steps, rapid and convenient, and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro screening method for HIV-1 fusion inhibitors. Background technique [0002] At present, there are two main categories of in vitro screening methods for HIV-1 fusion inhibitors: [0003] 1. Screening method based on the interaction between gp41 N-helix and C-helix [0004] The transmembrane protein gp41 located on the surface of the HIV-1 envelope is a trimeric protein, and the three C-helices of gp41 are stacked in the hydrophobic groove on the surface of the N-helix trimer in an antiparallel manner to form a stable six-helix The bundle structure pulls the viral envelope closer to the host cell membrane, leading to membrane fusion. Compounds that inhibit the interaction of the gp41 N-helix with the C-helix inhibit the formation of six-helix bundles, thereby inhibiting the occurrence of membrane fusion. Screening methods based on gp41 N-helix and C-helix int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/566G01N21/64
Inventor 王存新刘斌何红秋张小轶谭建军陈慰祖
Owner BEIJING UNIV OF TECH