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Method for filtering out natural products anti AIDS through activity guidence

A natural product and active technology, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., can solve problems such as complicated operation, difficult to be widely used, and inability to screen with crude extracts, and achieve wide application prospects , wide application range, convenient and quick application effect

Inactive Publication Date: 2011-08-10
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the interference of a large amount of endogenous biotin in the crude extract of natural products, the current methods for detecting the inhibitory activity of HIV-1 reverse transcriptase inhibitors cannot be used for the screening of crude extracts. Although some methods have little impact, However, due to the complexity of operation, it is difficult to widely use

Method used

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  • Method for filtering out natural products anti AIDS through activity guidence
  • Method for filtering out natural products anti AIDS through activity guidence
  • Method for filtering out natural products anti AIDS through activity guidence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Dilute anti-DIG in PBS buffer (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4), prepare a coating solution with a final concentration of 8 mg / l. Add 60 μl of coating solution to each well, and coat for 24 hours. Wash the plate 3 times with Buffer A (50mM Tris-HCl, pH 7.8, 0.15M NaCl, 0.05% Tween-20) and once with Wash Buffer B (50mM Tris-HCl, pH 7.8, 0.15M NaCl) . Each well was blocked with 75 μl of blocking solution (1% BSA, 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl) for 1 hour. Wash the plate 3 times with buffer A and once with buffer B. Prepare the reaction mixture (0.75 A 260nm / ml template / primer[poly[A] / poly[dT] 15 ], 10 μM dTTP, 5 μM biotin-dUTP, 5 μM DIG-dUTP, 45.8 mM Tris, 265.8 mM KCl, 27.5 mM MgCl 2 , 9.17mM DTT), HIV-1 RT was diluted in lysis buffer (50mM Tris-HCl, pH 7.8, 80mM KCl, 2.5mM DTT, 0.75mM EDTA, 0.5% TritonX-100), and the final concentration of preparation was 0.05~ 500mU / μl HIV-1 RT solution, 20μl reaction mixture, 20μl lysis buffer...

Embodiment 2

[0036] Anti-DIG was diluted in PBS buffer solution to prepare a coating solution with a final concentration of 8mg / l. Add 60 μl of coating solution to each well, and coat for 24 hours. Wash the plate 3 times with buffer A and once with buffer B. Each well was blocked with 75 μl blocking solution for 1 hour. Wash the plate 3 times with Wash Buffer A and once with Wash Buffer B. Prepare the reaction mixture (0.75 A 260nm / ml template / primer[poly[A] / poly[dT] 15 ], 10 μM dTTP, 5 μM biotin-dUTP, 5 μM DIG-dUTP, 45.8 mM Tris, 265.8 mM KCl, 27.5 mM MgCl 2 , 9.17mM DTT), HIV-1 RT was diluted in the lysis buffer, and the final concentration of the preparation was 50 mU / μl HIV-1 RT solution, and a certain amount of Efavirenz was dissolved in the lysis buffer, and the Efavirenz solution was prepared, and 20 μl of the reaction The mixture, 20 μl of Efavirenz solution and 20 μl of HIV-1 RT solution were added to the PCR tube, and the reverse transcription reaction was performed on a PC...

Embodiment 3

[0040] Dissolve Efavirenz in lysis buffer to a final concentration of 250 ng / ml. Dissolve biotin in lysis buffer to a final concentration of 2 μg / ml. Efavirenz and biotin were co-dissolved in lysis buffer to a final concentration of 250 ng / ml Efavirenz and 2 μg / ml biotin. These 3 kinds of solutions and the positive control were used for method testing, and the results were as follows: figure 2 and image 3 shown. The incorporation of biotin has almost no effect on the detection of positive controls and known inhibitors. According to literature reports, the method of using commercial kits can cause an inhibition rate of 77.78% when incorporation of 0.2 μg / ml biotin is detected, which is False positives caused by biotin affecting the method itself. Even when the method is mixed with 10 times the concentration of 2μg / ml biotin, it still has almost no effect on the determination, which shows that this method can effectively avoid the interference caused by endogenous biotin, ...

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Abstract

A method of utilizing active-targeting to screen anti-AIDS natural product includes doping biotin-dUTP and digoxigenin-dUTP into DNA randowly to form digoxigenin-biotin double labeled NDA, catching DNA by using anti-dogoxigenin antibody specificity, using fluorescent labeled chain avidin and biotin labeled NDA specificity to carry out detection, knowing amount of biotin in synthesized NDA according to intensity of fluorescence then deriving out activity of HIV-I reversed transcriptive enzyme.

Description

technical field [0001] The invention relates to a screening method for HIV-1 reverse transcriptase inhibitors, in particular to its application in the activity-oriented discovery of anti-AIDS compounds in natural product crude extracts. Background technique [0002] HIV-1 reverse transcriptase (HIV-1 RT) inhibitors are potential therapeutic drugs for acquired immunodeficiency syndrome (AIDS), and most of the current AIDS therapeutic drugs are reverse transcriptase inhibitors. Reverse transcriptase has outstanding target specificity and is currently a research hotspot in the treatment of AIDS. The discovery of new and efficient HIV-1 reverse transcriptase inhibitors is of great application value. Natural products contain a wide variety of compounds with novel structures, and are one of the important sources of new drugs. At present, the activity screening of natural products mostly focuses on the screening of pure compounds obtained after separation. The probability of findi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70G01N33/50G01N33/52G01N21/76
Inventor 张卫袁景利胡政靳艳王桂兰虞星炬金美芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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