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Hand-foot-and-mouth disease resistant drug activity detection method and kit

A technology of drug activity and detection method, which is applied in the field of medicine to achieve the effect of good repeatability, small workload, fast and accurate experiment

Active Publication Date: 2016-01-20
JIANGSU KANION PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, as of now, no drug based on this target has been officially approved for marketing

Method used

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  • Hand-foot-and-mouth disease resistant drug activity detection method and kit
  • Hand-foot-and-mouth disease resistant drug activity detection method and kit
  • Hand-foot-and-mouth disease resistant drug activity detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1 anti-hand, foot and mouth disease drug activity screening method

[0089] 1. Load the nucleotide sequence (CAAGGU) corresponding to the substrate Gln-Gly of 2A and 3C proteases into pGloSensor TM -10F plasmid (Promege Company), utilizes cell-free in vitro transcription and translation system to synthesize substrate recombinant luciferase;

[0090] 2. In vitro recombinant expression of 2A and 3C proteases of each hand, foot and mouth virus, including EV71, CoxA16, CoxA2, ​​CoxA4, CoxA5, CoxA7, CoxA9, CoxA10, CoxB1-5 and 2A and 3C proteases of ECHO virus;

[0091] 3. Incubate the obtained recombinant luciferase with 2A and 3C proteases and drugs at 30°C for 1-4 hours, and detect the bioluminescence intensity (RLU) of luciferin.

[0092] The protease reaction system is:

[0093] Substrate: 1~10μg

[0094] Protease: 10~100IU

[0095] Test substance: >0μmol / L

[0096] Hepes buffer (10~100mM,

[0097] pH7.0~8.0): Make up to 100μL

[0098] Taking no test su...

Embodiment 2

[0105] Embodiment 2 trans-epoxysuccinyl-L-leucyl amido (4-guanidino) butane (E-64) inhibition test

[0106] Using the recognized cysteine ​​protease inhibitor trans-epoxysuccinyl-L-leucyl amido (4-guanidino) butane to study its inhibitory effect on the activity of various hand, foot and mouth virus 2A and 3C proteases, verify reliability and applicability of this method.

[0107] 1. Materials and methods

[0108] 1.1 Substrate

[0109] In pGloSensor TM The CAAGGU fragment was inserted into the -10F plasmid, and a luciferase containing Gln-Gly amino acid residues was synthesized after in vitro cell-free transcription and translation, provided by Promege.

[0110] 1.2 Protease

[0111] The 2A and 3C proteases of EV71, CoxA16, CoxA2, ​​CoxA4, CoxA5, CoxA7, CoxA9, CoxA10, CoxB1-5, and ECHO virus were expressed and purified in vitro by Promega, with a purity of >85%.

[0112] 1.3 Reagents

[0113] Trans-epoxysuccinyl-L-leucylamino (4-guanidino) butane (E-64) was purchased fro...

Embodiment 3

[0125] Embodiment 3 investigates the inhibition of different antiviral drugs to EV71 virus 3C protease

[0126] Adopt the method described in the present invention, aim at the screening verification of existing antiviral compound inhibiting EV71 virus 3C protease activity

[0127] 1. Materials and methods

[0128] 1.1 Substrate

[0129] In pGloSensor TM The CAAGGU fragment was inserted into the -10F plasmid, and a luciferase containing Gln-Gly amino acid residues was synthesized after in vitro cell-free transcription and translation, provided by Promege.

[0130] 1.2 Protease

[0131] The 3C protease of EV71 virus was expressed and purified in vitro by Promega Company, with a purity of >85%.

[0132] 1.3 Reagents

[0133] The antiviral compounds ribavirin, oseltamivir, peramivir, favipiravir and rupintravir were all purchased from Bailingwei Technology Co., Ltd., and the luciferin detection kit Bright-Glo TM Purchased from Promega Company, the rest of the reagents were d...

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Abstract

The invention relates to the technical field of medicines and in particular relates to a hand-foot-and-mouth disease resistant drug activity detection method and kit. The detection method comprises the following steps: inserting a nucleotide sequence encoding Gln-Gly into a luciferase plasmid, thereby obtaining recombinant luciferase containing Gln-Gly by virtue of in-vitro acellular transcription and translation; performing in-vitro recombinant expression on hand-foot-and-mouth virus protease, wherein the hand-foot-and-mouth virus protease refers to 2A or 3C protease; carrying out an enzymatic reaction between the hand-foot-and-mouth virus protease, the recombinant luciferase containing Gln-Gly and a test substance, detecting the bioluminescence intensity of fluorescein, thereby obtaining the hand-foot-and-mouth disease resistant drug activity of the test substance. The detection method disclosed by the invention has the advantages of simplicity, trace amount and rapidness, is small in drug screening workload, rapid and accurate in experiment and high in experimental result repeatability and can be used for screening and developing hand-foot-and-mouth disease resistant drugs. The method refers to a bioluminescence detection method based on luciferase, the sensitivity is high, a few interference factors exist, and the compatibility of the test substance is high.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a method for detecting the activity of an anti-hand, foot and mouth disease drug and a kit thereof. Background technique [0002] Hand, foot and mouth disease is a pediatric disease caused by a virus and belongs to the legal category C infectious diseases in my country. Infants and young children aged 0-6 are susceptible to the disease, among which children aged 2-3 are most commonly infected. After infection, children first cause herpes or ulcers on the limbs and oral cavity. Most of the children are given timely and effective treatment. Heal within 1-2 weeks. However, the condition of a small number of children progresses rapidly, and central nervous system and cardiopulmonary infections may occur within 1-5 days of onset, which has a high risk of death, and sequelae may still remain in surviving cases. [0003] According to the report of the World Health Organization, there...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/37
Inventor 萧伟曹泽彧丁玥曹亮李娜丁岗王振中
Owner JIANGSU KANION PHARMA CO LTD
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