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Agonist and application of Toll-like receptors 7 and 8

An agonist and receptor technology, used in antiviral agents, medical preparations containing active ingredients, organic active ingredients, etc., can solve the problems of high price, difficult to use in combination, etc. The effect of enhancing the immune function of the body

Inactive Publication Date: 2013-03-27
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, some immunomodulators, such as tumor necrosis factor a (TNF-a), interleukin 12 (IL-12) and interleukin 6 (IL-6) and other cytokines, have been used in antiviral and antitumor therapy In the middle, the curative effect is relatively ideal, but the price is expensive and it is difficult to use in combination

Method used

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  • Agonist and application of Toll-like receptors 7 and 8
  • Agonist and application of Toll-like receptors 7 and 8
  • Agonist and application of Toll-like receptors 7 and 8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Toll-like receptor 7 and 8 agonist example and preparation

[0020] Agonist name: ssRNA120

[0021] Sequence structure: GUCUGAGUGUGUUCUUG

[0022] Modification structure: rG*rU*rC*rU*rG*rA*rG*rU*rG*rU*rG*rU*rU*rC*rU*rU*rG (* is sulfo modification, r is RNA)

[0023] Preparation method: chemical synthesis, entrusted to the United States Integrated DNA technologies (IDT) company. The initial yield was 250 nmoles of RNA oligo, and the purified amount was 45.2 nmoles, or 255.5 μg.

[0024] Configuration: 1) Dilute ssRNA120 with HBS buffer to 0.1 μg / μl;

[0025] 2) Cationic liposome DOTAP is diluted with HBS buffer solution at a ratio of 1:3;

[0026] 3) Mix the above-mentioned step 1 and step 2 reagents at a ratio of 1:2 as a working solution.

Embodiment 2

[0027] Example 2: Determination of ssRNA120's immune activation effect by TLR7 (ELISA method)

[0028] 2.1 Main materials, reagents and equipment:

[0029] 1) Cell line: RAW264.7 (ATCC, USA.).

[0030] 2) Cell culture medium: DMEM (Gibco, Inc., USA).

[0031] 3) Reference reagent: fakeRNA120 (5'-GACAGAGAGAGAACAAG-3')

[0032] ssRNA40 (5'-GCCGUCUGUUGUGUGACUC-3')

[0033] (IDT, Inc. Synthesis)

[0034] 4) Elisa kit: anti-mouse TNF-a (eBioscience, Inc., USA).

[0035] 5) Cell incubator: FORMA 3111.

[0036] 6) Microplate reader: full-wavelength multifunctional microplate reader (Thermo Scientific, USA).

[0037] 2.2 Experimental method: Dilute RAW264.7 cells to 1×10 6 / mL, added to a 96-well plate (200 μL / well), placed at 37 ° C, 5% CO 2 After culturing in the incubator for 2 hours, add 5 μg / ml ssRNA120 as the experimental group; 5 μg / ml ssRNA40 as the positive control group; 5 μg / ml fakeRNA120 as the invalid control group; DOTAP-HBS buffer as the ...

Embodiment 3

[0039] Embodiment 3: ssRNA120 is measured by the immune activation effect of TLR8 (ELISA method)

[0040] 3.1 Main materials, reagents and equipment:

[0041] 1) Cell line: THP-1 (ATCC, USA.).

[0042] 2) Cell culture medium: RPMI1640 (Gibco, Inc., USA).

[0043]3) Elisa kit: anti-human TNF-a and anti-human IL-12p40 (eBioscience, Inc., USA).

[0044] 4) Cell incubator: FORMA 3111.

[0045] 5) Microplate reader: full-wavelength multifunctional microplate reader (Thermo Scientific, USA).

[0046] 3.2 Experimental method: dilute THP-1 cells to 1×10 6 / mL, added to a 96-well plate (200 μL / well), placed at 37 ° C, 5% CO 2 After culturing in the incubator for 2 hours, ssRNA120 was added to the concentration gradient of 1.25, 2.5, 5, 10, and 20 μg / ml to set up the experimental group of each dose; Repeat hole. After culturing for 24 hours, the supernatant was collected, and the Elisa experiment was performed according to the operation instructions of the kit to detect the conce...

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Abstract

The invention relates to an agonist and application of Toll-like receptors 7 and 8, wherein the agonist contains a nucleotide sequence segment with 17basic group characteristics, and has the basic structure of GUCUGAGUGUGUUCUUG. The agonist can combine specificity with TLR7 / 8, so the immune cells of an organism are promoted to efficiently release tumor necrosis factor (TNF)-a, interleukin (IL)-12, IL-6 and other cell factors, the immune function is up-regulated, and the agonist can be used for antiviral antineoplastic adjuvant therapy.

Description

technical field [0001] The invention relates to an agonist, in particular to a Toll-like receptor 7 and 8 (TLR7 / 8) agonist and its application. Background technique [0002] For viruses and tumors, until now, people have no good drugs to deal with them. For non-self-limiting viruses and various tumors, the body's immune system is still the main defense force. In recent years, some immunomodulators, such as tumor necrosis factor a (TNF-a), interleukin 12 (IL-12) and interleukin 6 (IL-6) and other cytokines, have been used in antiviral and antitumor therapy Among them, the curative effect is relatively ideal, but the price is expensive and it is difficult to use them in combination. [0003] Since the discovery of Toll-like receptors (TLRs) in the late 1990s, people have gradually realized that a variety of TLR family members are at the front line of the body's immunity, can specifically recognize different pathogenic pattern molecules, and initiate innate immunity to block e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/117A61K31/7088A61P31/12A61P35/00
Inventor 李彦郑江周红祝元峰曹红卫
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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