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Application of TgCPC1 in preparation of drug for inhibiting inflammation

A technology to suppress inflammation and inflammatory response, applied in the field of biomedicine, can solve the problem that immunosuppressants cannot meet clinical needs

Active Publication Date: 2020-06-26
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available immunosuppressants are still far from meeting clinical needs

Method used

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  • Application of TgCPC1 in preparation of drug for inhibiting inflammation
  • Application of TgCPC1 in preparation of drug for inhibiting inflammation
  • Application of TgCPC1 in preparation of drug for inhibiting inflammation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The effect of TgCPC1 on the activity of NFκB promoter was detected by dual luciferase reporter gene method.

[0019] (1) Construction of TgCPC1 plasmid.

[0020] 1) Design of TgCPC1 gene primers.

[0021] Omega software was used to design primers for amplifying the open reading frame of TgCPC1 gene. The restriction sites at both ends of the primers were Not I and Kpn I respectively. The primers were synthesized by Guangzhou Aiji Biotechnology Co., Ltd. The sequences of the primers are as follows:

[0022] TgCPC1 forward primer: 5′-AAGGAAAAAAGCGGCCGCGATGGAGGGTATGGGGTCGCTGC-3′;

[0023] TgCPC1 reverse primer: 5'-CGGGGTACCTCACATTGTTTTACTTTTCAGAGCTTCAATAGC-3'.

[0024] 2) Amplify the TgCPC1 gene.

[0025] Toxoplasma gondii ME49 strain was extracted with RNA extraction kit (Omega) (Toxoplasma gondii ME49 strain has been described in the literature "Zhao YO, et al. death.PLoS pathogens.2009Feb; 5(2):e1000288.PubMed PMID:19197351.PubmedCentral PMCID:2629126.") total RNA wa...

Embodiment 2

[0057] The effect of TgCPC1 on the expressions of IL-1, IL-6, IL-8 and IL-12 downstream of NFκB transcription factors was detected by qPCR.

[0058] (1) Transfection

[0059] HEK293T cells were seeded into 24-well plates with a cell volume of 2×10 5 pcs / hole, CO 2 After culturing in a constant temperature incubator for 24 hours, when the cell density was 70%-80%, the plasmid was transfected with Lipo3000 liposome transfection kit. Set up the following test groups:

[0060] 1) Experimental group (CPC1 group): co-transfection of P65 (100ng / well) and 3×flag pCMV7.1-CPC1 plasmid (400ng / well);

[0061] 2) Blank control group: co-transfection of P65 (100ng / well) and p3XFLAG-CMV-7.1 plasmid (400ng / well).

[0062] After 24 hours of transfection, the cells were collected and RNA was extracted.

[0063] (2) RNA extraction

[0064] A. Add β-mercaptoethanol to the TRK lysate (OMEGA) at a ratio of 50:1, and add 350 μl of the prepared TRK and β-mercaptoethanol mixture to each well of ...

Embodiment 3

[0084] Western blot was used to detect the effect of TgCPC1 on NFκB signaling pathway proteins.

[0085] (1) Transfection

[0086] TLR4-HEK293T cells (invivogen) were seeded into 6-well plates with a cell volume of 8×10 5 pcs / hole, CO 2After culturing in a constant temperature incubator for 24 hours, when the cell density is 70%-80%, use Lipo3000 liposome transfection kit to transfect 2 μg / well of 3×flag pCMV7.1-CPC1 plasmid or p3XFLAG-CMV-7.1 plasmid . 24 hours after transfection, 100 ng of LPS was added to each well for stimulation, and the total protein was extracted at 0 min, 30 min, 1 h, 2 h, and 4 h after stimulation.

[0087] (2) Extraction of total protein

[0088] A. The total protein was extracted with western blot / IP cell lysate from Beyotime, and PMSF was added to the lysate to a final concentration of 1 mmol / L.

[0089] B. Aspirate the culture medium in the 6-well plate, wash it with PBS 3 times, add 100 μL of the lysate prepared in step A to each space, and ...

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Abstract

The invention discloses an application of TgCPC1 in preparation of a drug for inhibiting inflammation. The invention is obtained based on the achievement of the inventors for the first time that the transfection TgCPC1 can inhibit a natural immune NFkappaB signal pathway. The inventors provided by the invention find that the transfection TgCPC1 plasmid can inhibit the activity of a NFkappaB promoter induced by MyD88, TRAF6, IKKalpha, IKKbeta and P65, and the inhibitory effect increases with the increase of a TgCPC1 plasmid concentration; the transfection TgCPC1 plasmid can inhibit the expression of IL-1, IL-6, IL-8, IL-12 and TNFalpha at the downstream of the NFkappaB; and the transfection TgCPC1 plasmid can inhibit p65 phosphorylation. The results reveal that the TgCPC1 has an inhibitoryeffect on innate immunity, and lays a necessary foundation for the application of the TgCPC1 in preparation of the drug for inhibiting the inflammation.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of TgCPC1 in the preparation of drugs for inhibiting inflammatory reactions. Background technique [0002] The treatment of many diseases requires the use of drugs that suppress inflammatory responses, for example, patients with autoimmune diseases, organ transplant recipients, allergy patients, etc. However, currently available immunosuppressants are still far from meeting the clinical needs. Therefore, it is an urgent task for scientists to find highly efficient and low-toxic immunosuppressants. Contents of the invention [0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide the application of TgCPC1 in the preparation of medicines for inhibiting inflammatory reactions. [0004] The purpose of the present invention is achieved through the following technical solutions: the application of TgCPC1 ...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P29/00
CPCA61K38/1767A61K48/005A61P29/00Y02A50/30
Inventor 杨光刘宇美荆春霞张鲍欢区美玲叶晓红邹小倩
Owner JINAN UNIVERSITY
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