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Application of TgGRA12 to preparation of drugs for inhibiting inflammatory response

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

Pending Publication Date: 2020-07-21
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 TgGRA12 to preparation of drugs for inhibiting inflammatory response
  • Application of TgGRA12 to preparation of drugs for inhibiting inflammatory response
  • Application of TgGRA12 to preparation of drugs for inhibiting inflammatory response

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The effect of TgGRA12 on the activity of NF-κB promoter was detected by dual luciferase reporter gene method.

[0029] (1) Construction of TgGRA12 plasmid.

[0030] 1) Design of TgGRA12 gene primers.

[0031] Omega software was used to design primers for amplifying the open reading frame of the TgGRA12 gene. The restriction sites at both ends of the primers were Bgl II and XbaI, respectively. The primers were synthesized by Invitrogen. The sequences of the primers were as follows:

[0032] TgGRA12 forward primer: 5'-GGAAGATCTGATGAGGGCGATCGTGGCATCGA-3';

[0033] TgGRA12 reverse primer: 5'-TGCTCTAGATCAGTTGTGTTTGCTGCCTGCAGAGCC-3'.

[0034] 2) Amplify the TgGRA12 gene.

[0035] 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 was reverse-transcribed to obt...

Embodiment 2

[0079] The effects of TgGRA12 on the expressions of IL-6 and IFN-β downstream of NF-κB transcription factors were detected by qPCR.

[0080] (1) Transfection

[0081] Human embryonic kidney cells (HEK293T cells) were inoculated 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:

[0082] 1) Blank group: p3×Flag-CMV-7.1 plasmid (200ng / well).

[0083] 2) Control group: MyD88 (100 ng / well) and p3×Flag-CMV-7.1 plasmid (200 ng / well) were co-transfected.

[0084] 3) Experimental group (GRA12 group): co-transfect MyD88 (100ng / well) and p3×Flag-CMV7.1-GRA12 plasmid (200ng / well).

[0085] After 24 hours of transfection, the cells were collected for RNA extraction.

[0086] (2) RNA extraction

[0087] A. Add β-mercaptoethanol to the TRK lysate (OMEGA) at a volume rati...

Embodiment 3

[0107] Western blot was used to detect the effect of TgGRA12 on NF-κB signaling pathway proteins.

[0108] (1) Transfection

[0109] TLR4-HEK293T cells (invivogen) were seeded into 6-well plates with a cell volume of 8×10 5 pcs / hole, CO 2 After 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 of p3×Flag CMV7.1-GRA12 plasmid or p3×Flag-CMV-7.1 plasmid / hole. After 24 hours of transfection, 100ng of LPS was added to each well to stimulate, and at 0min, 15min, 30min, 1h, 2h, and 4h after stimulation, the cell pellet was collected to extract the total protein, or the nucleoplasmic protein was separated, and the cytoplasmic protein and nuclear protein were extracted respectively. protein.

[0110] (2) Extraction of total protein

[0111] a) 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...

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Abstract

The invention discloses application of TgGRA12 to preparation of drugs for inhibiting an inflammatory response. According to the application, it is discovered that GRA12 can inhibit the activity of anuclear factor kappa-B (NF-kappa-B) promoter, and meanwhile, it is discovered that transfection of TgGRA12 plasmids can inhibit the activity of the NF-kappa-B promoter induced by MyD88, TRAF2, TRAF6,TGF TAK1+TAB1, IKK-alpha, IKK-beta and P65 and the inhibiting effect is enhanced with the increase of the concentration of the TgGRA12 plasmids; transfection of the TgGRA12 plasmids can inhibit expression of downstream IL-6 and TNF-alpha of NF-kappa-B; and transfection of the TgGRA12 plasmids can inhibit activation of IKK-alpha / beta, degradation of I-kappa-beta and the phosphorylation of p65. Theresults reveal the inhibiting effect of TgGRA12 on innate immunity, and lay a necessary foundation for using TgGRA12 for preparation of drugs for inhibiting the inflammatory response.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of TgGRA12 in the preparation of medicines 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 TgGRA12 in the preparation of drugs for inhibiting inflammatory reactions. [0004] The purpose of the present invention is achieved through the following technical solutions: the application of TgGRA...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P29/00A61P37/02A61P17/02A61P37/06A61P37/08
CPCA61K48/005A61P29/00A61P37/02A61P17/02A61P37/06A61P37/08
Inventor 杨光区美玲荆春霞张鲍欢叶晓红邹小倩刘宇美
Owner JINAN UNIVERSITY
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