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Small molecule polypeptide tat-p53dm and its application in the preparation of medicines for treating or preventing ischemic stroke

A small molecule polypeptide, ischemic stroke technology, applied in the field of medical disease treatment drug research and development, can solve problems such as unobserved toxic side effects, reduce nerve cell death and brain damage, have no toxic side effects, and facilitate transformation and production.

Active Publication Date: 2015-10-28
WUHAN QR SCI & TECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current in vivo and in vitro tests have shown that HIV-TAT can pass through all tissue cells including neurons, and no obvious toxic side effects have been observed

Method used

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  • Small molecule polypeptide tat-p53dm and its application in the preparation of medicines for treating or preventing ischemic stroke
  • Small molecule polypeptide tat-p53dm and its application in the preparation of medicines for treating or preventing ischemic stroke
  • Small molecule polypeptide tat-p53dm and its application in the preparation of medicines for treating or preventing ischemic stroke

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthesis of TAT-p53DM

[0036] The sequence of TAT-p53DM is shown in SEQ ID NO.1, which was artificially synthesized by Wuhan Baiyixin Biotechnology Co., Ltd. The synthesis report is as follows, and the chromatogram is as follows figure 1 shown.

[0037] TAT-p53DM synthetic HPLC report

[0038]

[0039] Table 1

[0040]

[0041] After synthesis, the purity of the TAT-p53DM polypeptide is 99%, 5mg per piece, in the form of white powder, stored at -20 degrees in the dark, and completely soluble in water. Before use, it is prepared by diluting with sterile saline according to the specified concentration. Before using in cells or animals, preheat in a 37-degree incubator.

[0042] The control of TAT-p53DM is TAT-scramble-p53DM (TAT-s-p53DM), its sequence is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Cys- Pro-Gly-Glu-Cys-Val-Arg-Thr-Arg-Arg-Arg (YGRKKRRQRRR-CCPGECVRTRRR) was also synthesized by the company.

Embodiment 2

[0044] TAT-p53DM blocks the biological effect of the combination of neuronal DAPK1DD and p53DM, and inhibits the signaling downstream of DAPK1 that causes neuronal apoptosis and necrosis

[0045] After packaging the Flag-tagged DAPK1DD plasmid (Flag-DAPK1DD) and the GST-tagged p53DM plasmid (GST-p53DM) with lentivirus respectively, co-transfect the primary neurons on the 10th day of in vitro culture, and then transfer the 3rd day (primary On the 13th day of culture), TAT-p53DM synthetic peptide at a concentration of 5 μM or control TAT-s-p53DM or Vehicle was administered for incubation, and cell protein was extracted after 2 hours. Anti-Flag antibody was used to precipitate cell protein, and then anti-GST antibody was used to detect the precipitated protein. The detection of black blot on NC membrane indicated that DAPK1DD interacted with p53DM. The results showed that after the administration of TAT-p53DM, the anti-GST antibody could not detect the GST-p53DM protein on the NC...

Embodiment 3

[0047] Application of TAT-p53DM in cell model of ischemic stroke

[0048] (1) Primary neuron culture and construction of OGD cell model of ischemic stroke

[0049] The mice on the 20th day of the embryonic period were taken out from the uterus of the mother mice, quickly immersed in 75% ethanol, and after decapitation, the cerebral cortex tissue was taken out in the dissecting solution (D-Han k's Solution), and the papain dissociation kit was used to , Worthington Biochemical Corporation) digested and separated cortical cells, and the cells were seeded on 19 mm square coverslips coated with 30 μg / ml poly-lysine and laminin, and the cell seeding density was about 100–150 cells / The coverslips were placed in a 12-well plate containing fresh serum-free Neurobasal medium (21103, GIBCO) and 2% B27, and the same medium was replaced every 4 days. The immunocytostaining of the neuron marker β-tubulin III (Tuj1) on the cultured neurons proved that more than 85% of the cultured cells w...

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Abstract

The invention discloses a micro-molecule polypeptide TAT-p53DM and an application thereof to preparing a medicine for treating or preventing ischemic stroke. A fusion protein polypeptide TAT-p53DM of a TAT protein transduction domain and p53DM is artificially synthesized; TAT carries p53DM protein polypeptide to pass through a blood brain barrier through blood so as to be taken by nerve cells; used in an in-vitro and in-vivo ischemic stroke model, the micro-molecule polypeptide TAT-p53DM is capable of effectively playing a biological role in blocking binding between death domain of death associated protein kinase 1 (DAPK1DD) and tumor suppression protein p53DNA binding motifs (p53DM), inhibiting signals capable of causing neuronal apoptosis and necrosis at DAPK1 downstream, and reducing ischemic stroke brain injury; moreover, molecular targets are provided for further developing medicines for clinically treating ischemic stroke.

Description

technical field [0001] The present invention relates to the field of research and development of drugs for the treatment of medical diseases, in particular to a small molecule polypeptide TAT-p53DM, and also relates to the application of the small molecule polypeptide TAT-p53DM in the preparation of drugs for treating or preventing ischemic stroke. technical background [0002] Stroke is characterized by high morbidity, high mortality, and high disability rate, and is recognized as a common and refractory disease that seriously endangers human health and life safety. Ischemic stroke is caused by interruption of cerebral blood flow (ischemic stroke) ) is a serious neurological disease caused by thrombosis or embolism, and the patient will suddenly appear paralyzed, language function impairment or vision loss or even death. In the United States, there is usually one stroke every 40 seconds, and one death every four minutes. According to statistics in 2007, the mortality rate o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00A61K38/16A61P9/10A61P25/00
CPCA61K38/00C07K14/4746C07K2319/10
Inventor 鲁友明裴磊朱铃强徐传瑞
Owner WUHAN QR SCI & TECH DEV
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