Small molecular polypeptide inhibiting MIRI and vector and application thereof

A small molecule polypeptide and carrier technology, applied in the field of biomedicine, can solve problems such as long ischemia time and myocardial ischemia-reperfusion injury, and achieve inhibition of myocardial cell apoptosis, resistance to myocardial cell oxidative stress and apoptosis, and inhibition of Effects of MIRI on development

Inactive Publication Date: 2016-08-31
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heart can tolerate ischemia for a certain period of time, but if the ischemia time is long, the restoration of blood supply will easily lead to myocardial ischemia-reperfusion injury. At present, the main means of reducing reperfusion injury is reasonable drug treatment

Method used

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  • Small molecular polypeptide inhibiting MIRI and vector and application thereof
  • Small molecular polypeptide inhibiting MIRI and vector and application thereof
  • Small molecular polypeptide inhibiting MIRI and vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The cloning vector construction of embodiment 1 polypeptide analog

[0021] Rat mRNA was extracted, reverse-transcribed into cDNA, and cDNA was used as a template to successfully amplify the corresponding mRNA sequence by PCR technology (primer sequence: upstream-Forward: 5'-GCTGAATTCACCA TGGATGTAGAAAACGAGCAGA-3'; downstream-Reverse: 5' - TCGCTCGAGATCCAGG GGCAGAACCAC-3'), its sequence is shown in SEQ ID NO.1, and the corresponding expressed amino acid sequence is shown in SEQ ID NO.2. The amplified fragments are separated by horizontal electrophoresis, and the purified fragments are obtained using a recovery kit. The fragment purification product and pcDNA A3.1-Flag vector plasmid were digested with EcoR1\Xho1 enzyme for 2 hours at 37°C. Separation by horizontal electrophoresis, using a recovery kit to obtain fragment products with exposed cohesive ends. In a water bath at 16°C, the fragment and the carrier were ligated at a ratio of nucleic acid amount (3-10): 1 for ...

Embodiment 2

[0022] Example 2 Determination of the interaction between polypeptide analogs promoting PDCD4 and Nrf2

[0023] Flag-PDCD4-1 that will be constructed △ 1-180aa Transfected into rat cardiomyocytes, after 36-48 hours of transfection, the cells were collected and lysed with IP lysate (50mM Tris-HCl pH8.0, 150mM NaCl, 1% NP-40, 5mM EDTA, 20mM MgCl 2 , 1×Roche phosphatase&Protease inhibitor cocktail) to lyse the cells, centrifuge at 13000g, 4°C for 15min. Transfer the supernatant to a new tube, keep a part of the sample as Input, add 4 μg of the control Flag antibody and incubate overnight, then add 30 μL Protein A / G beads for immunoprecipitation. After washing 4 times with IP lysate, add 25 μL of loading buffer and boil for 5 min, and immunoblot to confirm the interaction between PDCD4 and Nrf2, such as Figure 1-3 as shown, figure 1 Figure A is the endogenous interaction map of myocardial tissue, indicating that in the rat myocardial ischemia-reperfusion model, there is an in...

Embodiment 3

[0025] 1) LDH detects the ability of peptide analogues to inhibit myocardial cell injury after myocardial ischemia-reperfusion: Cardiomyocyte H9c2 was seeded in a 96-well culture plate at a density of 20,000 / well, and the 265aa polypeptide eukaryotic expression vector and other The control group was transfected with H9c2 cells, and subjected to hypoxia / reoxygenation stimulation 36 hours later. The cell culture medium was taken, centrifuged at 13,000 rpm at 4°C, and detected with an LDH detection kit (Nanjing Jiancheng), and the absorbance was measured at 450 nm. The magnitude of the absorption value reflects the damage of the cells. According to the experimental data, the time is taken as the abscissa and the absorption value is plotted as the ordinate. LDH test results such as Figure 4 As shown, it shows that compared with the empty control group, the 60aa polypeptide eukaryotic expression experiment group obviously aggravated the cell damage.

[0026] 2) TUNEL assay to det...

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Abstract

The invention discloses small molecular polypeptide inhibiting MIRI and a vector and application thereof. A bioengineering technology is utilized, a section of sequence corresponding to polypeptide analogue is genetically recombined to a p3XFLAG-CMV eukaryotic expression vector; after it is proved that recombination is successful through enzyme digestion and sequential analysis, plasmid corresponding to the eukaryotic expression polypeptide analogue is transfected into myocardium cells, protein expression of polypeptide is proved through immunoblotting, and polypeptide recombination is achieved. It is proved through co-immunoprecipitation that the polypeptide analogue can promote interaction of PDCD4 and Nrf2, it is shown through cell biology experiments that the polypeptide analogue has the capacity of inhibiting myocardium cell apoptosis caused by myocardial ischemia and reperfusion injuries and the functions of resisting oxidative stress and apoptosis of the myocardium cells and inhibiting occurrence and development of MIRI.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a small molecular polypeptide for inhibiting the occurrence of MIRI, its carrier and application. Background technique [0002] In recent years, the morbidity and mortality rate of acute myocardial infarction (AMI) in the population has risen sharply, and it tends to become younger. Therefore, the effective prevention and treatment of AMI has become a hot spot of global concern. Myocardial ischemia is mainly due to the formation of thrombus on the basis of platelet activation and aggregation after the rupture of coronary atherosclerotic plaque, resulting in acute coronary occlusion. The most effective way to restore coronary blood flow is vascular recanalization. At present, various methods for the treatment of cardiac ischemic diseases are aimed at reducing myocardial ischemia-reperfusion injury. The heart can tolerate ischemia for a certain period of time, but if the isch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/85A61K38/17A61P9/10
CPCA61K38/00C07K14/4747
Inventor 沈爱国刘晓娟杨晨尤庆生
Owner NANTONG UNIVERSITY
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