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Polypeptide for constructing long QT syndrome animal model

A technology of QT syndrome and animal models, applied in the direction of polypeptides, hybrid peptides, and mammalian proteins containing positioning/targeting motifs, which can solve problems such as difficulty in functioning and limitations of biological macromolecules, and the process is simple and effective , easy to obtain, high modeling efficiency

Active Publication Date: 2020-11-06
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the role of the natural barrier of the cell membrane, biomacromolecules are limited in a large number of practical applications, and it is difficult to play a role in cells

Method used

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  • Polypeptide for constructing long QT syndrome animal model
  • Polypeptide for constructing long QT syndrome animal model
  • Polypeptide for constructing long QT syndrome animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0045] Example 2 Langendorff perfusion experiment.

[0046] Guinea pigs were injected with heparin (150U / kg / ip) and anesthetized with sodium pentobarbital (40mg / kg / ip). The trachea is connected to an artificial respirator. The aorta was cannulated in situ under artificial respiration. The heart is quickly removed and mounted on a perfusion set. Coronary arteries were retrogradely perfused with Krebs-Henseleit (K-H) solution (5.4mM glucose; 116.0mM NaCl; 3.6mM KCl; 23.0mM NaHCO 3 ; 1.16mM KH 2 PO 4 ; 1.2mM CaCl 2 ; 0.58mM MgSO 4 ; 0.3mM pyruvate; 2.8U L -1 insulin), and the heart was kept in a constant temperature room at 37°C. Coronary perfusion pressure was monitored during aortic cannulation and adjusted to a range of approximately 60-70 mmHg. Place the negative pole of the electrode in the right atrium, and the positive pole in the left ventricle (apex), and record the electrocardiogram (QT interval) using a biological experiment system. In this experiment, the is...

Embodiment 3

[0049] Example 3 GST Pull-Down experiment.

[0050] The concentration of polypeptide CM4 was 1 μg / ul. GST-CT1 (2-4ug) was immobilized on GS-4B, incubated in 300μL system for 4h, 4℃, CM4 concentration gradient (0.1, 0.3, 1.0, 3.0, 10.0μM), system calcium ion concentration (0, 100nM , 10 μM, 2 mM). Calculated by WEBMAXC v2.10 software [Ca 2+ ]. After incubation, with the corresponding Ca 2+ Gently wash the GST-4B-GST-CT1 system twice with buffer, and discard the supernatant for the last time. Then 15μl 5×SDS Loading buffer was used for elution treatment. The supernatant was taken, and the combination was detected by 15% SDS-PAGE electrophoresis. Coomassie brilliant blue R (Coomassie brilliant blue R, CBB) staining and decolorization, such as Figure 4 shown. like Figure 4 In order to successfully detect the combination of CM4 and CT1 ([Ca 2+ ]≈free, 100nM, 10μM, 2mM), correspondingly labeled GST-CT1 and CM4 at molecular weights of 54 and 4kDa. CM4 binds to CT1 with p...

Embodiment 4

[0051] Example 4 Whole animal model experiment.

[0052] The mice were randomly divided into three groups: (1) Control group: 0.9% NaCl; (2) CM4 group: 21 mg / kg CM4; (3) CM4-R group: 21 mg / kg CM4-R. All mice were intraperitoneally injected twice a day at 8:00 and 20:00. After 3 weeks, mice in each group were anesthetized with isoflurane (3%, inhalation). To determine the effect of CM4 on the QT interval in mice, we first detected the ECG using a biological experiment system (Chengdu Taimeng Software Co., Ltd., Chengdu, China). The two ECG electrodes are placed in the standard lead II direction. To calculate the QT interval, the formula for calculating QTc is: Bazett's formula (QTcB[ms]=QT[ms] / (RR[s])1 / 2), such as Figure 5 and Image 6 shown.

[0053] Figure 5 Electrocardiograms were monitored 3 weeks after administration of the polypeptides to the mice. The results showed that the electrocardiogram waveform of the Control group (0.9% NaCl) was stable and the cycle was...

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Abstract

The invention belongs to the field of animal experiment modeling methods, and relates to a polypeptide for constructing a long QT syndrome animal model and a method for establishing an animal model for an animal experiment. According to the polypeptide for constructing the long QT syndrome animal model, an amino acid sequence of the polypeptide is GRKKRRQRRRGEVDEMIREADIDGDGQVNYEGFVQMM, and the polypeptide is named as CM4. The method for establishing the long QT syndrome animal model comprises the step of administering the polypeptide CM4 to an animal to obtain the long QT syndrome animal model. The model establishing method provided by the invention is simple and reliable, and is convenient for researchers to establish a long QT syndrome mouse or guinea pig isolated heart model and carry out related experimental researches. The modeling process is simple and effective, meanwhile, a corresponding control group can be set, and electrocardiogram changes before and after amino acid mutation are compared and analyzed. The CM4 is given to induce the QT interval of isolated hearts of mice and guinea pigs to be prolonged, the CM4 is easy to obtain and high in purity, the CM4 can penetratethrough cell membranes to enter cells to play a role, the activity of myocardial calcium channels is adjusted, the molding efficiency is high, the cost is low, and research of related experiments is facilitated.

Description

technical field [0001] The invention belongs to the field of animal experiment modeling methods, and relates to a polypeptide for constructing an animal model of long QT syndrome and a method for establishing an animal model for animal experiments, in particular to an animal model of long QT syndrome. Background technique [0002] Long QT syndrome (long Q-T syndrome, LQTS), refers to the delayed ventricular repolarization caused by the prolonged QT interval on the electrocardiogram, which is the main cause of sudden death and syncope, but there is no obvious abnormality in the heart structure. Divided into two categories: congenital and acquired. Studies have shown that Na + , K + , Ca 2+ Gene mutations of ion channels or calmodulin (CaM) can lead to long QT syndrome. [0003] CaM passes through with Ca 2+ Binding is activated to regulate cardiac Ca V 1.2 The opening and closing of the channel, CaM gene mutation can weaken the Ca V 1.2 channel Ca 2+ CDI-dependent ina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A01K67/02
CPCA01K67/02C07K14/4728C07K2319/10
Inventor 郝丽英苏敬阳
Owner 中国医科大学
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