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Preparation method of active peptide Apelin

A technology of active peptide and solid-phase synthesis method, applied in the field of biomedicine, can solve the problems of complex biotechnology and difficult large-scale production, and achieve the effect of prolonging half-life and improving activity

Active Publication Date: 2021-04-06
温州医科大学慈溪生物医药研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its preparation requires relatively complex biotechnology and is difficult to produce on a large scale

Method used

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  • Preparation method of active peptide Apelin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: The solid-phase synthesis of 2-(4-chlorophenyl)-2,2-difluoroacetic acid-QRPRLSHKGPMPF

[0018] (1) Weigh 1mmol of 2-cl-Trt resin in a solid-phase synthesizer, add 15mL of anhydrous dichloromethane (hereinafter referred to as DCM), place on a shaker and shake for 5min to fully swell the 2-Cl-Trt resin ;

[0019] (2) DCM is removed from the solid-phase synthesizer equipped with 2-Cl-Trt resin with ear washing ball;

[0020] (3) Dissolve 0.75 mmol of Fmoc-Phe in 10 mL of anhydrous DCM, add 0.75 mmol of DIPEA, then transfer to the above-mentioned solid-phase synthesizer, add 0.75 mmol of DIPEA, and react at room temperature for 1 h;

[0021] (4) Sealing: remove the reaction liquid in the solid-phase synthesizer with ear washing balls, then wash with 10 mL of anhydrous DCM, each time for 1 min, and wash 5 times in total, add the prepared volume ratio of anhydrous DCM: DIPEA: methanol =17:1:2 solution 20mL, react at room temperature for 10min;

[0022] (5) Re...

Embodiment 2

[0027] Example 2: Regulation of target active peptides on intracellular cAMP content

[0028] Using unmodified [Pyr1]-Apelin-13 as a control, the regulation of intracellular cAMP content by the target active peptide of the present invention was determined. The HEK293 cell line was transfected along with a luciferase reporter gene [cAMP response element (CRE, 4X)-luciferase] to stably express full-length human hAPLNR (amino acids 1-380 of accession number NP_005152.1). The resulting cell line HEK293 / CRE-luc / hAPLNR was cultured in DMEM containing 10% FBS, NEAA, penicillin / streptomycin and 100 μg / mL hygromycin B. HEK293 / CRE-luc / hAPLNR cells were seeded on 96-well assay plates at 20,000 cells / well in 80 μL OPTIMEM supplemented with 0.1% FBS and penicillin / streptomycin / L-glutamine and incubated at 37°C for 5 %CO 2 Incubate for 16 hours. Serial dilutions (1 :3) of unmodified [Pyrl]-Apelin-13 and the active peptide of interest were then mixed with forskolin in assay buffer (5 μΜ f...

Embodiment 3

[0030] Embodiment 3: Experiment of the biological half-life of the target active peptide in rats

[0031] (1) Experimental animal information

[0032] SPF grade SD rats, 16 males, weighing 190g to 210g, were provided by the Experimental Animal Center of Wenzhou Medical University.

[0033] (2) Dosing regimen and plasma sample collection and processing

[0034] Intravenous administration, the dosage was 10mg / kg, the rats were randomly divided into positive control [Pyr1]-Apelin-13 solution group, experimental group 2-(4-chlorophenyl)-2,2-di Fluoroacetic acid-QRPRLSHKGPMPF solution group, a total of 2 groups (n=8), fixed rats, administered through the tail vein. At different time points after administration, 0.2 mL of whole blood was collected through the jugular vein cannula and placed in a 1.5 mL LEDTA-2K anticoagulant centrifuge tube. ) to seal the tube for smooth collection at the next time point. The blood sample was centrifuged at 4000g for 5 minutes at 4°C, the plasma w...

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Abstract

The invention relates to a preparation method of an active peptide Apelin, which comprises the steps of preparing Apelin by adopting a solid-phase synthesis method, and further connecting 2-(4-chlorphenyl)-2,2-difluoroacetic acid by adopting the solid-phase synthesis method. The active peptide Apelin (2-(4-chlorphenyl)-2,2-difluoroacetic acid-QRPRLSHKGPMPF) is obtained through the solid-phase synthesis method, and the activity of the active peptide Apelin obtained on the basis of the method can be further improved while the half-life period of the active peptide Apelin can be prolonged.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of active peptide Apelin. Background technique [0002] Apelin is an important functional peptide in the human body. Under the action of protease in vivo, the Apelin propeptide containing 77 amino acids is cleaved into active Apelin fragments, which can be divided into Apelin-13 (thirteen peptides) and pyroglutamic acid-type Apelin-13 ( [Pyrl]-Apelin-13), Apelin-17 (17-peptide), Apelin-36 (36-peptide), Apelin-55 (55-pentadepeptide), etc. (European Journal of Pharmacology 2015, 763: 149-159), Herein, the above-mentioned Apelin fragments are collectively referred to as "Apelin". [0003] Apelin is the endogenous ligand of the angiotensin II-like-1 receptor (APJ). APJ receptor, also known as Apelin receptor, is a member of the orphan G-protein-coupled receptor (GPCR) family. The structure of the APJ receptor is similar to the angiotensin II t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/04C07K1/06C07K1/107A61K38/10A61K47/54
CPCC07K14/57563A61K38/10A61K47/542Y02P20/55
Inventor 孔晓霞张宏宇肖健张海邻
Owner 温州医科大学慈溪生物医药研究院
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