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Multifunctional fusion polypeptide and preparation method and application thereof

A fusion polypeptide, multi-functional technology, applied in the preparation method of peptide, fusion polypeptide, hybrid peptide and other directions, can solve the problems of single target, easy to produce drug resistance, lack of specific drugs for the treatment of pulmonary fibrosis, etc. The method is simple, the effect of increasing the activity of anti-pulmonary fibrosis and anti-pulmonary infection, and good application prospect

Active Publication Date: 2016-06-29
NANJING ANJI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of existing polypeptide drugs such as single target, easy drug resistance, and lack of specific drugs for the treatment of pulmonary fibrosis, the present invention provides a multifunctional fusion polypeptide and its preparation method and application, which can treat human pulmonary fibrosis , lung tissue lesions, lung cancer and other tumors

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  • Multifunctional fusion polypeptide and preparation method and application thereof
  • Multifunctional fusion polypeptide and preparation method and application thereof
  • Multifunctional fusion polypeptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation and inspection of the multifunctional polypeptide of the present invention

[0043] In this example, polypeptides I-VI were all synthesized by solid-phase synthesis, separated and purified by preparative HPLC, and the purity of the polypeptide was determined by analytical high-performance liquid chromatography.

[0044] Polypeptide I-VI solid-phase synthesis method uses solid-phase carrier Fmoc-wang-resin (Gill Biochemical Co., Ltd.) as the starting material, and then uses protected amino acids to inoculate dipeptide to nonacupeptide in sequence, and fully washes the peptide after the work is completed. , Peptide cutting, and post-treatment to obtain the crude product of the polypeptide; dissolve the crude product, purify twice with preparative high-performance liquid phase, concentrate and freeze-dry to obtain the pure product, and finally obtain the refined product of the polypeptide through the third purification. The method can not only ensure the synthe...

Embodiment 2

[0073] In this example, polypeptides I-VI were all synthesized by liquid phase synthesis, separated and purified by preparative HPLC, and the purity of the polypeptide was determined by analytical high performance liquid chromatography. The following are the synthesis steps of polypeptide I, and the synthesis steps of polypeptide II-VI are the same as those of polypeptide I.

[0074] Polypeptide I synthesis steps are as follows:

[0075] 1. According to the sequence of polypeptide I, in 10 mL of dichloromethane, 1 mg of the first amino acid Pro and 1 mg of the second amino acid D-Pyr are connected through an amide bond, and the inactive groups of the amino acids participating in the reaction are modified with Fmoc.

[0076] 2. Add 10 mL of ammonia water to the above reaction system to remove the Fmoc group.

[0077] 3. Repeat the reaction of the first step, add the third amino acid D-Cys according to the sequence of the synthesized polypeptide, and modify the inactive group o...

Embodiment 3

[0081] Establishment of In Vitro Pulmonary Fibrosis Model

[0082] Human non-small cell lung cancer cell A549 was cultured in DMEM medium containing 10% (volume fraction) fetal bovine serum, cultured in a 5% carbon dioxide incubator at 37°C, and the medium was changed every other day, depending on the cell density. Take A549 cells in the logarithmic growth phase, digest with 0.25% trypsin to make cell suspension, adjust the cell concentration to 1×10 9 pcs / L, and according to 4×10 5 piece / cm 2 Density seeded at 90cm 2 In the cell culture dish, they were randomly divided into two groups: (1) control group: cultured with DMEM containing 10% (volume fraction) fetal bovine serum; (2) model group: containing 10% (volume fraction) fetal bovine serum, DMEM culture of transforming growth factor (TGF-β1, final concentration 5 μg / L). Place them in an incubator at 37°C with a volume fraction of 5% carbon dioxide, change the medium every other day, and passage as scheduled depending o...

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Abstract

The invention discloses a multifunctional fusion polypeptide and a preparation method and application thereof, and belongs to the field of biological pharmacy. The fusion polypeptide has the structural domains of Pro-(D-Pyr)-(D-Cys)-Bip-Arg-Gly-Glu, Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro, Arg-Gly-Asp and Gly-Gly-Gly-Gly; the fusion polypeptide can treat human lung fibrosis, pathological changes of lung tissue, lung cancers and other tumors, and in a lung fibrosis cell model, the fusion polypeptide can significantly reduce the hydroxyproline content in model group cells and inhibit the process of lung fibrosis. It is shown through MTT tests that the fusion polypeptide can inhibit proliferation of multiple kinds of anthropogenic tumor cells; the fusion polypeptide is prepared through an artificial synthesizing method, the preparation method is simple, and a good application prospect is achieved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and more specifically relates to a new multifunctional fusion polypeptide and its preparation method and application. Background technique [0002] Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF) has long been considered as a progressive and basically irreversible pathological change. The five-year mortality rate of IPF patients after diagnosis reaches 65%, which seriously threatens public health. . Due to the minimal efficacy of current treatment measures, IPF has not even formed a recognized treatment plan. The most common symptom of pulmonary fibrosis is: difficulty breathing. In mild pulmonary fibrosis, dyspnea often occurs with strenuous activity and is therefore often overlooked or misdiagnosed as another disorder. When pulmonary fibrosis progresses, dyspnea also occurs at rest, and patients with severe pulmonary fibrosis may develop progressive dyspnea. [0003] A v...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K5/09C07K7/06C07K1/02C07K1/06A61K38/16A61P11/00A61P31/00A61P35/00
CPCC07K5/0817C07K7/06A61K38/00C07K2319/01A61K38/16C07K1/02C07K1/04C07K1/06C07K14/00C07K19/00A61P11/00A61P35/00C07K14/8146C07K2319/00
Inventor 康梦实
Owner NANJING ANJI BIOLOGICAL TECH CO LTD
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