Fusion protein Slit2D2 (C386S)-HAS and application thereof in treating fibrosis diseases

一种融合蛋白、蛋白的技术，应用在生物医药领域，能够解决半衰期短等问题，达到分子量小、提高稳定性、组织渗透性好的效果

Active Publication Date: 2017-03-29
SICHUAN ASCLEPIUS BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Protein drugs with a molecular weight of less than 20kD are easily filtered by the glomerulus during metabolism, resulting in a short half-life in the body. In order to achieve therapeutic effects, frequent or large doses of drugs are often required, which brings great inconvenience to patients.

Method used

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  • Fusion protein Slit2D2 (C386S)-HAS and application thereof in treating fibrosis diseases
  • Fusion protein Slit2D2 (C386S)-HAS and application thereof in treating fibrosis diseases
  • Fusion protein Slit2D2 (C386S)-HAS and application thereof in treating fibrosis diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of fusion protein Slit2D2(C386S)-HSA

[0073] According to the known Slit2 sequence [GenBank:EAW92793.1], the second structural domain Slit2D2 of Slit2 was designed and constructed, and Slit2D2 (C386S) was designed as shown in SEQ ID NO: 1, and then Slit2D2 (C386S) and Slit2D2 (C386S) were designed - The coding sequences of HSA are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 4.

[0074] The coding sequence of Slit2D2(C386S)-HSA was obtained by whole gene synthesis, and inserted into the expression vector pCDNA3.4 (brand: Thermo, catalog number: A14697) by T / A cloning, and the recombinant vector pCDNA3.4-Slit2D2(C386S)- HSA profile such as figure 1 shown. The above-mentioned recombinant expression vector was transformed into Escherichia coli TOP10, and then transferred to a solid medium containing ampicillin (AMP) for propagation, positive clones were screened, and the success of vector construction and species preservation were confirmed by...

Embodiment 2

[0077] Example 2 Determination of the affinity of the fusion protein to the target protein Robo1 by SPR

[0078] Utilize the SPR (Surface Plasmon resonance BIAcore200) method to detect the affinity constant between the protein and the Robo1 protein, bind the Robo1 (ORIGEN company, product number: TP327713) protein on the CM5 chip, and analyze the fusion protein Slit2D2(C386S)-HSA (Example 1), the interaction between Slit2D2-HSA (see patent application PCT / CN2015 / 092079) and the receptor protein Robo1. Kinetic measurements were performed according to the protocol of Canziani et al. (2004, Anal. Biochem. 325:301-307). At the same time, the affinity between Slit2N protein (Slit2N is a protein with a molecular weight of about 120 KDa at the N-terminal of slit2 protein) and Robo1 protein was also determined by the same method. The results are shown in Table 1.

[0079] Table 1 The results of SPR determination of the affinity of the fusion protein to the receptor Robo1

[0080] ...

Embodiment 3E

[0082] Embodiment 3ELISA measures protein stability

[0083] 1. Reagents:

[0084] Neutroavidin-HRP dilution;

[0085] Coating buffer: -0.16%Na 2 CO 3 ;-0.3% NaHCO 3 ;-pH9.8;

[0086] Washing buffer: PBS containing -0.1% Tween20;

[0087] Blocking buffer: washing buffer containing -1% Goat Serum;

[0088] TMB: purchased from Shanghai Biyuntian Biotechnology Co., Ltd.;

[0089] Stop solution (Stop solution): purchased from Shanghai Biyuntian Biotechnology Co., Ltd.;

[0090] NOTE: All antibodies are diluted in blocking buffer.

[0091] 2. Experimental process:

[0092] Robo1 protein was diluted to 1 μg / ml, coated with 100 μl / well, and left overnight at 4°C. Wash the plate 3 times with Washing buffer. Blocking buffer (200μl / well) was blocked at room temperature for 2 hours. Wash the plate 3 times with Washing buffer. The samples to be tested (fusion protein Slit2D2(C386S)-HSA (prepared in Example 1), Slit2D2-HSA (see patent application PCT / CN2015 / 092079) 100 μl) wer...

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PUM

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Abstract

The invention relates to the technical field of biological medicine, in particular to a fusion protein Slit2D2 (C386S)-HAS and application thereof in treating and / or preventing fibrosis diseases. In the fusion protein, amino acid residues are subjected to mutation on the basis of the Slit2D2 structural domain, and stability of the fusion protein is improved compared with un-mutation proteins. The fusion protein is obtained through fusion of Slit2D2 (C386S)-HAS protein and HAS protein, and the internal metabolism time of medicine is prolonged while stability of the medicine is improved. The fusion protein provided by the invention has a better effect on the prevention and treating aspect of the fibrosis diseases, in particular to pulmonary fibrosis, than positive contrast medicine and shows good druggability of the medicine.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fusion protein Slit2D2(C386S)-HSA and its application in treating and / or preventing fibrotic diseases. Background technique [0002] Fibrosis can occur in a variety of organs. The main pathological changes are the increase of fibrous connective tissue and the reduction of parenchymal cells in organ tissues. Continuous progress can lead to organ structural damage, functional decline, and even failure, which seriously threaten human health and life. As long as any reason can cause tissue cell damage, it can lead to tissue cell degeneration, necrosis, and inflammatory response. If the damage is small, normal parenchymal cells around the damaged cells will undergo hyperplasia and repair, and this repair can completely restore normal structure and function. However, if the damage is large or repeated damage exceeds the regeneration capacity of the parenchymal cells around the da...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C07K19/00C12N15/62A61K38/17A61K47/64A61P11/00A61P31/00
CPCC07K14/4702A61K38/00C07K2319/31A61K38/1709A61P9/00A61P11/16C12N15/62A61K9/0019A61K9/19A61K9/08C07K14/765C07K14/47C12N15/85
Inventor 任宝永李华顺刘鹏
Owner SICHUAN ASCLEPIUS BIOTECHNOLOGY CO LTD
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