Artificial fusion protein and application thereof

A fusion protein and artificial technology, applied in the field of biomedicine, can solve the problems of reduced biological activity, low reaction yield, difficult administration of interferon-α2, etc., and achieve the effect of increasing half-life

Inactive Publication Date: 2018-08-10
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current PEGylated interferon has disadvantages such as low reaction yield, difficulty in controlling the binding site and conjugation stoichiometry, and severely reduced biological activity.
By fusing human serum albumin, the circulating half-life of interferon can be effectively improved and the modification site can be effectively controlled, but the activity remains only 1%, and the effect of clinical trials is not obvious
[0003] Therefore, how to effectively solve the problems such as difficult administration of interferon-α2 and unsatisfactory therapeutic effect is a key issue for researchers to be solved

Method used

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  • Artificial fusion protein and application thereof
  • Artificial fusion protein and application thereof
  • Artificial fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Physicochemical Characterization of IFNα-Sup35

[0098] The molecular weight of IFNα and IFNα-Sup35 was determined by quadrupole / ionization time-of-flight mass spectrometry (Q-TOF). The samples were first separated by nanoACQUITY high-performance liquid chromatography system, and then separated by SYNAPT-G2-Si mass spectrometer. The mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. Afterwards, the samples were placed in an autosampler for electrospray ionization and analyzed in a Q-TOF (SYNAPT G2-Si, Waters company) mass spectrometer. image 3 The Q-TOF analysis results are shown.

[0099] The hydration radius of IFNα and IFNα-Sup35 was determined by dynamic light scattering (DLS) method on Malvern Zetasizer Nano-zs90. Samples were diluted in PBS buffer and filtered through a 0.22 μm pore size filter before testing. As tested by DLS, the hydrated diameter of IFNα is 7nm, while the ...

Embodiment 2

[0103] Example 2 In vitro biological activity measurement and biological safety of IFNα-Sup35

[0104] The anti-cell proliferation activity and biological safety of IFNα-Sup35 in the present invention are determined by MTT method. We chose human Burkitt's B lymphoma cells (Daudi B) to test the anti-proliferation activity of IFNα-Sup35NP, because the cells are highly sensitive to IFN-α2. Human mammary epithelial cells (L929) and mouse fibroblasts (MCF-10) Daudi B cells were used to test the biological safety of IFNα-Sup35NP. After the cells were cultured in RMPI-1640 containing 10% FBS, 50 U / mL penicillin and 50 μg / mL streptomycin for a period of time (L929 and MCS-10 were cultured in DMEM medium), a certain concentration of The cell suspension (50 μL / well, 104 cells), the IFNα and IFNα-Sup35NP samples were serially diluted, 50 μL each was added to a 96-well culture plate, and a negative control (without IFN-α2) and a blank control (only cultured solution), 37°C, 5% CO2 for 7...

Embodiment 3

[0107] Example 3 Pharmacokinetic test of IFNα-Sup35NP

[0108] All the following animal experiments were completed under the guidance of Tsinghua University's regulations on animal experiments. In the present invention, the BABL / C athymic female nude mouse model was used to inject IFNα or IFNα-Sup35NP through the tail vein, and the change of interferon concentration in blood over time was measured, and the data was analyzed by DAS software. Before drug treatment, six 8-week-old male BABL / C with a body weight of about 20 g were observed for a period of time and randomly divided into two groups. IFNα and IFNα-Sup35NP were injected into the tail vein at a dose of 50 μg / 20 g, and then 10 μL of blood was collected by docking the tail at the set time point, left at room temperature for 30 min, and the supernatant serum was collected by centrifugation at 4°C and 3000×g. Store in a -80°C low-temperature refrigerator. The content of IFN-α2 in the serum was measured with the human IFN...

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Abstract

The invention provides a method for improving the half life of a hydrophilic substance. The method comprises connecting the hydrophilic substance to a self-assembled polypeptide, wherein and the connection site is located in a non-interference region of the active site of the hydrophilic substance. According to the invention, the self-assembled polypeptide is connected with the non-interfering region of the active site of the hydrophilic substance, and the formed artificial fusion protein can form nano particles under the self-assembly mediation of the self-assembled polypeptide; in addition,compared with the hydrophilic substance, the nano particles have greatly improved stability, pharmacokinetics and biodistribution in an organism, and the half life is obviously improved.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to artificial fusion proteins and their uses. More specifically, the present invention relates to methods for increasing the half-life of hydrophilic substances, artificial fusion proteins, artificial nucleic acids, constructs, recombinant cells, obtained Methods, nanoparticles and pharmaceutical compositions for artificial fusion proteins. Background technique [0002] Interferon-α2 (IFN-α2) has anti-viral replication, anti-tumor proliferation and immunomodulatory effects, and has been successfully used to treat viral diseases (such as hepatitis B, hepatitis C, genital warts, etc.) and related cancers (such as leukemia, kidney cancer, malignant melanoma, multiple sclerosis, etc.). However, after systemic injection, IFN is easily degraded by proteases in the body and excreted by the kidneys. The half-life of the circulation is very short, and frequen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62B82Y30/00B82Y5/00A61K38/21A61K47/42A61K9/51A61P31/12A61P35/00A61P37/02
CPCA61K9/5169A61K38/00A61P31/12A61P35/00A61P37/02B82Y5/00B82Y30/00C07K14/56C07K2319/31
Inventor 高卫平徐志坤
Owner TSINGHUA UNIV
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