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Fusion protein IFN-ELP and application thereof

A fusion protein and protein technology, applied in the field of biomedicine, can solve the problems of difficult control of binding sites and coupling stoichiometry, prolonging the half-life of interferon circulation, and ineffective clinical test effects, and achieves improved pharmacokinetics, in vivo and other problems. The effect of prolonging the average retention time and improving the stability of the drug

Inactive Publication Date: 2017-05-10
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.
In the prior art, the circulating half-life of interferon can be effectively extended and the modification site can be effectively controlled by fusing human serum albumin, but the activity remains only 1%, and the effect of clinical trials is not obvious

Method used

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  • Fusion protein IFN-ELP and application thereof
  • Fusion protein IFN-ELP and application thereof
  • Fusion protein IFN-ELP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Prokaryotic expression and purification of embodiment 1, IFN-ELP and transpeptidase Sortase

[0083] 1. Preparation of recombinant plasmid pET-25b-IFN-ELP

[0084] Artificially synthesized recombinant plasmid pET-25b-IFN-ELP (double-stranded circular plasmid). The nucleotide sequence of the recombinant plasmid pET-25b-IFN-ELP is shown in sequence 1 of the sequence listing. There is an expression cassette A in the recombinant plasmid pET-25b-IFN-ELP.

[0085] The nucleotide sequence of the expression cassette A is shown in the 5121 to 7314 positions from the 5' end of Sequence Listing Sequence 1, wherein the 5121 to 5140 positions from the 5' end are T7 promoters, and the 5209 to 5706 positions are interferon- In the coding gene of α2, positions 5728 to 5745 are the recognition region of transpeptidase A, positions 5812 to 7161 are genes encoding elastin-like polypeptide, and positions 7265 to 7314 are T7 terminators. Interferon-α2 is hereinafter referred to as IFN. ...

Embodiment 2

[0119] Obtaining of embodiment 2, IFN and ELP

[0120] The steps to obtain IFN and ELP are as follows:

[0121] 1. Take 10 mL of the 100 μM SrtA solution prepared in step 3 of Example 1, add CaCl 2 And make the concentration 10mM to obtain a solution.

[0122] 2. Take 10 mL of the 200 μM IFN-ELP solution prepared in step 3 of Example 1, add 5 mM triglycine (product of sigma company) to it, then mix it with 10 mL of the solution obtained in step 1, and react overnight at room temperature to obtain the mixed solution 1 . The mixed solution 1 was passed through a HiPrep 26 / 10 Desalting column (product of GE Company) with pH 7.4 and 10 mM PBS buffer to remove small molecular impurities to obtain a solution, and the mixed solution 2 was obtained.

[0123] 3. After completing step 2, the mixed solution 2 was purified by anion exchange chromatography (HiTrap Capto Q 5 mL) on an AKTA protein purification system (AKTA Purifier 10, product of GE Company) to obtain an ELP solution.

...

Embodiment 3

[0128] The physicochemical characterization of embodiment 3, IFN-ELP

[0129] 1. Determination of molecular weight

[0130] The molecular weights of IFN-ELP, IFN and ELP obtained in the above steps were determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF), and the instrument was 4800Plus MALDI-TOF / TOF TM Analyzer (AB SCIEX), for specific measurement methods and steps, please refer to the instruction manual that comes with the instrument.

[0131] the result shows( Figure 5 ), IFN, ELP and IFN-ELP were purified by SrtA catalysis and anion exchange column (AEX). The measured molecular weight of IFN-ELP was 58236.1, and that of IFN was 20338.1, both of which were basically consistent with the theoretical values.

[0132] 2. Hydration radius

[0133]Dissolve the protein to be tested (IFN-ELP or IFN) with pH 7.4, 10 mM PBS buffer, and then filter through a 0.22 μm pore size filter to obtain the sample to be tested. The samples...

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Abstract

The invention discloses a fusion protein IFN-ELP and an application thereof. The provided fusion protein is the protein of b1) or b2), wherein b1) comprising fusion protein through fusion of functional protein and elastin-like polypeptide; and b2) the tagged protein by connecting N terminal or C terminal of b1) with a label. The invention also provides an application of the fusion protein in product preparation; and the product has the functions for inhibiting tumor cell proliferation / or treating tumor. Experiment proves that the fusion protein IFN-ELP is taken as a protein drug so as to increase the conservation rate of biological activity, prolong the in-vivo half life, prolong the in-vivo average persistence time, and can effectively inhibit tumor.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to fusion protein IFN-ELP and its application. 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 frequent administration is required to maintain a high blood concentration, which leads to serious side effects and brings heavy economic costs to patients. burden. Modification of IFN-α2 with polyethylene glycol (PEG) can effectively improve its pharmacokinetics, drug distribution and curative effect. However, the current PEGylated inte...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/21A61K47/48A61P35/00
CPCA61K38/21C07K19/00C12N5/10C12N15/62C12N15/63
Inventor 高卫平胡瑾王贵林
Owner TSINGHUA UNIV
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