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Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof

A technology of polyethylene glycol and alpha interferon, which is applied in the direction of pharmaceutical formulations, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve the problem of reducing clearance, reducing renal clearance rate, reducing protein, cell And bacteria adsorption and other problems, to achieve the effect of long half-life in vivo, strong liver accumulation capacity, and low plasma clearance rate

Inactive Publication Date: 2014-04-30
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This protective shell reduces the clearance of these materials in biological systems (such as the human body), greatly reduces the adsorption of proteins, cells and bacteria, and reduces the clearance rate of the kidneys

Method used

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  • Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof
  • Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof
  • Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Preparation of linear polyethylene glycol-carrying marker protein recombinant porcine interferon-alpha

[0026] Dilute the interferon to 0.1mg / ml with 5mM potassium dihydrogen phosphate buffer solution pH3.0-5.0; add the calculated amount of interferon, adjust the pH value to 8.0 with NaOH solution, and use the interferon:mPEG ratio of 1:20 Add 1 / 4 amount of mPEG-SC (Mw=20KD) to the amount of substance, react at 0-4°C, then add 1 / 4 amount of mPEG-SC every 2h, continue to react for 4h after adding, add 0.75 M glycine 1ml to stop the reaction; then the above reaction solution was diluted with 5 times the volume of sodium acetate buffer solution with a concentration of 50mM pH7.2, and then applied to a carboxymethylcellulose column (Waterman CM-52), with After washing the column with sodium acetate buffer solution of pH 7.2, it was eluted with sodium acetate buffer solution containing 0.5M NaCl, and the eluate containing PEG-interferon was collected, concentrated, and dete...

Embodiment 2

[0027] Embodiment 2: the detection of product by SDS-PAGE electrophoresis

[0028] The concentrated elution peaks were detected by SDS-PAGE electrophoresis, the separation gel was 10%, and the stacking gel was 5%. After electrophoresis, fix the colloid (fixative solution: 30% methanol, 5% acetic acid) and shake at room temperature for 1 hour (staining solution: 0.05% Coomassie brilliant blue, 30% methanol, 5% acetic acid); finally transfer the colloid into the decolorization solution (decolorization solution) Solution: 1% glutaraldehyde, 30% methanol, 5% acetic acid) at room temperature shaking and soaking until the bands are clear.

Embodiment 3

[0029] Example 3: 100TCID can be inhibited on WISH cells for purified polyethylene glycol-carrying recombinant porcine alpha interferon of tagged protein 50 The result of the multiplication of the VSV virus is carried out titer determination;

[0030] WISH cells were treated with different doses of modified interferon, discarded after 24 hours, and then inoculated with 100TCID 50 The VSV virus; the results showed that the recombinant porcine interferon-alpha modified by polyethylene glycol could significantly inhibit the cytopathy caused by VSV, and the activity of the obtained single-modified product had reached more than 25% through titer determination.

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Abstract

The invention discloses a polyethylene glycol-recombinant porcine interferon alpha conjugate and a preparing method thereof. The preparation method comprises the steps: under a weakly alkaline condition, coupling polyethylene glycol with amino of a recombinant porcine interferon alpha, and purifying to obtain the target product. The modification rate of the polyethylene glycol modified recombinant porcine interferon alpha is about 50%, the purity is about 97% or more, the in-vivo retention time is long, and the blood plasma half-life period reaches 16-38 hours. The conjugate can be applied in preparation of animal antiviral drugs.

Description

technical field [0001] The invention relates to the fields of biomedicine and chemical industry, in particular to a preparation method of a recombinant porcine alpha interferon-polyethylene glycol conjugate carrying a marker protein, and its use in the preparation of veterinary antiviral drugs. Background technique [0002] PEG (polyethylene glycol) is a neutral, water-soluble, non-toxic polymer. It is one of the few FDA-approved polymers that can be used on body surfaces and in vivo, and is widely used in food, cosmetics, personal healthcare and pharmaceutical industries. The characteristics of PEG are revealed by its water-soluble behavior. In aqueous solution, long-chain PEG molecules have high hydration (that is, water molecules are bound to its surface). An important property of PEG is its ability to attach to other molecules and surfaces, providing a biocompatible, protective shell. This protective shell reduces the clearance of these materials in biological systems ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K38/21A61P31/12
Inventor 王明丽李增赵俊王彩玲
Owner ANHUI JIUCHUAN BIOTECH
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