Human recombinant [beta]-interferon fusion protein

A fusion protein and interferon technology, applied in the field of biomedicine, can solve the problems of short plasma half-life of beta interferon, unbearable for patients, long action time, etc.

Inactive Publication Date: 2015-10-14
GUANGZHOU FULENGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, beta interferon has a short plasma half-life, and only frequent dosing can achieve satisfactory efficacy
However, such frequent administration combined with a long treatment cycle makes it unbearable for patients, and there is an urgent need to develop interferon beta with a long acting time

Method used

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  • Human recombinant [beta]-interferon fusion protein
  • Human recombinant [beta]-interferon fusion protein
  • Human recombinant [beta]-interferon fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0049] Example 2. Yeast expression and protein purification

[0050] The plasmid pPicZα-6His-Zipper1-CTHS-IFN was transformed into Pichia pastoris, and a single clone was picked and inoculated into a 1L separator shake flask containing 100ml of BMGY medium. Grow in a shaker at 28-30°C, 250-300rpm to OD600=2-6, centrifuge at 1500-3000g for 5min to collect the cells, remove the supernatant, and resuspend the cells with 1 / 5-1 / 10 of the original medium volume of BMMY (about 10-20ml), inoculated in a l00ml septum shaker flask, covered the mouth of the bottle with 2 layers of sterilized gauze or cheese cloth, and put it in a shaker to continue culturing. Every 24 h, add methanol to a final concentration of 0.5% to induce.

[0051] After the induction, centrifuge at 12,000rpm for 30min, take the supernatant, filter it with a filter membrane with a pore size of 0.45μm, and then perform Ni-NTA affinity chromatography with continuous gradient elution (equilibrium solution: 50mM Tris (p...

Embodiment 3

[0054] Example 3. Cytopathic effect inhibition (CPEI) of beta interferon

[0055] Add beta-interferon samples of different dilutions to a 96-well flat-bottomed culture plate, 100 μl / well; add standard products of different dilutions to a 96-well flat-bottomed culture plate, 100 μl / well; add 4X106 / ml Wish cells to each well, 100 μl / well; except the Wish cell control well, add VSV virus to each well, 50 μl / well; incubate in a 5% CO2, 37°C incubator for 48-52 hours; observe under the microscope that the virus control well shows cytopathy, discard the supernatant , add crystal violet staining solution for staining for 5 minutes; rinse the culture plate with running water until the washing solution is colorless, and dry the culture plate; add 150 μl / well of 70% ethanol to each well. Fully dissolve the crystal violet; compare the color on a microplate reader, and read the value at a wavelength of 600nm.

[0056] Avonex (beta-interferon produced by U.S. BIOGEN company) was used as a...

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Abstract

The invention relates to human recombinant [beta]-interferon fusion protein, which is a fusion protein of human [beta]-interferon and an anti-human serum albumin heavy chain single-domain antibody. The amino acid sequence of the fusion protein is represented as the Seq ID No.1 and the amino acid sequence of the anti-human serum albumin heavy chain single-domain antibody is represented as the Seq ID No.2. In the invention, by means of construction of the fusion expression cloning of the anti-human serum albumin heavy chain single-domain antibody and the [beta]-interferon and introduction into a host cell for expression, a heavy chain single-domain antibody-[beta]-interferon fusion protein having a long acting time is obtained.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a human recombinant beta interferon fusion protein. Background technique [0002] Beta interferon (also called beta interferon, Interferon-β, IFN-β) is a cytokine secreted by fibroblasts and macrophages in the body. It is a glycoprotein with 166 amino acids and a molecular weight of about 20. 500 Daltons, has anti-inflammatory function, anti-virus, anti-proliferation and immunomodulatory activities, and it can be widely used as a drug for anti-infection, anti-tumor and antagonism of autoimmune diseases. However, beta interferon has a short plasma half-life, and only frequent dosing can obtain satisfactory curative effect. However, such frequent dosing combined with a long treatment cycle makes it unbearable for patients, and there is an urgent need to develop interferon beta with a long acting time. [0003] Human serum albumin (HSA) is a natural material delivery carrier in human blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
Inventor 李华平张金阔钱柳青范名俊朱苗苗
Owner GUANGZHOU FULENGEN
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