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A recombinant β-hngf-fc fusion protein, preparation method and application

A fusion protein, -hngf-fc technology, applied in the field of recombinant biology, can solve problems such as inability to carry out protein post-translational modification

Active Publication Date: 2019-10-15
SINOBIOWAY BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of the growing demand for injectable mouse nerve growth factor, today's NGF products have two potential drawbacks: 1. It is potentially immunogenic because it is a mouse protein; 2. It requires a large number of qualified male mice Submandibular gland, which has become a major bottleneck to ensure production requirements
However, the recombinant expression of NGF through prokaryotic expression systems (such as E.coli) has the disadvantage of being unable to perform protein post-translational modification

Method used

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  • A recombinant β-hngf-fc fusion protein, preparation method and application
  • A recombinant β-hngf-fc fusion protein, preparation method and application
  • A recombinant β-hngf-fc fusion protein, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of pCHO1.0-hNGF-hFc expression vector

[0046] The hNGF-hFc nucleic acid sequence is shown in SEQ ID NO: 2, and the translated amino acid sequence is shown in SEQ ID NO: 1.

[0047] SEQ ID NO:1 is as follows:

[0048]

[0049] Among them, 1-18aa is the signal peptide, 19-121aa is the leader peptide, 122-239aa is the mature peptide, 240-251aa is the connecting short peptide, and 252-478aa is the Fc fragment. The structure of the hNGF-hFc fusion protein obtained by recombinant secretion expression is Mature peptide-linked short peptide-hFc.

[0050] SEQ ID NO: 2 is as follows:

[0051]

[0052]

[0053] Among them 1-54 bp is the hNGF signal peptide sequence; 55-363 bp is the hNGF leader peptide sequence; 364-717 bp is the hNGF mature peptide sequence; 718-753 bp is the linking short peptide sequence, which is lysine-threonine-glycine- Glycine-glycine-serine-glycine-glycine-glycine-serine-valine-glutamic acid; 754-1437bp is the hIgG Fc segment sequence.

[0...

Embodiment 2

[0060] Example 2: Construction and screening of β-hNGF-Fc recombinant cell strain

[0061] Plasmid purification: Use the plasmid large extraction kit to extract and purify the plasmid pCHO1.0-hNGF-hFc, and use Bio-Spec-Nano to determine the concentration and purity of the plasmid. The concentration of the plasmid used for transfection must be ≥1.0μg / μl, A260 / A280=1.7-1.9. If the concentration is insufficient, use isopropanol to precipitate the plasmid and concentrate to an appropriate concentration.

[0062] CHO cell activation: remove the frozen CHO cells from liquid nitrogen, remove the protective solution, and add appropriate amount of CD FortiCHO medium to a cell density of 5×10 5 , In a 125ml shake flask, 37℃, 100r / min, 5% CO2 conditions. Cell density to 2×10 6 When, use appropriate amount of medium to dilute to 5×10 5 , Until the cell viability reaches more than 90%.

[0063] Transfection: On the day of transfection, the cell density is 1×10 6 , The vitality should be greater...

Embodiment 3

[0068] Example 3: Recombinant expression, separation, purification and characterization of β-hNGF-Fc

[0069] Recombinant expression

[0070] The β-hNGF-Fc recombinant cell strain obtained in Example 2 was fed into culture. Seed cell culture to a density of 2×10 6 , The vitality is above 90%, the fermentation can be inoculated, and the initial density of fermentation is 5×105. Measure the cell density, viability and glucose content in the culture medium every day, and supplement FeedC medium to make the final concentration of glucose in the culture medium 4g / L. The fermentation ends when the cell viability is ≤70%. The culture fluid was collected by centrifugation for separation and purification.

[0071] Protein A affinity chromatography separation and purification of β-hNGF-Fc

[0072] use Purifer100 chromatography system for affinity chromatography, the column is HiTrapMabSelect 1mL. The operation of the instrument was carried out according to the operating instructions, the A...

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Abstract

The invention discloses a recombinant beta-hNGF-Fc fusion protein as well as a preparation method and application. The recombinant beta-hNGF-Fc fusion protein sequentially consists of the amino acid sequences shown by human beta NGF, an oligopeptide linker and human IgG Fc. On the mole basis, the recombinant beta-hNGF-Fc fusion protein has in-vitro bioactivity equivalent to or higher than that of human NGF. The invention also discloses a recon and a cell strain comprising the recombinant beta-hNGF-Fc fusion protein. he recon and cell strain are applied to the recovery of peripheral neuropathy and nerve injury as well as the long-acting application of nerve growth factor.

Description

Technical field [0001] The present invention relates to the field of recombinant biology technology, in particular to design a recombinant β-hNGF-Fc fusion protein, a preparation method and application. Background technique [0002] Nerve growth factor (NGF) has a wide range of effects, and β subunit is the active area of ​​NGF. Currently commercialized β-mNGF (NOBEX) is obtained from the submandibular gland of adult male mice. It is widely used in the treatment of diseases such as ophthalmology, neurosurgery, orthopedics, neurology, pediatrics, endocrinology, neuroatrophy, neurodegeneration, and trauma repair. Because of the increasing demand for mouse nerve growth factor for injection, today’s NGF products have two potential defects: 1. Because it is a mouse-derived protein, it has potential immunogenicity; 2. It requires a large number of qualified male mice The submandibular gland, which has become a major bottleneck to ensure production demand. However, the recombinant e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/85A61P25/02
Inventor 张文宇黄奋飞章永垒白羊陈星阮卡陈胜亮葛平辉邹有土马燕玲王明灶
Owner SINOBIOWAY BIOMEDICINE