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Recombinant beta-hNGF-Fc fusion protein as well as 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: 2016-10-12
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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  • Recombinant beta-hNGF-Fc fusion protein as well as preparation method and application
  • Recombinant beta-hNGF-Fc fusion protein as well as preparation method and application
  • Recombinant beta-hNGF-Fc fusion protein as well as 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 nucleic acid sequence of hNGF-hFc 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 shown below:

[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 hNGF-hFc fusion protein structure obtained by recombinant secretory expression is Mature peptide-linked short peptide-hFc.

[0050] SEQ ID NO: 2 is shown below:

[0051]

[0052]

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

Embodiment 2

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

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

[0062] CHO cell activation: Take out the frozen CHO cells from liquid nitrogen, remove the protective solution, and add an appropriate amount of CD FortiCHO medium to a cell density of 5×10 5 , in a 125ml shake flask, cultured at 37°C, 100r / min, 5% CO2. Cell density up to 2 x 10 6 , dilute to 5×10 with an appropriate amount of medium 5 , until the cell viability reached more than 90%.

[0063] Transfection: On the day of transfection, the cell density was 1×10 6 , the vitality sho...

Embodiment 3

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

[0069] recombinant expression

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

[0071] Separation and Purification of β-hNGF-Fc by Protein A Affinity Chromatography

[0072] use Purifer100 chromatography system was used for affinity chromatography, and the chromatographic column was HiTrapMabSelect 1mL. The operation of the instrument was car...

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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 invention relates to the field of recombinant biotechnology, in particular designing a recombinant β-hNGF-Fc fusion protein, its preparation method and application. Background technique [0002] Nerve growth factor (NGF) has a wide range of effects, and the β subunit is the active region of NGF. Currently commercially available β-mNGF (NOBEX), which was isolated and purified 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, neurodegeneration, trauma repair and other diseases. Due to 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 r...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/85A61P25/02
CPCC07K14/48C07K2319/30C12N15/62
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