Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

NGF mutant

A nerve growth factor, growth factor technology, applied in the direction of nerve growth factor, growth factor/inducing factor, nervous system diseases, etc., can solve the problems of severe NGF, pain, limited use, etc., to improve biological activity, reduce pain and side effects Effect

Active Publication Date: 2022-05-27
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
View PDF10 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although recombinant human NGF has avoided some potential risk of pathogenicity, there are still big problems in the actual use process: 1) Maintain the biological activity of NGF. Like other proteins, the biological activity of NGF depends on its secondary and tertiary proteins. Therefore, it is particularly important to maintain its biological activity during preparation, purification, storage, and administration; 2) NGF will cause severe pain during use, which some patients cannot tolerate limit
However, such a low dose limits the application of NGF, and also limits the expansion of its indications, such as use in the central nervous system

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • NGF mutant
  • NGF mutant
  • NGF mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Plasmid construction of wild-type hNGF and its mutants

[0062] 1. Construction of the DNA sequence expression plasmid of wild-type hNGF

[0063] Synthesize the DNA sequence of wild-type hNGF (SEQ ID NO: 1 in the sequence listing), and use primers (F: GGAATTCATGTCCATGTTG (SEQ ID NO: 41 in the sequence listing), R: CAAGCTTTCAGGCTCTTCT (SEQ ID NO: 42 in the sequence listing)) to carry out PCR on the target sequence Amplification, after digestion with EcorI (NEB #R0101S), the digestion product was subjected to secondary digestion with HindIII (NEB #R0104S). The pcDNA3.1(-) expression vector was digested using the same method. Carry out agarose gel electrophoresis on the digested vector and the PCR-amplified target fragment, cut out the target fragment, and use a DNA gel recovery kit (TIANGEN, #DP209-03) to recover the digested vector and target DNA fragment respectively. DNA ligase kit (Takara / 6022) was ligated at 16°C for 1 h to complete the plasmid construct...

Embodiment 2

[0066] Example 2. Plasmid transformation and extraction of hNGF and its mutants

[0067] 1. Conversion:

[0068] The hNGF and its mutant plasmids constructed in the above Example 1 were subjected to heat shock transformation. transform. 2ul plasmid was added to it, mixed by flicking, and ice-bathed for 30min. Dry bath at 42°C for 90s, do not shake the centrifuge tube during this period, and immediately place it on ice for 2min. Add 500ul of anti-anti-LB / SOC medium, 150rpm / min, and incubate at 37°C for 45min. Pour all the liquid in the centrifuge tube onto the LB plate and spread it evenly. After the plates were air-dried, they were placed upside down in an incubator for 16 h.

[0069] 2. Plasmid lift

[0070] Pick a single colony obtained in the above 2.1 experiment and inoculate it in 500ul LB liquid medium, cultivate at 37°C for 7h, and send the bacterial liquid to the detection sequence at the same time. Shake the bacteria with the correct sequencing in a large amoun...

Embodiment 3

[0071] Example 3. Expression of wild-type hNGF and its mutants

[0072] The wild-type hNGF and its mutant plasmids obtained in Example 2 above were transfected into 293F cells, and the expression supernatant was collected and quantified on the fourth day after transfection.

[0073] Experimental steps:

[0074] 1. One day before transfection, inoculate 293F cells, 0.5×10 6 / ml, a total of 900ml, 300ml / bottle.

[0075] 2. Count on the day of transfection, and the cell density is about 1.0×10 6 / ml, the viability rate is over 99%.

[0076] 3. Transfection: Take 36ml of cell culture medium into a 125ml culture flask; add 360ug plasmid, mix well; add 1080ug PEI, mix well. Let stand at room temperature for 15 minutes; mix with cells, about 12.3ml / vial; 37℃, 8%CO 2 , 120RPM culture.

[0077] 4. The cell supernatant was collected on the fourth day after transfection, and centrifuged at 10,000g for 20 minutes.

[0078] 5. Collect the supernatant and filter with 0.45um to obtain...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The nerve growth factor mutant is the amino acid sequence shown in any one of SEQ ID No: 3 to SEQ ID No: 21 in the sequence table. The advantage is that the nerve growth factor mutation can reduce pain side effects, and belongs to the field of biopharmaceuticals.

Description

technical field [0001] The invention relates to a mutant of nerve growth factor and belongs to the field of biopharmaceuticals. Background technique [0002] According to its neurophysiological mechanism, pain can be divided into two categories: receptive pain and neuropathic pain. The former is directly caused by noxious stimuli and is related to tissue damage or inflammatory response, also known as inflammatory pain; the latter is caused by damage to the somatosensory nervous system or Chronic pain as a direct result of disease. [0003] Nerve Growth Factor (NGF) is the first neurotrophic factor discovered in mouse sarcoma cells by Italian scientist Levi-Montlcini in 1953. NGF is a dual biological function of neuron nutrition and neurite outgrowth. It plays an important role in regulating the development, differentiation, growth, regeneration and expression of functional properties of central and peripheral neurons. NGF contains three subunits, α, β, and γ. The β subunit...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/48A61K38/18A61P25/00
CPCC07K14/48A61K38/00A61K38/18A61P25/00C07K2319/30C12N15/85A61K38/185
Inventor 王超马磊杜天飞
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com