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Nerve growth factor mutants

一种神经生长因子、突变体的技术，应用在生物制药领域，能够解决疼痛、NGF严重、使用受限等问题，达到减轻疼痛副作用、生物活性提高的效果

Inactive Publication Date: 2022-06-28
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Plasmid construction of wild-type hNGF and its mutants

[0063] 1. Construction of the DNA sequence expression plasmid of wild-type hNGF

[0064] 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

[0067] Example 2. Plasmid transformation and extraction of hNGF and its mutants

[0068] 1. Conversion:

[0069] 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.

[0070] 2. Plasmid lift

[0071] 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

[0072] Example 3. Expression of wild-type hNGF and its mutants

[0073] 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.

[0074] Experimental steps:

[0075] 1. One day before transfection, inoculate 293F cells, 0.5×10 6 / ml, a total of 900ml, 300ml / bottle.

[0076] 2. Count on the day of transfection, and the cell density is about 1.0×10 6 / ml, the viability rate is over 99%.

[0077] 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.

[0078] 4. The cell supernatant was collected on the fourth day after transfection, and centrifuged at 10,000g for 20 minutes.

[0079] 5. Collect the supernatant and filter with 0.45um to obtain...

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Abstract

The invention relates to a nerve growth factor mutant, and belongs to the field of biological pharmacy. The nerve growth factor mutant is an amino acid sequence as shown in any one of SEQ ID No: 3 to SEQ ID No: 21 in a sequence table. The invention has the advantage that the nerve growth factor mutation can relieve the side effect of pain.

Description

[0001] This application is a divisional application of the patent application with the application number of 201780017019.9 and the invention name of "Nerve Growth Factor Mutant". technical field [0002] The invention relates to a mutant of nerve growth factor and belongs to the field of biopharmaceuticals. Background technique [0003] According to its neurophysiological mechanism, pain can be divided into two types: 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. [0004] 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 devel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/48C12N15/12A61K38/18A61P3/04A61P25/00
CPCC07K14/48A61K38/00A61K38/18A61P25/00C07K2319/30C12N15/85A61K38/185
Inventor 王超马磊杜天飞
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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