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Low-pain nerve growth factor mutant

A nerve growth factor and mutant technology, applied in the field of biopharmaceuticals, can solve the problems of severe NGF, pain, limited use, etc.

Active Publication Date: 2017-10-24
STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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  • 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

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Example 1. Plasmid construction of wild-type hNGF and its mutants

[0137] 1. Construct the DNA sequence expression plasmid of wild-type hNGF

[0138] Synthesize the DNA sequence of wild-type hNGF (SEQ ID NO: 1 in the sequence table), use primers (F: GGAATTCATGTCCATGTTG (SEQ ID NO: 50 in the sequence table), R: CAAGCTTTCAGGCTCTTCT (SEQ ID NO: 51 in the sequence table) to carry out PCR amplification of the target sequence Increase, use EcorI (NEB#R0101S) to carry out enzyme digestion, use HindIII (NEB#R0104S) to carry out the second enzyme digestion of the digestion product. Use the same method to carry out digestion of pcDNA3.1 (-) expression vector. Enzyme Carry out agarose gel electrophoresis after cutting the vector and amplifying the target fragment by PCR, excise the target fragment, use the DNA gel recovery kit (TIANGEN, #DP209-03) to recover the digested vector and the target DNA fragment respectively, and use DNA ligation Enzyme kit (Takara / 6022) was ligated at...

Embodiment 2

[0141] Plasmid transformation and extraction of embodiment 2, hNGF and its mutants

[0142] 1. Conversion

[0143] The hNGF and its mutant plasmids constructed in the above Example 1 were subjected to heat shock transformation, and the Top10 competent cells (Tiangen / CB104-02) were taken out from the -70°C refrigerator and immediately thawed on ice, and 50ul was used for transform. Add 2ul of plasmid to it, flick to mix well, and ice bath for 30min. Dry bath at 42°C for 90s, do not shake the centrifuge tube during the period, immediately place it on ice for 2min after taking it out. Add 500ul of anti-antibody-free LB / SOC medium, culture at 150rpm / min, shaker at 37°C for 45min. Pour all the liquid in the centrifuge tube onto the LB plate and spread evenly. After the plates were dried, they were placed upside down in an incubator for 16 h.

[0144] 2. Plasmid extraction

[0145] Pick a single colony obtained from the above transformation step and inoculate it in 500ul LB li...

Embodiment 3

[0146] Example 3. Expression of wild-type hNGF and its mutants

[0147] 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.

[0148] Experimental steps:

[0149] 1. One day before transfection, inoculate 293F cells, 0.5×10 6 / ml, 900ml in total, 300ml / bottle.

[0150] 2. Count on the day of transfection, the cell density is about 1.0×10 6 / ml, the activity rate is above 99%.

[0151] 3. Transfection: Take 36ml of cell culture medium into a 125ml culture bottle; add 360ug of plasmid, mix well; add 1080ug of PEI, mix well. Stand at room temperature for 15 minutes; mix with cells, about 12.3ml / bottle; 37°C, 8% CO 2 , 120RPM cultivation.

[0152] 4. Collect the cell supernatant on the fourth day after transfection, and centrifuge at 10,000 g for 20 minutes.

[0153] 5. Collect the supernatant and filter at 0.45um to obtain th...

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Abstract

The invention relates to a low-pain nerve growth factor mutant, and belongs to the field of biological pharmacy. The low-pain nerve growth factor mutant is any one amino acid sequence in sequence tables SEQ ID No:3 to SEQ ID No:49. The low-pain nerve growth factor mutant has the advantages that the nerve growth factor mutant has low pain performance; the pain side effect can be reduced.

Description

technical field [0001] The invention relates to a low pain nerve growth factor mutant and belongs to the field of biopharmaceuticals. Background technique [0002] According to its neurophysiological mechanism, pain can be divided into two types: sensory 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 neuropathic pain. Chronic pain as a direct result of disease. [0003] Nerve Growth Factor (NGF) is the first neurotrophic factor discovered by Italian scientist Levi-Montlcini in mouse sarcoma cells in 1953. NGF is a dual biological function of neuron nutrition and promotion of neurite growth. It is a nerve cell growth regulator, which plays an important role in regulating the development, differentiation, growth, regeneration and expression of functional properties of central and periphera...

Claims

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Application Information

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IPC IPC(8): C07K14/48C12N15/12C12N5/10A61K38/18A61P25/00A61P3/04A61P27/02A61P15/00A61P37/02A61P19/08
CPCC07K14/48A61K38/00
Inventor 陈志王超马磊杜天飞
Owner STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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