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Method for extracting nerve growth factor

A technology of nerve growth factor and extraction method, which is applied in the field of extraction of nerve growth factor, can solve problems such as complex steps, unfavorable large-scale preparation, low yield, etc., achieve simple extraction method, improve sample yield and sample activity, avoid adverse effects

Pending Publication Date: 2019-05-17
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human nerve growth factor extraction and preparation mainly come from human placenta and recombinant expression. The known preparation methods have problems such as complicated steps and low yield, which makes it unfavorable for large-scale preparation

Method used

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  • Method for extracting nerve growth factor
  • Method for extracting nerve growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1, the extraction of human nerve growth factor in the submandibular gland of transgenic mice

[0058] 1. Homogenate: Take the submandibular gland of transgenic mice expressing human nerve growth factor, mix the submandibular gland with purified water pre-cooled to 4°C at a mass volume ratio of 1g:4ml, and fully crush it with a high-speed tissue grinder to obtain a homogenate. The homogenate was centrifuged at 11000×g and 4°C for 30 min, and the centrifuged supernatant was collected.

[0059] 2. Acidification centrifugation: Add acid buffer 1mol / L acetate buffer solution (pH3.8) to the centrifugal supernatant obtained in step 1 to quickly reduce the pH to 4.0, then add NaCl solid to make the final concentration of NaCl 0.2mol / L, after standing for about 5 minutes, centrifuge at 10,000×g for 30 minutes at 4°C to obtain the acid hydrolysis supernatant.

[0060] 3. Cation exchange chromatography: CM Sepharose Fast Flow packing column, fully equilibrated with 0.05m...

Embodiment 2 and 3

[0088] Repeat the steps of Example 1, the difference is only that the parameters provided in Table 3 are used to carry out cation exchange chromatography and hydrophobic chromatography:

[0089] table 3

[0090]

[0091]

Embodiment 4

[0097] Repeat the steps of Example 1, the only difference is that the following steps are inserted after the hydrophobic chromatography step:

[0098] The Superdex 75 prep grade chromatographic column was fully balanced with pH6.8, 0.05mol / L phosphate-0.15mol / L sodium chloride buffer solution, and the sample was loaded, and the collection began when the number displayed at 280nm on the ultraviolet detector began to rise, and the drop Stop collecting the target protein peak at baseline and obtain a gel filtration chromatography sample.

[0099] The index detection of the hNGF extract of test example 3, embodiment 4

[0100] According to the method of Test Example 1, various indicators of the hNGF extract of Example 4 were detected, and the results are shown in Table 5.

[0101] table 5

[0102] Test items

[0103] It can be seen from Table 5 that compared with Example 1, the process of Example 4 has significantly reduced residual host protein and DNA residues in the...

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Abstract

The invention discloses a method for extracting a nerve growth factor, wherein the method includes the steps: acidizing and centrifuging a supernatant containing the nerve growth factor to obtain an acidolysis supernatant, carrying out cation-exchange chromatography on the acidolysis supernatant, and collecting a target elution peak containing the nerve growth factor, to obtain a protein liquid 1;and carrying out hydrophobic chromatography on the protein liquid 1, and collecting a target elution peak containing the nerve growth factor, to obtain a target protein liquid. The method does not include the step of cationic reverse adsorption chromatography. Compared with the prior art, the method has simple steps and is safer and more reliable, and the purity, yield and sample activity of theobtained nerve growth factor sample are improved. The method not only is suitable for extracting the mouse nerve growth factor, but also is suitable for extracting the human nerve growth factor, and provides a new way for large-scale separation and purification of the nerve growth factor.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the extraction of nerve growth factors. Background technique [0002] Since the discovery of nerve growth factor (nerve growth factor, NGF) by Italian scientist Levi-Montalcini in 1953, the scientific community has opened the prelude to the study of nerve growth factor. Nervous system diseases are common clinical diseases, but there is a lack of effective prevention and treatment methods. The discovery of nerve growth factor has brought new dawn to the treatment of nervous system diseases. The results of a large number of pharmacodynamic experiments and clinical experiments show that nerve growth factor has potential or obvious curative effect on peripheral nerve injury, peripheral neuropathy, peripheral neurotoxic disease, traumatic brain injury, and optic nerve injury. [0003] The complete nerve growth factor is composed of three subunits α, β, and γ through non-covalent bonds to α ...

Claims

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Application Information

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IPC IPC(8): C07K14/48C07K1/18C07K1/20C07K1/16
Inventor 张世贤马磊张增伟
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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