Method for producing human brain-derived neurotrophic factor NDNF by using methanol yeast (pichia pastoris) and its application

A neurotrophic factor, methanol yeast technology, applied in biochemical equipment and methods, applications, nervous system diseases, etc., can solve the problems of low BDNF expression, cumbersome purification, and high cost

Inactive Publication Date: 2004-05-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

Because the expression level of BDNF expressed in Escherichia coli is low, the purification is cumbersome, complex denaturation and renaturation are required to restore biological activity, and the renaturation rate is not high (mature BDNF contains three pairs of disulfide bonds, and it is easy to form incorrect disulfide bonds. connect)
These issues make the cost prohibitive and it's no surprise that the price is so expensive

Method used

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  • Method for producing human brain-derived neurotrophic factor NDNF by using methanol yeast (pichia pastoris) and its application
  • Method for producing human brain-derived neurotrophic factor NDNF by using methanol yeast (pichia pastoris) and its application
  • Method for producing human brain-derived neurotrophic factor NDNF by using methanol yeast (pichia pastoris) and its application

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Embodiment Construction

[0032] The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0033] (1) Extraction and purification of genomic DNA. Take 0.6 g of human fetal brain tissue cryopreserved in liquid nitrogen, grind it, add it to a centrifuge tube filled with 5 ml of extraction lysate (3M guanidine isothiocyanate, 0.01M sodium acetate), shake gently in a shaker at 30°C for 1 hour. Use a large-bore straw to add the brain tissue lysate below the liquid level in a centrifuge tube filled with 18ml of ethanol, and stir slowly with a glass rod until completely mixed. The DNA was recovered, dissolved in 2ml TE after dripping dry with ethanol, and purified by DEAE micro-column.

[0034] (2) Amplification, cloning and sequencing of BDNF gene. According to the BDNF gene sequence reported in the literature, a primer for the 5' end of the BDNF gene was designed,

[0035] Primer I: 5′-ACT AT...

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Abstract

The present invention belongs to the field of gene engineering medicine, in particular, its relates to a method for producing and preparing human brain-derived neurotrophic factor (BDNF) by utilizing engineering bacterium pichia pastoris and its application in preparation of medicine for promoting development, growth and differentiation of several sensory neurons of peripheral neural crest and basal plate source, maintaining nerve center basal forebrain, cholinergic neuron, GABA neuron and mesencephalic substantic nigra dopaminergic neuron and curing some neurogenic disease, such as progerine dementia, parkinsonism and injury and peripheral nerves.

Description

technical field [0001] The invention belongs to the field of genetic engineering drugs, in particular, it relates to the production and preparation of human brain-derived neurotrophic factor BDNF by using engineering bacteria Pichia pastoris, and in the preparation and treatment of maintaining and promoting various sensations derived from peripheral neural crest and substrate Development, growth and differentiation of neurons, nerve center basal forebrain, cholinergic neurons, GABAergic neurons, midbrain substantia nigra dopaminergic neurons and some nerves such as Alzheimer's disease, Parkinson's disease, peripheral nerve injury, etc. application in disease medicine. Background technique [0002] Research status of BDNF abroad: In 1989, Leibrock first cloned and expressed porcine BDNF in monkey kidney COS cells. In 1991, Rosenthal et al. expressed hBDNF in CHO cells through anionic DEAE column, cationic S-Sepharose column, and S-300 Sephacryl gel column and C 4 Reverse-ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61P25/02A61P25/16A61P25/28C07H21/04C12N15/12C12N15/70C12N15/79C12P21/02C12Q1/68
Inventor 康良仪彭毅杨希才
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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