Nerve growth factor cDNA, clone and expresion of Chinese cobra

A nerve growth factor, Chinese cobra technology, applied in nerve growth factor, growth factor/inducing factor, biochemical equipment and methods, etc., can solve the problem of unmanned use of recombinant snake NGF and so on

Inactive Publication Date: 2005-03-23
郝文学 +3
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  • Abstract
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  • Application Information

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Problems solved by technology

In recent years, it has been found that brain-derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF) can reduce blood sugar in experimental animals by regulating the metabolism of the body, b

Method used

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  • Nerve growth factor cDNA, clone and expresion of Chinese cobra

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning of NGF cDNA:

[0032] a. After decapitating the Chinese cobra, separate the venom gland as soon as possible, and put it into liquid nitrogen for freezing.

[0033] b. Take 0.1-1g tissue, add 1-5ml TRIzol reagent, chloroform, shake for 15-30sec, and place on ice for 5-10min. Centrifuge at 10000-30000xg for 10-30min at 4°C, and take the supernatant. Transfer the supernatant to another centrifuge tube, add 0.5-2ml of isopropanol, and place it on ice for 10-20min. Then centrifuge again under the above conditions. Add 75% ethanol to the precipitate to wash, mix well, and then centrifuge at 4°C to obtain RNA precipitate. After air-drying, add an appropriate amount of TE solution or RNase-free water for later use.

[0034] c. RT-PCR: RNAPCR Kit (AMV) was used for reverse transcription PCR. The basic point is: add MgCl to the reaction solution 2 , RNA PCR Buffer, Deoxymononucleotide Mixture, Ribonuclease Inhibitor, Random 9-mer, Sample RNA. The total a...

Embodiment 2

[0070] Embodiment 2: the expression of recombinant NGF in Escherichia coli:

[0071] Construction into plasmid pET23b: use the above double enzyme digestion to construct the identified gene fragment into the multiple cloning site of plasmid pET23b, and amplify it in Escherichia coli DH5α, prepare competent cells by calcium chloride method, and use PCR after construction and double-digestion fragment electrophoresis for identification, and the above-mentioned method was used to transform pET23b containing NGF cDNA into Escherichia coli BL21 (DE 3 ) expressed NGF protein under the induction of IPTG.

Embodiment 3

[0072] Embodiment 3: expression of recombinant NGF in Pichia pastoris:

[0073] Construction and screening of target gene into yeast expression plasmid pPIC9K. PCR was performed on pET23b containing the target gene using primers containing SnaBI and Eco521 double restriction sites, and the PCR product was identified by electrophoresis and purified using a kit provided by Qiagen. Then use these two enzymes to digest pPIC9K and insert the purified target gene. Thus, pPIC9K containing the target gene was obtained.

[0074] Primers containing His tag:

[0075] F5'-GCTACGTAGAAGATCATCCTGT-3'

[0076] R5'-TTCGGCCGGTGGTGGTGGTGGTGGTGCGCAAGCTT-3'

[0077] Primers without His tag:

[0078] F5'-GCTACGTAGAAGATCATCCTGT-3'

[0079] R5'-TTCGGCCGGTGGTGGTGGTGGTGGTGCGCAAGCTT-3'

[0080] The pPIC9K plasmid containing the target gene was transformed into Pichia pastoris GS115 by electroporation. The conditions of electroporation were 2.5KV, 800-1000Ω, 25μF, and the time was 14-21 milliseco...

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Abstract

The invention is cDNA, cloning and expression of Chinese cobra neural growth factor (NGF), relating to cDNA and cloning of recombinant Chinese cobra neural growth factor, expression of neural growth factor, a method of using its expression carrier to prepare it and pharmacological use of the mentioned protein, separate-extracting total RNA from the poison gland of Chinese cobra, using RT-PCR method to make reverse transcription to get total cDNA, using the primer designed by us to provide a specific Cdna sequence at a length of 348 bp polynucleotide sequence and also providing a small-molecular weight protein, the molecular weight about 13000, successfully expressed in pichia pastoris. The recombinant neutral growth factor has pharmaceutical use in the aspects of reducing blood sugar and improving pancreas islet as well as curing peripheral polyneuritis caused by diabetes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the cDNA and clone of recombinant Chinese cobra (Chinesecobra [Naja naja atra]) nerve growth factor (nerve growth factor, NGF), the expression of the nerve growth factor and the method for preparing the nerve growth factor by using its expression vector and pharmaceutical uses of said protein. technical background [0002] Nerve growth factor (NGF) is one of the most important biologically active proteins in the nervous system. It mainly acts on the nervous system, such as sympathetic neurons, sensory neurons and forebrain cholinergic neurons. NGF participates in regulating their development and differentiation, maintaining their normal functions, and promoting their repair after injury. At present, NGF has shown important clinical application value in promoting the repair of nerve injury and treating some neuron degenerative diseases such as Alzheimer's disease. At present, NGF has ...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61P3/10C07K14/48C12N15/12
Inventor 郝文学赵宝昌田晓光爱萍
Owner 郝文学
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