Chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and preparation method thereof

A technique for expression of neurotrophic factors and chloroplasts, applied in the field of genetic engineering, can solve the problems of unfound tissue sources and low natural content of growth factors

Inactive Publication Date: 2018-07-06
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the natural content of growth factors in the human body is extremely low, and only micrograms of growth factors can be extracted per kilogram of tissue, and no rich tissue source has been found so far

Method used

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  • Chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and preparation method thereof
  • Chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and preparation method thereof
  • Chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Tobacco chloroplast expression vector pWYP23402 (BDNFb)

[0031] 1) Optimize the design of the nucleotide sequence of the human brain-derived neurotrophic factor b (human Brain-derived neurotrophic factorb, BDNFb) gene in the NCBI database by using modern bioinformatics technology. Under the premise of keeping the amino acid sequence unchanged, use Gene Designer to replace all codons of the coding gene with the optimal coding method of tobacco chloroplast codons, and then pass the modified gene sequence through DNAMAN7.0 and VectorNTI 10.0 The software analyzes the enzyme cutting site information, and changes the coding codon of the 104th amino acid Tyr of the brain-derived neurotrophic factor b coding gene from the first optimal codon TCT to the second optimal codon TCA to eliminate Nsi I enzyme cutting site, the nucleotide sequence of modified human brain-derived neurotrophic factor b (human Brain-derived neurotrophic factorb, BDNFb) gene is shown in SEQ ID ...

Embodiment 2

[0035] Example 2 Tobacco chloroplast transformation and homogenization screening of BDNFb gene

[0036] The gold powder was coated with vector pWYP23402 for gene gun bombardment. For specific operations, please refer to the patented human basic fibroblast growth factor, tobacco chloroplast expression vector and production method (application number: 201510310239.3, application date: 2015-06-08, publication number: CN104946656A).

[0037] A. Tobacco explant preparation

[0038] Tobacco seeds soaked in 70% ethanol for 30sec, shake gently during the period; carefully discard the ethanol, add 0.1% HgCl 2 , shake gently for 2min; carefully discard HgCl 2 Solution, rinse with sterile water 3 times, 1 min each time. After the last rinsing, leave a small amount of water, suspend the seeds, pour them on several layers of sterile filter paper, blot the water, and carefully inoculate the seeds on 1 / 2 MSO medium with tweezers. The cycle was cultured in a culture room with light 16h / da...

Embodiment 3

[0087] Example 3 In vivo detection of protein label green fluorescent protein GFP

[0088] According to the method of a patented human basic fibroblast growth factor, tobacco chloroplast expression vector and production method (application number: 201510310239.3, application date: 2015-06-08, publication number: CN104946656A), the whole plant of the transgenic material Fluorescence analysis.

[0089] Fluorescent detection of the GFP tag at the N-terminus of the target protein expressed in the transgenic plants. Under the ultraviolet light of 365nm, the transgenic plants emitted strong green fluorescence in the dark room, indicating that the green fluorescent protein was expressed in large quantities, while the control plants showed spontaneous red fluorescence ( image 3 ).

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Abstract

The invention discloses chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and a preparation method thereof. The invention further discloses the chloroplast-expressed brain-derivedneurotrophic factor hBDNFb protein of which the amino acid sequence is shown as a SEQ ID NO.2. The preparation method of the protein comprises the following steps: constructing a tobacco chloroplastexpression vector pWYP23402; performing tobacco chloroplast transformation and homogenization screening on an hBDNFb gene to obtain a homologous plant of the tobacco chloroplast transformed hBDNFb gene; analyzing the hBDNFb expression quantity in a chloroplast transgenic tobacco plant; purifying the brain-derived neurotrophic factor hBDNFb protein to obtain the chloroplast-expressed brain-derivedneurotrophic factor hBDNFb protein. According to the invention, GFP fusion protein of the hBDNFb is highly expressed in a tobacco chloroplast, and a commercial production condition is available.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and a preparation method thereof. Background technique [0002] Growth factors are a type of active protein synthesized and secreted by cells. In animal tissues, cells, growth factors and extracellular matrix are in a dynamic environment. functioning microenvironment. Through the research on the development of hematopoietic cells, it is found that growth factors can act on the proliferation and differentiation of cells by inducing or regulating the expression of genes. As various growth factors and their biological characteristics are continuously revealed, the research data on growth factors in improving various physiological and pathological conditions, promoting tissue regeneration, wound repair and healing process continue to accumulate, and play a role in the application fields such as me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/475C07K1/36C07K1/34C07K1/22C07K1/14C12N15/12C12N15/82C12N15/65
CPCC07K14/475C07K2319/60C12N15/65C12N15/8257C12N2800/22
Inventor 王云鹏韦正乙邢少辰范杰英
Owner JILIN ACAD OF AGRI SCI
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