Chloroplast-expressed human epidermal growth factor (hEGF) protein and preparation method thereof

An epidermal growth factor and chloroplast expression technology, applied in the field of genetic engineering, can solve the problems of undetectable, poor expression level of exogenous protein, and no fusion GUS gene EGF expression, etc.

Inactive Publication Date: 2018-05-15
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In the existing literature reports, although the expression level of exogenous genes expressed by plant chloroplasts is better than that of nuclear transgenic plants, there are still some cases that prove that the expression level of exogenous proteins in transgenic plants obtained by this technology is not good.
Most of the tests can improve the situation by replacing the appropriate promoters, terminators, 5`UTR and other regulatory elements, but it has been reported that the EGF gene (Wirth, S., M.E.Segretin, A.Mentaberry and F.B.Almonacid.2006.Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transform edtobacco plants is higher in the dark than in the light. Biochemical & Biophysical Research Communications, 125(2):159-172.) Under the condition of fusion GUS gene, the expression of fusion protein The amount is only 0.001% of the total soluble protein, and there is no expression of EGF fusion GUS gene, even undetectable

Method used

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  • Chloroplast-expressed human epidermal growth factor (hEGF) protein and preparation method thereof
  • Chloroplast-expressed human epidermal growth factor (hEGF) protein and preparation method thereof
  • Chloroplast-expressed human epidermal growth factor (hEGF) protein and preparation method thereof

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Embodiment 1

[0031] The construction of the tobacco chloroplast expression vector pWYP23404 (EGF) of embodiment 1 epidermal growth factor

[0032] 1) The nucleotide sequence of the human epidermal growth factor (human Epidermal growth factor, hEGF) gene in the NCBI database is optimally designed using modern bioinformatics technology. Under the premise of keeping the amino acid sequence unchanged, Gene Designer was used to replace all codons of the coding gene with the optimal coding method of tobacco chloroplast codons, and the modified gene sequence was passed through DNAMAN7.0 and Vector NTI10 .0 software analyzes the enzyme cutting site information therein, and changes the coding codon of the 35th amino acid Ile of the epidermal growth factor coding gene from the first optimal codon TAT to the second optimal codon TAC to eliminate EcoR I As for the enzyme cutting site, the nucleotide sequence of the modified human epidermal growth factor (human Epidermal growth factor, EGF) gene is sho...

Embodiment 2

[0036] Embodiment 2: Tobacco chloroplast transformation and homogenization screening of EGF gene

[0037] The gold powder was coated with vector pWYP23404 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).

[0038] A. Tobacco explant preparation

[0039] 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 / ...

Embodiment 3

[0089] Example 3 In vivo detection of protein label green fluorescent protein GFP

[0090] For the whole plant fluorescence analysis of transgenic materials, please refer to the patent for a human basic fibroblast growth factor, tobacco chloroplast expression vector and production method (application number: 201510310239.3, application date: 2015-06-08, publication number: CN104946656A).

[0091] Fluorescent detection of the GFP tag at the N-terminus of the target protein expressed in the transgenic plants. Under 365nm ultraviolet light, 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 auto-red fluorescence ( image 3 ).

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Abstract

The invention discloses a chloroplast-expressed human epidermal growth factor (hEGF) protein and a preparation method thereof. The amino acid sequence of the protein is shown as SEQ ID NO.2. The invention further discloses a tobacco chloroplast expression vector pWYP23404 for producing the epidermal growth factor hEGF protein. The preparation method of the protein comprises the following steps: building the tobacco chloroplast expression vector pWYP23404, performing tobacco chloroplast transformation and homogenization screening on an EGF (Epidermal Growth Factor) gene to obtain a homogenization plant of a tobacco chloroplast-transformed hEGF gene, and analyzing the hEGF expression amount in the chloroplast-transformed gene tobacco plant; purifying the hEGF protein to obtain the chloroplast-expressed epidermal growth factor hEGF protein. By adopting the chloroplast-expressed hEGF protein and the preparation method thereof, a GFP (Green Fluorescent Protein)-fused protein of the hEGF isexpressed efficiently in a tobacco chloroplast, and commercial production conditions are realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a chloroplast-expressed epidermal growth factor hEGF 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 medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/485C12N15/82C07K1/34C07K1/22
CPCC07K14/485C12N15/8251
Inventor 王云鹏邢少辰韦正乙范杰英
Owner JILIN ACAD OF AGRI SCI
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