Chloroplast expressed fibroblast growth factor 21 protein and preparation method thereof

A fibroblast 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: 2018-05-04
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 inchloroplast-transformed tobacco 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 level of fusion protein also Only 0.001% of total soluble protein without even detectable expression of EGF fused to the GUS gene

Method used

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  • Chloroplast expressed fibroblast growth factor 21 protein and preparation method thereof
  • Chloroplast expressed fibroblast growth factor 21 protein and preparation method thereof
  • Chloroplast expressed fibroblast growth factor 21 protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Tobacco chloroplast expression vector pWYP23406 (hFGF21)

[0031] 1) Optimal design of the nucleotide sequence of the human fibroblast growth factor 21 (human Fibroblast growth factor, hFGF21) 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 the modified FGF21 gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0032] 2) Using chemical synthesis to syn...

Embodiment 2

[0035] Example 2 Tobacco chloroplast transformation and homogenization screening of fibroblast growth factor 21 (hFGF21) 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...

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 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 chloroplast expressed fibroblast growth factor 21 protein and a preparation method thereof. The amino acid sequence is shown as SEQ ID NO. 2. The preparation method of the protein comprises the following steps: constructing a tobacco leaf chloroplast expression vector pWYP23406; converting tobacco leaf chloroplast of a fibroblast growth factor 21 gene and carrying out homogenization screening to obtain a homogenized plant of the tobacco leaf chloroplast converted fibroblast growth factor 21 gene; analyzing an expression amount of the fibroblast growth factor 21 in chloroplast transgenic tobacco leaf plants; purifying the fibroblast growth factor 21 to obtain the chloroplast expressed fibroblast growth factor 21. According to the chloroplast expressed fibroblast growth factor 21 protein and the preparation method thereof, GFP fusion protein of the fibroblast growth factor 21 is efficiently expressed in the tobacco leaf chloroplast and commercial production conditions are provided.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a chloroplast-expressed fibroblast growth factor 21 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 medicine. play...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/50C07K1/36C07K1/34C07K1/22C12N15/12C12N15/82C12N15/65
CPCC07K14/50C07K2319/60C12N15/65C12N15/8257C12N2800/22
Inventor 韦正乙王云鹏范杰英邢少辰
Owner JILIN ACAD OF AGRI SCI
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