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Application of plant serving as host in expressing epidermal growth factor

An epidermal growth factor, plant technology, applied in the direction of epidermal growth factor, growth factor/inducing factor, plant gene improvement, etc., can solve problems such as hindering the development of plant exogenous protein drugs and increasing costs.

Pending Publication Date: 2019-03-12
山东益华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tobacco has a high fiber content and potentially toxic compounds, such as the alkaloid nicotine, which significantly increase the cost in the downstream purification process, greatly hindering the further development of plant exogenous protein drugs

Method used

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  • Application of plant serving as host in expressing epidermal growth factor
  • Application of plant serving as host in expressing epidermal growth factor
  • Application of plant serving as host in expressing epidermal growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The construction of embodiment 1 plant transient expression vector

[0051] In order to provide high-efficiency expression of foreign protein in plants, the construction method of the expression vector provided by the present invention is as follows: Human EGF (GenBank Accession No.: A00372.1) was designed and synthesized by GenScript (Nanjing). Xbal was added at the 5' end of the EGF sequence, and a Sacl site was added at the 3' end, and cloned into the pUC57-EGF vector by GenScript (Nanjing). The growth factor gene fragment EGF was isolated from the pUC57-EGF cloning vector by Xbal / Sacl, and cloned into the binary plant vector pCam35S to generate the transient expression vector p35S-EGF respectively. The three plant expression constructs were individually transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany). The resulting bacterial strains were evenly spread on selective LB plates containing kanamycin ...

Embodiment 2

[0053] Example 2 Agrobacterium-mediated vacuum infiltration

[0054] The invention optimizes the method of vacuum infiltration of Agrobacterium. The prepared Agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. The lettuce kept in this laboratory was turned upside down (core up) and gently swirled in the bacterial suspension, and the desiccator was sealed. The vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was seen in the leaf tissue. Maintain the pressure state for 30-60 seconds. The system is quickly opened to release the pressure and allow permeate to seep into the spaces within the tissue. This process was repeated 2 to 3 times until it was clearly visible that the permeate diffused significantly in the lettuce tissue. The lettuce tissue was then gently removed from the permeate and rinsed three times consecutively with distilled water before being transferred to a container covered with plastic f...

Embodiment 3

[0056] Example 3 Protein Extraction and Separation

[0057] Lettuce samples vacuum-infiltrated with Agrobacterium were stirred with a stirrer and extracted with extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM at a ratio of 1:1 by volume). mercaptoethanol) high-speed homogenization in a blender for 1-2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 min at 4°C to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated with shaking on ice for 60 minutes. Centrifuge again (10,000 g) for 15 min at 4°C. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, shaken and suspended on ice for 60 min, and centrifuged again at 10,000 g for 15 min at 4°C. Then, the supernatant was discarded, and the protein precipitated from the treated samples was dissolved in 5 mL of buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β-me...

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Abstract

The invention relates to the technical field of biology, in particular to a plant, in particular to an application of a plant serving as a host in expressing an epidermal growth factor. The human epidermal growth factor (abbreviated as EGF) can be expressed by using a recombinant vector as well as an agrobacterium-mediated vacuum permeation method. The expression system determines that the plant foreign proteins can be collected after being impregnated with agrobacteria for four days. The successful expression of the recombinant EGF is determined by using an SDS-PAGE method and a western method. The cell proliferation assay proves that EGF produced by the plant and particularly by lettuce has bioactivity. The method provided in the invention is low in cost, convenient, and capable of producing a great amount of active recombinant human EGF.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of plants as hosts in expressing epidermal growth factor. Background technique [0002] Growth factors are naturally occurring substances that stimulate cell growth, proliferation, healing, and cell differentiation. A growth factor is usually a protein or a steroid hormone. Growth factors play a very important role in regulating a variety of cellular processes. Growth factors generally act as intercellular signaling molecules that promote cell differentiation and maturation. However, the effect of each growth factor is different. For example, bone morphogenic protein stimulates osteocyte differentiation, and fibroblast growth factor and vascular endothelial growth factor stimulate blood vessel differentiation (angiogenesis). [0003] Epidermal growth factor (EGF) stimulates cell growth and differentiation by binding to its receptor, EGFR. Human epidermal growth f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/485C12N15/84
CPCC12N15/8205C07K14/485
Inventor 王跃驹马洁焦顺昌
Owner 山东益华生物科技有限公司
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