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Application of lettuce used as host to expressing growth factors

A growth factor and lettuce technology, applied in the direction of growth factor/inducing factor, fibroblast growth factor, application, etc., can solve problems such as hindering the development of plant exogenous protein drugs and increasing costs

Inactive Publication Date: 2017-10-17
SAGACITY FAITHFUL CONVERGENCE HEALTH TECH LTD
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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 lettuce used as host to expressing growth factors
  • Application of lettuce used as host to expressing growth factors
  • Application of lettuce used as host to expressing growth factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The construction of embodiment 1 plant transient expression vector

[0050] In order to provide high-efficiency expression of foreign aid proteins in plants, the present invention optimizes the codons of human FGF-1 (GenBank Accession No.: NM_001257210.1) and FGF-2 (GenBank Accession No.: M27968.1) to plant-preferred codons. Gene Art TM GeneOptimizer TM(ThermoFisher) designed and synthesized. Add Xbal and Kpnl restriction sites at the 5' end of the optimized FGF-1 and FGF-2 sequences, add Sacl and Pacl sites at the 3' end, and clone them into 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T vectors by ThermoFisher . Growth factor gene fragments FGF-1 and FGF-2 were isolated from 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T by Kpnl / Sacl, and cloned into the binary plant vector pCam35S to produce transient expression vectors p35S-FGF1 and p35S-FGF2, respectively. The two plant expression constructs were transformed separately into Agrobacterium tumefaciens GV3101 by electro...

Embodiment 2

[0052] Example 2 Agrobacterium-mediated vacuum infiltration

[0053] The present invention optimizes the method of Agrobacterium vacuum infiltration ( figure 2 ). 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 co...

Embodiment 3

[0055] Example 3 Protein Extraction and Separation

[0056] 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 to 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 sample was dissolved in 5 mL buffer (20 mM KPi, pH 7.8; 2 mM EDTA; -mercapt...

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Abstract

The invention relates to the field of biotechnologies, in particular to application of lettuce used as a host to expressing growth factors. The human acidic fibroblast growth factors (FGF-1) and the basic fibroblast growth factors (FGF-2) are expressed by the aid of recombinant vectors and agrobacterium tumefaciens mediated vacuum infiltration methods. Expression systems can be collected after the fact that plant exogenous proteins are infected in agrobacterium tumefaciens for 4 d is confirmed. Successful expression of recombinant FGF-1 and FGF-2 is confirmed by the aid of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) methods and western blotting methods (Western methods). The biological activity of the FGF-1 and the FGF-2 which are produced by the lettuce is provided by cell proliferation experiments. The application has the advantage that methods for producing large quantities of the active recombinant human FGF-1 and FGF-2 are low in cost and are convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of lettuce as a host in expressing growth factors. Background technique [0002] Fibroblast growth factors (FGFs) comprise a large family of genes involved in growth and differentiation. Fibroblast growth factor is found in both invertebrates and vertebrates. FGFs have been shown to play important roles in the development, metabolism and repair of various tissues and organs. Human fibroblast growth factor was originally identified as a protein capable of promoting fibroblast proliferation and is now known to contain 22 members. Due to their potential biological functions, FGF has been used to regenerate damaged tissues, including skin, blood vessels, muscles, fat, tendons / ligaments, cartilage, bones, teeth and nerves. A promising source of FGF for tissue regeneration was then used with the recombinant human FGF family. In fact, many previous studies applied FGFs d...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/12C12N15/66
CPCC07K14/501C07K14/503C12N15/8257
Inventor 王跃驹李文焦顺昌
Owner SAGACITY FAITHFUL CONVERGENCE HEALTH TECH LTD
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