Application of plant as host in expressing vaccine of Middle East Respiratory Syndrome

A technology for respiratory syndrome and plants, applied in the direction of vaccines, medical preparations containing active ingredients, microorganisms, etc., can solve the problems of high price, low production capacity of animal cells, and low safety, and shorten the production cycle and production cost , reduce biosafety issues, and facilitate the effect of protein purification

Inactive Publication Date: 2017-08-18
SAGACITY FAITHFUL CONVERGENCE HEALTH TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal cell culture requires expensive culture medium, strict factory conditions, complex operations, a time period of at least two weeks, and low production capacity of animal cells, resulting in extremely high costs
Sometimes the virus carried by animal cells can infect humans, resulting in low safety

Method used

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  • Application of plant as host in expressing vaccine of Middle East Respiratory Syndrome
  • Application of plant as host in expressing vaccine of Middle East Respiratory Syndrome
  • Application of plant as host in expressing vaccine of Middle East Respiratory Syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0051] In order to improve the expression and translation of the protein in the lettuce system, the present invention redesigned CTB-S377-588-Fc to preferentially match the codon frequency found in plants (the sequence is shown in SEQ ID No.7). Cholera toxin B subunit (CTB) can be shown to increase antigen uptake and potently induce mucosal responses. To improve the immunogenicity of intranasal vaccine, we fused CTB (Genbank ID: AY475128.1) to RBD (Genbank ID: KM027288.1)-Fc (Genbank ID: BC156864.1). CTB-S377-588-Fc optimized codon will base by GeneArt TM GeneOptimizer TM (ThermoFisher) designed and synthesized.

[0052]Add Kpnl restriction site at the 5' end of CTB-S377-588-Fc and optimized sequence, add Sacl and Pacl sites at the 3' end, and generate pWT-CTB-RBD-Fc and pOP-CTB- by ThermoFisher RBD-Fc vector. The gene fragments were isolated by Kpnl / Sacl, and cloned into the binary plant expressi...

Embodiment 2

[0054] Example 2 Agrobacterium-mediated vacuum infiltration

[0055] The prepared Agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. The first 10% of the lettuce was removed with a knife, turned upside down (core up) and gently swirled in the bacterial suspension, the desiccator was sealed. The vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate for approximately 25-45 s until bubble formation on the leaf space was observed. And it can be seen that the permeate is in the leaf tissue. Maintain the pressure state for 30-60 seconds. The pressure is quickly released to allow the penetrant to seep into the spaces within the tissue. This process was repeated 2 to 3 times until the penetration of the permeate into the lettuce tissue was clearly visible. 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 plast...

Embodiment 3

[0056] Example 3 Protein Extraction and Separation

[0057] Lettuce samples subjected to vacuum infiltration by Agrobacterium were stirred with a stirrer, and homogenized at high speed in a blender for 1 to 2 minutes with an extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM β-mercaptoethanol) with a volume ratio of 1:1. 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 min. 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 of buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β-m...

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Abstract

The invention relates to the technical filed of biology, in particular to application of a plant as a host in expressing a vaccine of Middle East Respiratory Syndrome. According to the technical scheme, lettuce is utilized to instantly express the vaccine to deal with Middle East Respiratory Syndrome, and high-content protein can be produced within a short time (4d). The biology safety problem is reduced to the largest limit, the lettuce basically contains no botanical toxic substances, and the fiber of the lettuce itself is little, which is conducive to protein purification in the downstream. As test results show, through the adoption of a lettuce system to produce the vaccine, the production period can be sharply shortened, and the production cost can be sharply reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of plants as hosts in expressing Middle East Respiratory Syndrome vaccines. Background technique [0002] Middle East Respiratory Syndrome (MERS), caused by a novel coronavirus (MERS-CoV), emerged in Saudi Arabia in April 2012. People infected by MERS-CoV develop severe acute respiratory illness with symptoms including high fever, cough, and shortness of breath. Pneumonia and gastrointestinal symptoms have also been reported. The first widespread outbreak outside the Arabian Peninsula occurred in South Korea in May 2015, when the virus spread among people in close contact with each other, causing public panic. As of May 13, 2017, MERS-CoV had infected 1,952 patients and caused 693 deaths; the high mortality rate was about 35%. More than 27 countries have reported cases of MERS-CoV. [0003] MERS-CoV poses a threat to the public. Although the virus has temporarily...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/62C07K14/165
CPCC12N15/8258C07K14/005C07K2319/55C07K2319/30C12N2770/20022C12N2770/20034C12N2800/22C12N15/8257A61K39/12A61P31/14A61K2039/6037A61K2039/6056A61K2039/543C12N15/8205C12N15/62C07K14/165
Inventor 王跃驹李文焦顺昌唐顺学周卫斌
Owner SAGACITY FAITHFUL CONVERGENCE HEALTH TECH LTD
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