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A kind of soluble human ige receptor protein truncated protein and its preparation method and application

A technology of receptor protein and truncated protein, which is applied in the field of bioengineering, can solve the problems of different natural proteins, no public expression level, and unidentified activity of proteins, so as to increase expression level, resist protease hydrolysis, correct folding, and improve protein solubility Effect

Active Publication Date: 2021-06-15
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned products have some disadvantages: for example, Miltenyi’s goat antibody and mouse antibody contain exogenous proteins, and their safety is poor
[0005] In 2016, Xing Huihui of Ningxia Medical University and others used the prokaryotic expression vector PET-32a to transfer the entire extracellular region gene of FCεRIα into the prokaryotic expression host BL21(DE3), and expressed a soluble FCεRIα protein with a molecular weight of 42KDa, but the article pointed out that the sequencing results There are 2 amino acid mutations, the conformation of the protein may be different from the native protein
Professor Jia Lingyun of Dalian University of Technology (CN 102660569 B) expressed FCεRIα-D2 with E. coli PET23 plasmid, obtained soluble protein through inclusion body renaturation, and linked the protein to a solid phase carrier. The adsorption capacity of the synthetic adsorbent for IgE was 285 -355IU / mL gel, the adsorption effect is poor, indicating that the activity of the protein containing only the D2 region is low
Wang Qingwen of Peking University Shenzhen Hospital and others published a patented Escherichia coli vector PET32M, which expressed a soluble receptor protein in vitro, but the expression level was not disclosed, and the activity was not identified

Method used

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  • A kind of soluble human ige receptor protein truncated protein and its preparation method and application
  • A kind of soluble human ige receptor protein truncated protein and its preparation method and application
  • A kind of soluble human ige receptor protein truncated protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Construction of Escherichia coli expression strain

[0034] The plasmid PET SUMO and Rosetta gami 2 competent cells containing the target gene shown in SEQ ID NO: 2 (synthesized by Guangzhou Aiji Biotechnology Co., Ltd.) were thawed on ice. After complete thawing, mix the two thoroughly in the ultra-clean workbench (50-100ul (10ng / ul) for plasmids, 100-200ul for competent cells, place on ice for 30min, heat shock at 42 degrees for 90s, quickly place on ice for 2 ~5min, add 800~1000μL SOC medium, 37℃, 180rpm culture for 1h, 5000rpm, centrifuge 5~10min, remove most of the supernatant, leave about 50~100μL medium to resuspend the bacteria, evenly spread on ( In AMP+) LB medium, cultivate overnight at 37°C. Pick positive clones with normal colony morphology as bacterial species. After expanding the culture with LB medium, take a small amount of culture fluid and send it to Sangon Bioengineering Shanghai (Stock) Co., Ltd. Extraction and sequencing of the plasm...

Embodiment 2

[0035] Example 2: Expression and purification of receptor protein truncated protein

[0036] Prepare sterilized LB liquid medium (add 2g / L glucose), add kanamycin (final concentration is 100μg / ml), pick a single colony with the tip of a pipette, culture at 37°C, 180rpm, every Measure OD every 1 hour 600 , record the data, and add the inducer IPTG (final concentration is 1mol / L) in the rapid growth period, continue to cultivate, and measure the OD every 1h 600 , record data, finally get its growth curve, measure OD 600 When it was observed that the absorbance value did not change significantly, the cultivation was stopped, and the cells were collected by centrifugation at 5000 rpm for 10 minutes. In an ice bath, 1 g of bacteria was added to 5 mL of PBS solution, the power of ultrasonic crushing was 300 W, and the crushing time was 4 seconds and 4 seconds, the total time was 5 minutes, and then centrifuged at 8000 rpm for 5 minutes to collect the crushed supernatant. The nick...

Embodiment 3

[0037] Example 3: Synthesis of IgE Immunosorbent

[0038] Take 5mL of agarose GE Sepharose 6FF, add 8mL of 2M sodium hydroxide and 5mL of bisglycidyl ether, 40°C, 180rpm, 5h, after the reaction, filter the filler, and wash the filler with about 100mL of purified water until neutral. The activated agarose was added to 5 mL of receptor protein solution with a concentration of 15 mg / mL, and reacted at 20° C. and 180 rpm for 15 h. After the reaction is completed, use about 100ml of purified water to pump and wash the packing until neutral. Finally, 10 mL of 1M ethanolamine (pH=8.0) solution was added and mixed, and reacted at 20°C and 180 rpm for 10 h. Wash the filler with about 100ml of purified water to obtain the IgE immunosorbent.

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Abstract

The present invention provides a soluble human IgE receptor protein truncated protein and its preparation method and application, which relate to the field of bioengineering and blood purification technology, and efficiently express the receptor protein FcεRIα‑D1 with high binding force with IgE through recombinant gene technology ‑D2, as the ligand of the blood adsorbent, the adsorbent prepared by immobilizing it on the solid phase carrier can be used to remove excess IgE in the blood of allergic patients and treat severe allergic diseases.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method and application of a truncated protein of soluble human IgE receptor protein. Background technique [0002] Allergic diseases are increasing year by year all over the world, and the incidence rate has increased by at least three times in the past 30 years. It is estimated that in the next 20 years, 50% of the population in industrialized countries will suffer from allergies. The mechanism of allergic diseases is IgE-mediated immune allergy. Common diseases include: allergic asthma, seasonal allergic rhinitis, atopic dermatitis, drug-induced interstitial pneumonia, bronchopulmonary aspergillosis, leprosy, Pemphigus and certain parasitic infections. Allergic diseases are a series of immune allergies caused by the combination of IgE and receptor protein (FcεR) in the human body. FcεR is located on the surface of mast cells and basophils, and also exis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/735C07K1/36C07K1/34C07K1/22C12N15/12C12N15/70A61K38/17A61P37/08
CPCA61K38/00A61P37/08C07K14/70535C12N15/70
Inventor 张海珍杨正根余波光陈臻毅王云喜林大鸿陈校园
Owner GUANGZHOU KONCEN BIOSCI
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