Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein

A technology of recombinant protein and receptor protein, applied in the field of application, can solve the problems of high cost, increase of IgE concentration, aggravated allergic reaction, etc., and achieve the effect of reducing production cost, ensuring safety, and alleviating the disease

Active Publication Date: 2012-09-12
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main defect that exists is: (1) the antibody of IgE is the serum polyclonal antibody that obtains by immune sheep, and the content in serum is low, purification process is complicated, so cost is high, expensive; (2) the molecular weight of IgG is big, reaches 150kDa , causing the immobilized goat anti-human IgG to fall off easily, and foreign proteins entering the patient's body will become new allergens and lead to aggravated allergic reactions. In order to reduce this risk, the author connected a human IgE as the The adsorption column of the ligand is used to absorb the falling IgG ligand, which greatly increases the application cost of the adsorbent, and the coupled IgE on the adsorbent also has the problem of falling off. Once falling off, the IgE concentration in the blood of the patient is increased. concentration
The main disadvantages of the adsorbent are: (1) the adsorbent also has a high removal rate for IgG, and the se

Method used

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  • Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein
  • Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein
  • Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction and Identification of pET23a / FcεR I α-D2 Recombinant Plasmid

[0029] (1) PCR amplification of the target gene fragment

[0030] Search the National Center for Biological Information (NCBI) in the United States to obtain the complete gene sequence of human FcεRI (NCBI Reference Sequence: NM 002001.3), and select the gene sequence of the D2 domain (87-173 amino acids of FcεRIα) with IgE affinity properties of human FcεRIα . EcoR I and Xho I were used as upstream and downstream restriction sites respectively, and a 6×His tag was added to the C-terminus. Shanghai Xuguan Biotechnology Development Co., Ltd. was commissioned to synthesize the gene sequence (SEQ ID NO: 1) and the target gene Linked to pET 28a expression vector, named pET 28a / FcεRIα-D2.

[0031] Using the pET 28a / FcεRIα-D2 genomic DNA as a template, design primers and add enzyme cutting sites. The sequence shown in the upstream primer sequence SEQ ID NO: 2, the sequence shown in the d...

Embodiment 2

[0058] Embodiment 2: the shaking flask culture of recombinant bacterium

[0059] Pick a single colony of E.coli BL21 (DE3) containing the recombinant plasmid pET23a / FcεRIα-D2 on the LB solid medium plate in Example 1, inoculate it in 20 mL of LB liquid medium containing 1 mg / mL ampicillin, and shake it at a constant temperature Cultivate overnight in bed at 37°C, 170rpm. Inoculate the activated bacteria into 100mL LB liquid medium containing 1mg / mL ampicillin according to the inoculum amount of 1%, cultivate at 37°C and 170rpm for 3-4 hours, and wait until the OD of the bacteria 600nm When reaching 0.5-1, add IPTG to a final concentration of 0.1 mM, and continue culturing for 5 hours. SDS-PAGE electrophoresis detection, the results are attached figure 2 As shown in the middle lane 3, the recombinant bacteria expressed FcεRIα-D2 at about 13kDa, which was consistent with the theoretical molecular weight of 12950.19Da. The soluble detection of the target protein is carried ou...

Embodiment 3

[0060] Example 3: Renaturation of recombinant protein FcεRIα-D2 inclusion body and separation and purification of protein

[0061] (1) Extraction and preliminary purification of inclusion bodies (cloning, prokaryotic expression and purification of human FcγR I receptor extracellular region gene [J]. Journal of Cell and Molecular Immunology. 2010, (002): 178-180.): Will The obtained inclusion bodies were washed 5 times with a washing buffer, and then washed once with a resuspension buffer to obtain preliminarily purified inclusion bodies containing the recombinant protein FcεRIα-D2. The pH of the washing buffer is 8.0, containing 0.01M Tris-HCl, 0.1M NaCl, 0.01M EDTA, 2mM DTT and 10% Triton-100; the pH of the resuspension buffer is 8.0, containing 0.01M Tris-HCl, 0.1M NaCl, 0.01M EDTA and 2mM DTT.

[0062] (2) Renaturation of the recombinant protein FcεRIα-D2: Denaturation and renaturation of the initially purified inclusion bodies containing the recombinant protein FcεRIα-D2....

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Abstract

The invention discloses a method for preparing recombinant human IgE receptor protein and application of the recombinant human IgE receptor protein, and belongs to the field of biological engineering and blood purification technology. Fc epsilon RI alpha-D2 with high binding force with IgE is highly expressed by means of gene recombination technology, recombinant protein is obtained by means of purification and used as a genin of an adsorbent, and the prepared adsorbent fixed onto a solid phase can be used for binding the IgE in a high-affinity manner. The method solves the problems of low safety and high cost of the prior art in the field of IgE removal by means of the blood purification technology. The method has an excellent application prospect in terms of removing excessive IgE in blood of an allergic reaction patient by means of the blood purification technology, treating allergic reaction mediated by the IgE, purifying the IgE and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to the expression and preparation of a blood purification adsorbent ligand-the extracellular second region (FcεRIα-D2) of the recombinant human IgE receptor protein FcεRIα chain, and its application in the field of blood purification and antibody Applications such as purification. Background technique [0002] Allergies have an enormous prevalence, affecting more than 25% of the world's population. Among them, type I allergy (Allergic inflammation, Al), also known as immediate hypersensitivity reaction, is an immune system disease caused by IgE, such as allergic asthma, allergic urticaria, allergic rhinitis and penicillin-induced allergy. Sexual shock, etc. [0003] The mechanism of allergic reaction is that the allergen first invades the allergic body through the respiratory tract, digestive tract mucosa and skin, stimulating the body to produce specific IgE against the a...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C07K14/735C07K1/36C07K1/34C07K1/16C07K16/28C07K16/00C07K1/22A61K38/17A61P37/08C12R1/19
Inventor 贾凌云滕婷婷徐丽任军谢健
Owner DALIAN UNIV OF TECH
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