Immunoglobulin binding protein, and preparation method and application thereof

An immunoglobulin and binding protein technology, applied in the field of immunoglobulin binding protein and its preparation, can solve the problems of unsatisfactory treatment effect of autoimmune diseases, large toxic and side effects, cross infection, etc., and achieves high affinity and stable protein quality. , the effect of prolonging the service life

Active Publication Date: 2020-04-24
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because it inhibits the production of all antibodies in the body, it has relatively large toxic and side effects, and the treatment effect on many autoimmune diseases and their symptoms is not ideal; the latter is to eliminate the variation in the body by regularly replacing the patient's body plasma with normal human plasma immunoglobulin
Due to the transfusion of allogeneic plasma, it may cause: 1) cross-infection; 2) allergic reaction; 3) bleeding tendency, etc.

Method used

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  • Immunoglobulin binding protein, and preparation method and application thereof
  • Immunoglobulin binding protein, and preparation method and application thereof
  • Immunoglobulin binding protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Vector Construction and Expression of Immunoglobulin Binding Proteins

[0044] Artificially synthesized amino acid sequences such as SEQ ID No: 1-13, the recombinant plasmid PET30a (Novagen) that can encode the gene sequence of the amino acid sequence of interest was ligated with restriction sites NdeI and XhoI, and then transformed into competent Escherichia coli strain BL21 (DE3) (merck millipore). Put 50mL of LB medium in a 250mL Erlenmeyer flask with a final concentration of kanamycin of 50 μg / mL, culture at 37°C and 180 rpm for 4 hours, then add IPTG with a final concentration of 50 μg / mL to induce expression, and culture for another 5 hours. Collect bacteria.

Embodiment 2

[0045] The crushing treatment of embodiment 2 thalline

[0046] Get 1 kg of thalline containing immunoglobulin binding protein, add the thalline to the PBS buffer solution of pH=7.2 according to the ratio of mass:volume=1kg:10L, break it with a high-pressure homogenizer after stirring evenly, and break it with a pressure of 800bar 2 Once, centrifuge at 4°C, 8000rpm for 15min to collect about 9.5L of supernatant. Add hydrochloric acid to the obtained solution to adjust the pH to 3.0, centrifuge at 4°C, 8000rpm for 15min, collect the supernatant and adjust the pH to 7.0 with sodium hydroxide at 8000rpm, centrifuge for 15min to obtain the supernatant of the broken cells.

Embodiment 3

[0047] Example 3 Purification of Immunoglobulin Binding Proteins

[0048] (1) Affinity chromatography: equilibrate the chromatographic column packed with TALON superflow metal ion chelating filler with 10mMbis-tris[bis(2-hydroxyethyl)triimino(hydroxymethyl)methane], pH 7.2 5 times the column volume, then load the broken supernatant obtained in Example 2, equilibrate 5 times the column volume with a solution containing 10mM bis-tris, pH=7.2 after loading the sample, wash 1 times the column volume with 0.01M NaOH, The column volume was then equilibrated for 5 times with a solution containing 10 mM bis-tris, pH=7.2, and finally the collected protein was eluted with a solution containing 180 mM imidazole and 10 mM bis-tris, pH 7.2. The main purpose of affinity chromatography is to capture proteins. Using metal ion chelating fillers and the alkali-resistant properties of proteins to be purified, proteins with weak binding forces are washed away with low-concentration sodium hydroxi...

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Abstract

An immunoglobulin binding protein provided by the invention comprises variants of the B structural domain of a protein A, the C2 structural domain of a protein G and the B3 structural domain of a protein L or a combination of any one to three of the variants. The protein A, the protein G and the protein L have different binding characteristics and certain complementarity, so the immunoglobulin binding protein is an alkali-resistant multifunctional IBP molecule with wide reactivity and high affinity. The protein is purified by a three-step chromatography method, so that the protein quality canbe stabilized, the protein purity is more than 97%, the endotoxin level is lower than 1 Eu/mg, and the requirement of clinical protein is met. The stability of an immunoadsorption filler synthesized from the purified immunoglobulin binding protein is improved, so the use frequency of the filler can be effectively increased, and the service life of the filler is prolonged.

Description

technical field [0001] The invention belongs to the field of biomedical materials and blood purification, and in particular relates to an immunoglobulin binding protein and its preparation method and application. Background technique [0002] Autoimmune diseases are a series of symptoms and diseases caused by the mutation of the body's own antibodies against its own tissues, causing damage to the tissues and organs of the whole body. It is currently one of the major diseases that are difficult to deal with and threaten human health and life. There has been no effective treatment. At present, the main treatment methods for autoimmune diseases are the application of immunosuppressive drugs and plasma exchange. The former is to suppress the body's immune system through drugs, and reduce the damage caused by antibodies in the body against its own tissues and organs, so as to achieve the purpose of alleviating symptoms and delaying the progress of the disease. Because it inhibi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C07K1/20C07K1/18C07K1/22C07K1/36C07K1/34B01J20/24A61K38/16A61P37/02C12R1/19
CPCC07K14/31C07K14/315C12N15/70B01J20/24A61P37/02C07K2319/00C12N2800/22A61K38/00
Inventor 张海珍杨正根余波光王云喜林大鸿罗丽华陈校园
Owner GUANGZHOU KONCEN BIOSCI
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