A kind of immunoglobulin binding protein and its preparation method and application

A technology of immunoglobulin and binding protein, which is applied in the field of immunoglobulin binding protein and its preparation, which can solve the problems of unsatisfactory treatment effect, large toxic and side effects, allergic reactions and other problems of autoimmune diseases, and achieve high affinity and stable protein quality , wide reactivity effect

Active Publication Date: 2021-09-07
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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  • A kind of immunoglobulin binding protein and its preparation method and application
  • A kind of immunoglobulin binding protein and its preparation method and application
  • A kind of immunoglobulin binding protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Vector Construction and Expression of Immunoglobulin Binding Proteins

[0050] 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

[0051] The crushing treatment of embodiment 2 thalline

[0052] Take 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, stir it evenly and crush it with a high-pressure homogenizer at a pressure of 800bar Twice, 4°C, 8000rpm, centrifuge for 15min to collect about 9.5L 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

[0053] Example 3 Purification of Immunoglobulin Binding Proteins

[0054] (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 present invention includes the B domain of protein A, the C2 domain of protein G, the variant of the B3 domain of protein L or any combination of 1-3 variants thereof, through protein A The different binding properties of protein G and protein L have certain complementarity, and they are alkali-resistant multifunctional IBP molecules with wider reactivity and higher affinity. In the present invention, the three-step chromatography is used to purify the protein, which can stabilize the protein quality, ensure the protein purity is greater than 97%, and the endotoxin level is lower than 1Eu / mg, meeting the requirements of clinical protein. The stability of the immunoadsorption filler synthesized by the purified immunoglobulin binding protein is increased, which can effectively increase the use times of the filler and prolong the service life of the filler.

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 Patents(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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