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Affinity peptide and application thereof

An affinity peptide, affinity technology, applied in the direction of peptides, specific peptides, peptide preparation methods, etc., can solve the problem of unsatisfactory separation methods and media, inability to effectively separate κ-type antibodies and λ-type antibodies, and easy inactivation. and other problems, to achieve the effect of cheap and easy-to-obtain quality control, easy media preparation, and easy quality control

Active Publication Date: 2020-02-21
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, Protein A / G / L affinity purification medium is used for the separation and purification of antibodies in the industry. However, the Protein A / G affinity purification medium cannot effectively separate κ-type antibodies and λ-type antibodies; although Protein L affinity purification medium It can adsorb κ-type antibodies, but Protein A / G / L generally has problems such as expensive media, easy inactivation, difficult cleaning, ligand shedding and pollution, etc.
However, other separation methods and media are not satisfactory, and there are problems such as non-specific adsorption, ligand immunogenicity and toxicity.

Method used

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  • Affinity peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: the synthesis of polypeptide

[0020] 1) Activated resin: Weigh 1000mg of Fmoc Cys-wang Resin, soak in N,N-dimethylformamide (DMF) for 30min to swell.

[0021] 2) Deprotection: remove DMF by pressure filtration, add the DMF solution containing 20% ​​piperidine and blow with nitrogen to react for 15min, and remove by pressure filtration; wash the resin three times with isopropanol, wash three times with DMF, and then use the ninhydrin method to detect the resin Fmoc Remove status.

[0022] 3) Condensation reaction: Weigh 1.4mmol FMOC-Tyr, add O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU), 1-hydroxybenzotriazole The DMF mixed solution of oxazole (HOBt) and N,N-diisopropylethylamine (DIEA) was used as the reaction solution, and the reaction was carried out under nitrogen blowing at room temperature for 2 hours; after the reaction, the resin was washed three times with isopropanol, and then washed with DMF Three times, detected by...

Embodiment 2

[0026] Embodiment 2: the preparation of affinity medium

[0027] 1) Dissolve 10 mg of antibody affinity peptide (amino acid sequence shown in SEQ ID No.1) in 10 mL of 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) buffer, pH 7.4.

[0028] 2) Take 1 mL of Sephadex activated by sulfhydryl groups.

[0029] 3) The compounds in step 1) and 2) were mixed and reacted for 12 hours.

[0030] 4) After the reaction is completed, the gel particles are washed with Hepes buffer to obtain an affinity medium.

Embodiment 3

[0031] Example 3: Affinity peptide separation and purification of kappa chain antibody

[0032] 1) Prepare 10mmol / L Hepes buffer, pH 7.0, NaCl content 0.15M, set flow rate 1mL / min.

[0033] 2) Using the liquid prepared in step 1) as the mobile phase, run the AKTA protein purification system.

[0034] 3) Take 1 mL of the affinity medium and 1 mL of the Protein L medium in Example 2 to fill the separation column respectively.

[0035] 4) Add 1 mL of antibody solution and collect 3 mL of the effluent.

[0036] 5) Prepare 0.1M HCl-Gly buffer at pH 2 as the eluent.

[0037] 6) Load 3 mL of the eluate and collect 3 mL of the eluate.

[0038] 7) Measure the content and purity of the kappa chain antibody in the effluent and eluent before adsorption, after adsorption, respectively, and calculate the total extraction rate. The results are shown in Table 1 below.

[0039] Table 1. Comparison of adsorption characteristics between affinity peptide medium and Protein L medium

[0040] ...

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Abstract

The invention relates to the field of bioseparation, in particular to affinity peptide and an application thereof. The amino acid sequence of the affinity peptide is Ala Glu Phe Tyr Cys. The affinitypeptide can be specifically bound with a kappa chain antibody, the effect of purifying the kappa chain antibody is comparable to that of Protein L, and the affinity peptide is cheap and available andhas good safety.

Description

technical field [0001] The invention relates to the field of biological separation, in particular, the invention relates to an affinity peptide and its application. Background technique [0002] Antibody is a kind of immunoglobulin secreted by plasma cells that can specifically bind to antigen. Because of its specific binding ability, it can be used as a drug for treating diseases, a reagent for disease diagnosis, and a medium for affinity separation, etc. It has a wide range of applications in the fields of biology, medicine, and chemical industry, and has great social and economic benefits. [0003] Natural antibody molecules contain four heterologous polypeptide chains, among which, the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (Light chain, L). According to the configuration of the light chain, it can be divided into kappa (κ) chain and lambda (λ) chain, corresponding to ...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K16/00C07K1/22
CPCC07K7/06C07K16/00C07K1/22
Inventor 韦宇平张曼焦朋飞宋梦煜孙海翌
Owner NANYANG NORMAL UNIV
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