Micromolecule antibody affinity peptide and applications thereof

A small molecule antibody and affinity peptide technology, applied in the field of molecular biology, can solve the problems of protein A being unstable, unusable, and increasing operational complexity.

Inactive Publication Date: 2014-12-17
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1) Expensive: Because the protein A ligand needs to be produced and purified by genetic engineering, the production cost is high, and its price reaches $10,000/liter of affinity medium;
[0005] 2) There is a lack of effective cleaning methods. Since protein A is unstable in an alkaline environment, general sodium hydroxide in-situ cleaning methods cannot be used;
[0006] 3) Since the ligand is also a protein, it is easily hydrolyzed by the protease present in the raw material solution, thereby reducing the separation effect;
[0008] 5) The ligand is easy to fall off, and the fallen protein A will cause human immune response and has been clin...

Method used

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  • Micromolecule antibody affinity peptide and applications thereof
  • Micromolecule antibody affinity peptide and applications thereof
  • Micromolecule antibody affinity peptide and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Polypeptide Synthesis

[0032] 1) Activation of the resin: Weigh 1000mg of FMOC Cys-wang Resin, add 10-15mL of DMF and soak for 30min (soak all the resin), to make it fully swell.

[0033] 2) Deprotection: Press filtration to remove the DMF soaked in the resin, add 10mL of DMF solution containing 20% ​​piperidine, blow and boil with nitrogen for 15min, then press filter to remove the DMF solution containing 20% ​​piperidine, and remove the FMOC group that protects the amino group Wash the resin three times with 10mL isopropanol and three times with 10mL DMF, and then use the ninhydrin method to detect that the resin should turn black or purple.

[0034] 3) Condensation reaction: connect the first amino acid R 5 , the amount of FMOC-amino acid (His or Lys) weighed is 1.4mmol / g resin, 910mg O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid (TBTU), Add 10mL DMF and 0.45g 1-hydroxybenzotriazole (HOBt) and mix well, then add 0.52mL DIEA to form a...

Embodiment 2

[0039] Example 2 Determination of IgG affinity characteristics

[0040] 1) Prepare 10mmol / L Hepes buffer, pH 7.4, NaCl content 150mmol / L.

[0041] 2) Use the liquid prepared in 1) as the mobile phase, and run the BiacoreT200 analyzer.

[0042] 3) Insert the detection chip CM5 and set the flow rate to 30 μL / min.

[0043] 4) Inject 100 μL each of EDC and NHS in sequence to activate the chip.

[0044] 5) Dissolve IgG in sodium acetate buffer, pH 4.5, IgG 100μg / mL.

[0045] 6) Inject 100 μL of the IgG solution in 5), and immobilize IgG on the surface of the chip.

[0046]7) Small molecule antibody affinity peptide 1 (amino acid sequence shown in SEQ ID No.11), small molecule antibody affinity peptide 2 (amino acid sequence shown in SEQ ID No.34) and natural ligand (ProG and ProA), dissolved in 1) to prepare liquid, content 100μg / mL.

[0047] 8) Sequentially inject different concentrations of peptides and natural ligands (concentrations from high to low are 500 μm, 250 μm, 125...

Embodiment 3

[0053] Example 3 Comparison of adsorption properties of small molecule antibody affinity peptides with IgG and HAS

[0054] 1) Prepare 10mmol / L Hepes buffer, pH 7.4, NaCl content 150mmol / L.

[0055] 2) Use the liquid prepared in 1) as the mobile phase, and run the BiacoreT200 analyzer.

[0056] 3) Insert the detection chip CM5 and set the flow rate to 30 μL / min.

[0057] 4) Inject 100 μL each of EDC and NHS in sequence to activate the chip.

[0058] 5) Dissolve IgG in sodium acetate buffer, pH 4.5, IgG 100μg / mL. Also dissolve HSA in sodium acetate buffer, pH 4.5, HSA 100 μg / mL.

[0059] 6) Inject 100 μL of the IgG solution in 5), and immobilize IgG on the surface of the chip.

[0060] 7) Inject 100 μL of the HSA solution in 5), and immobilize HSA on the surface of another chip.

[0061] 8) Dissolve small molecule antibody affinity peptide 3 (amino acid sequence shown in SEQ ID No.1) and small molecule antibody affinity peptide 4 (amino acid sequence shown in SEQ ID No.4) ...

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PUM

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Abstract

The invention discloses a micromolecule antibody affinity peptide and applications thereof. The amino acid sequence of the peptide is represented by any one of SEQ ID No. 1-112, and the peptide can combine with the Fc section of human immune globulin IgG, and does not combine with human serum albumin HAS. The provided micromolecule antibody affinity peptide can be taken as the affinity ligand to achieve the high efficient, safe, and economic IgG separation and purification.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular, the present invention relates to a small molecular antibody affinity peptide and its application. Background technique [0002] Immunoglobulin IgG, that is, an antibody is a special protein molecule, which is used as a reagent for in vitro diagnosis, a drug for treating diseases, a ligand for immunoaffinity chromatography, etc., and has a wide range of applications in the fields of life science research, biotechnology and medicine. Applications. [0003] At present, the separation and purification of IgG mostly use the natural affinity ligand Protein A / G of the antibody. Due to the ability of Protein A / G to specifically recognize the Fc fragment of the antibody, the protein A / G affinity chromatography is used to purify the antibody, and the obtained The purity of the product is high. However, since Protein A / G is also an active biological substance, there are also some pr...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K5/093C07K5/113C07K1/22A61M1/34A61M1/18
Inventor 徐霞韦宇平
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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