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Specific affinity polypeptide for polyurethane interface and screening method of specific affinity polypeptide

A screening method and polyurethane technology, applied in the fields of peptides, material testing products, biological testing, etc., can solve the problems of being unsuitable for large-scale industrial application, complex preparation process, high environmental and equipment requirements, and achieve the effect of fast, efficient and stable combination.

Inactive Publication Date: 2015-03-25
陕西卓阳瑞信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem with covalently immobilizing enzyme proteins on the surface of polyurethane coatings is that the preparation process is complicated and requires high requirements on the environment and equipment, which is not suitable for large-scale industrial applications.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0021] (1) Preparation of water-based polyurethane coating: 3 mL of aliphatic water-based polyurethane PU-116 (purchased from Anhui Anke Fine Chemicals) was added to a cell culture dish (35 mm X 12 mm purchased from Fisher Scientific), and placed horizontally Cured at room temperature for 24 hours, then placed at 70 o C further solidified in the oven for 4 hours and took it out for use. Preparation of solvent-based polyurethane coating: polyacrylic acid resin (Hydroxyl-bearing polyacrylate, Desmophen A870) and polyisocyanate resin (Hexamethylene diisocyanate, Desmodur N3600) were purchased from Bayer. Weigh 2.1 g polyacrylic resin Desmophen A870 into a 20 mL glass bottle, add 0.5 mL n-butyl acetate, stir vigorously (1500 rpm) for 1 minute, add 0.8 g polyisocyanate resin Desmodur N3600, and continue stirring for 1 minute. Transfer the mixture to a cell culture dish placed horizontally, put the coating in a ventilated place for 10 minutes, and then put it in 70 o C oven, place...

Embodiment

[0030] 1. Experimental materials

[0031] 1) Random dodecapeptide phage display library 100 μl, 1.5×1013 pfu / ml. Store in TBS solution containing 50% glycerol. Complexity ~2.7×10 9 a transformant.

[0032] 2) -28 gIII sequencing primer 5'-HOGTA TGG GAT TTT GCT AAA CAA C-3', 100 pmol, 1 pmol / μl

[0033] 3) -96 gIII sequencing primer 5'-HOCCC TCA TAG TTA GCG TAA CG-3', 100 pmol / μl, 1 pmol / μl

[0034] 4) E.coli ER2738 host strain F' lacIq Δ(lacZ)M15 proA+B+ zzf::Tn 10 (TetR) / fhuA2 supE thi Δ(lac-proAB) Δ(hsdMS-mcrB)5 (rk – m k – McrBC –). The strain is provided in the form of bacterial culture containing 50% glycerol, non-competent cells. Store at -70°C.

[0035] 5) Streptavidin, freeze-dried powder 1.5 mg.

[0036] 6) Biotin, 10 mM 100 μl Ph.D.TM is a registered trademark of New England Biolabs, Inc.

[0037] 7) Antibodies.

[0038] 8) Water-based aliphatic polyurethane PU-116 was purchased from Anhui Anke Fine Chemicals

[0039] 9) Solvent-based two-component polyure...

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PUM

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Abstract

The invention discloses a specific affinity polypeptide for a polyurethane interface and a screening method of the specific affinity polypeptide, and relates to the technical field of biological materials. The specific affinity polypeptide contains affinity peptide of a VHWDFRQWWQPS specific amino acid sequence. The screening method comprises the following steps: respectively adopting water-based polyurethane and solvent-based polyurethane coating materials as target interfaces; screening the peptide which is in specific affinity to the target interfaces by using a phage display technology, including affinity selection of a phage 12 peptide library and phage-cloned ELISA identification; and finally preparing positive phage clone DNA and carrying out sequence determination. Short peptide molecules can be rapidly, stably and efficiently combined on the surface of a polyurethane coating material through direct infiltration contact and can be stably combined on the surface of the solvent-based polyurethane material; a foundation is laid for further integration of active enzyme protein molecules and development of a rapid immobilization process of zymoprotein on the polyurethane surface; and a good method is also provided for development of the polyurethane material with biological activity.

Description

Technical field: [0001] The invention relates to the technical field of biological materials, in particular to a short peptide specifically bound to a polyurethane material interface and a screening method thereof. Background technique: [0002] The phage random peptide library display technology is to insert the DNA sequence encoding the foreign peptide into the gene encoding the coat protein on the surface of the phage, so that a large number of random peptides are expressed on the surface of the phage in the form of fusion. On the basis of this technology, peptides with specific affinity to target proteins, cells, tissues, materials, etc. can be screened through repeated binding-elution-monoclonal amplification. [0003] The full name of polyurethane is polyurethane, which is a general term for macromolecular compounds containing repeated carbamate groups in the main chain. It is formed by addition polymerization of organic diisocyanate or polyisocyanate and dihydroxy...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68
Inventor 杨柳张立挺
Owner 陕西卓阳瑞信息科技有限公司
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