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A kind of preparation method of the functional silk fibroin membrane that is beneficial to cell adhesion

A silk film and cell technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, animal/human peptides, etc., can solve the problems of few research reports, no research reports, no application, etc., and achieve single molecular weight , promote adhesion and spreading, and reduce damage

Active Publication Date: 2018-01-02
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Silkworm silk is a good natural biological material, domestic and foreign studies mainly focus on silkworm silk fibroin, and there are few reports on the use of wild silkworm silk such as tussah silk or celestial silk as a biological material (Journal of Wuhan University of Technology: Materials Scinece , 2011, 26(6): 1044-1048; Biomed Mater, 2006, 1: 181-187), because it cannot be domesticated, the yield is very low
But it is worth noting that there are a large number of unit repeating fragments containing RGD tripeptide in the amino acid sequence of tussah silk or cepebromin, which comes from the function of the unit sequence and repeating sequence containing RGD in natural wild silk fibroin No research reports yet, let alone applications

Method used

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  • A kind of preparation method of the functional silk fibroin membrane that is beneficial to cell adhesion
  • A kind of preparation method of the functional silk fibroin membrane that is beneficial to cell adhesion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Will carry (-RGD-) 4 The prokaryotic system expression vector of the polypeptide gene was transfected into Escherichia coli (BL21) cells, coated on Luria-Bertani solid medium containing ampicillin, and placed upside down in a biochemical incubator at 37°C for 14-16 hours. Pick a single colony and put it into 4mL of fresh Luria-Bertani liquid medium containing ampicillin, insert an air shaker with a shaking speed of 200r / min at 37°C for 8-10 hours with shaking. The cultured bacterial liquid was amplified in 1 L of fresh Luria-Bertani liquid medium containing ampicillin at a ratio of 1:100. When the bacterial liquid density reaches OD 600 When = 0.3-2.1, 0-1.0 mM isopropyl-β-D-thiogalactoside was added to induce culture for 1-8 hours, and the bacterial cells were collected by centrifugation at 4°C.

[0022] 2. Resuspend and mix the collected bacterial cells with the binding buffer of glutathione transferase (GST) affinity chromatography, and put them on ice to sonica...

Embodiment 2

[0029] 1. Will carry (-RGD-) 8 The prokaryotic system expression vector of the polypeptide gene was transfected into Escherichia coli (BL21) cells, coated on Luria-Bertani solid medium containing ampicillin, and placed upside down in a biochemical incubator at 37°C for 14-16 hours. Pick a single colony and put it into 4 mL of fresh Luria-Bertani liquid medium containing ampicillin, and insert it into an air shaker with a shaking speed of 200 r / min at 37°C for 8 to 10 hours. The cultured bacterial liquid was amplified in 1 L of fresh Luria-Bertani liquid medium containing ampicillin at a ratio of 1:100. When the bacterial liquid density reaches OD 600 When = 0.3-2.1, 0-1.0 mM isopropyl-β-D-thiogalactoside was added to induce culture for 1-8 hours, and the bacterial cells were collected by centrifugation at 4°C.

[0030] 2. Resuspend and mix the collected bacterial cells with the binding buffer of glutathione transferase (GST) affinity chromatography, and put them on ice to ul...

Embodiment 3

[0037] 1. Will carry (-RGD-) 4 The prokaryotic system expression vector of the polypeptide gene was transfected into Escherichia coli (BL21) cells, coated on Luria-Bertani solid medium containing ampicillin, and placed upside down in a biochemical incubator at 37°C for 14-16 hours. Pick a single colony and put it into 4 mL of fresh Luria-Bertani liquid medium containing ampicillin, and insert it into an air shaker with a shaking speed of 200 r / min at 37°C for 8 to 10 hours. The cultured bacterial liquid was amplified in 1 L of fresh Luria-Bertani liquid medium containing ampicillin at a ratio of 1:100. When the bacterial liquid density reaches OD 600 When = 0.3-2.1, 0-1.0 mM isopropyl-β-D-thiogalactoside was added to induce culture for 1-8 hours, and the bacterial cells were collected by centrifugation at 4°C.

[0038] 2. Resuspend and mix the collected bacterial cells with the binding buffer of glutathione transferase (GST) affinity chromatography, and put them on ice to ul...

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Abstract

The invention discloses a preparation method of a functional silk fibroin film which is beneficial to cell adhesion. The steps include: transfecting Escherichia coli with a prokaryotic system expression vector carrying (‑RGD‑)4 polypeptide or (‑RGD‑)8 polypeptide gene Cells, induced and cultured for several hours; centrifuge the cell culture liquid, collect and crush the bacterial cells, purify the crushed protein supernatant, collect by enzymatic digestion in one step, and freeze-dry to obtain (‑RGD‑)4 polypeptide powder or (‑RGD ‑)8 Polypeptide powder; degumming, dissolving, dialysis, filtering and concentrating silkworm silk or silkworm cocoons, pouring them into a container with a certain area and air-drying to obtain silkworm fibroin film; immersing the silkworm silk film in excess cross-linking agent solution Then add (‑RGD‑)4 polypeptide or (‑RGD‑)8 polypeptide and react overnight at 4°C to obtain a functional silk fibroin film. The functional silk fibroin membrane prepared by the invention has excellent cell adhesion performance, belongs to biomedical materials, has no cytotoxicity, has recognition sites of cell adhesion proteins, and is beneficial to cell adhesion, growth, and promotion of defect tissue healing.

Description

technical field [0001] The invention relates to a preparation method of a silk fibroin material applied in the field of biomedicine, in particular to a preparation method of a functional silk fibroin film favorable for cell adhesion. Background technique [0002] The RGD sequence is composed of arginine, glycine and aspartic acid, and widely exists in a variety of extracellular matrices, such as collagen, fibronectin, laminin, vitronectin, elastin, etc. A variety of integrins specifically bind, contribute to cell adhesion, and promote the adhesion and growth of adherent cells. Silk membranes chemically modified by RGD and other modified materials such as polymers can improve the adhesion, diffusion and proliferation of cells on materials (such as J Biomed MaterRes A, 2003, 67 (2), 559-570; Biomaterials, 2002, 23: 4315-4323, etc.). RGD peptide can also competitively inhibit the binding of various adhesion proteins such as fibrin to platelets and the adhesion of platelets to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08J5/18C08L89/00C12N15/12C12N15/70C07K14/435
Inventor 王建南武明扬裔洪根
Owner SUZHOU UNIV
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