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Collagen for preparing hydrogel and preparation method of collagen

A collagen and hydrogel technology, applied in the field of bioengineering, can solve the problems of poor adhesion and unsuitable for in vivo application, etc.

Active Publication Date: 2021-03-19
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been reported artificial silk elastin-like hydrogels, which are mainly based on the physical cross-linking between silk protein peptide units to form a gel network, which can be quickly and gently oxidized into a gel, and endow the hydrogel with a redox response. However, the poor adhesion between these hydrogels and cells makes them unsuitable for in vivo applications.

Method used

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  • Collagen for preparing hydrogel and preparation method of collagen
  • Collagen for preparing hydrogel and preparation method of collagen
  • Collagen for preparing hydrogel and preparation method of collagen

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Synthesis of embodiment 1 vector plasmid

[0034] The control protein sequence VCL was designed according to the codon preference of the expression host Escherichia coli BL21 (DE3), and its specific sequence is shown in SEQ ID No.5. Then according to figure 1 The design mechanism shown in the sequence design sequence S-VCL-S, S-VCL-S1, S-VCL-S2, S-VCL-S3, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and connected to the pET-28a vector plasmid.

[0035] A collagen S-VCL-S for preparing a hydrogel, the sequence of which is shown in SEQ ID No.1, and cysteines are respectively connected at both ends;

[0036] A collagen S-VCL-S1 used for preparing hydrogel, its sequence is shown in SEQ ID No.2, except that cysteine ​​is connected at both ends, and cysteine ​​is inserted into the sequence at the same time.

[0037] A collagen S-VCL-S2 used for preparing hydrogel, its sequence is shown in SEQ ID No.3, except that cysteines are connected at both ends, and two cys...

Embodiment 2

[0039] The preparation of embodiment 2 collagen

[0040] The designed sequence contains the Nco I restriction site and the Bam HI restriction site, and the recombinant plasmid pET-28a containing the polymer gene of the target length was obtained through double restriction screening verification. The verification results are as follows: figure 2 As shown, it proves that it successfully connects the target gene sequence.

[0041] (1) Take Escherichia coli BL21(DE3) competent cells and thaw on ice, add all 20 μL of recombinant plasmids to 100 μL competent cells, gently pipette evenly, and place on ice for 30 minutes. After the ice bath, the competent cells were heat-shocked in a water bath at 42°C for 90 seconds, and quickly placed on ice to cool for 2 minutes after removal. Then, add 500 μL of fresh LB medium without antibiotics to the test tube, and incubate in a constant temperature shaker at 37°C for 1 hour, then spread the bacterial solution on the LB plate containing Kann...

Embodiment 3

[0046] The preparation of embodiment 3 collagen hydrogels

[0047] Take 90 μL of 4.5% w / v one of the four collagen solutions at the bottom of the glass tube, add 10 μL of 0.1% H 2 o 2 Mix gently; place in a 37°C water bath and incubate for 30min. The VCL protein without cysteine ​​was used as the control group, and 0.1% H 2 o 2 , and incubated at 37°C for 30min. After the incubation is complete, remove the glass tube and place it upside down. If the polymer molecules are cross-linked to form a hydrogel, its fluidity is limited and still exists at the bottom of the glass tube; otherwise, it will flow down the tube wall, which can be used to detect whether the gel is formed or not.

[0048] The results showed that VCL could not successfully form hydrogels, while S-VCL-S, S-VCL-S1, S-VCL-S2, and S-VCL-S3 could successfully prepare hydrogels.

[0049] Figure 5 It is the SEM picture of S-VCL-S collagen hydrogel. It can be seen from the picture that the hydrogel exhibits a u...

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Abstract

The invention relates to collagen for preparing hydrogel and a preparation method of the collagen. A collagen sequence containing a bioactive structure GFPGER region is designed, cysteine with an oxidative crosslinking characteristic is introduced into a plurality of sites of the collagen sequence, and four kinds of cysteine-containing collagen with higher purity are obtained through heterologousexpression of escherichia coli. Hydrogen peroxide is used as a crosslinking agent for crosslinking the collagen to prepare the hydrogel. The prepared collagen has good thermosensitivity, can form stable hydrogel construction in an oxidation environment, has no toxicity to cells, and provides a good basis for the application of a drug controlled release carrier.

Description

technical field [0001] The invention relates to collagen for preparing hydrogel and a preparation method thereof, belonging to the technical field of bioengineering. Background technique [0002] Collagen is an important class of biopolymer materials, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for about 30% of the total protein. Collagen has good biocompatibility, low immunogenicity and tissue degradable absorbability. It can stop bleeding and heal wounds, fill, repair and reconstruct defective tissues, and diagnose, treat and repair organisms, as well as drug carriers and tissues. It is widely used in engineering and other aspects, and is an ideal medical biomaterial. [0003] Natural collagen has a wide range of sources, mainly in animal tendons, bones, skin, ligaments and other tissues, and is the main source of collagen raw materials. The methods of extracting collagen include a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/70C12N5/00A61K47/42A61L27/24A61L27/52
CPCC07K14/78C12N15/70C12N5/00A61K47/42A61L27/24A61L27/52C12N2533/54
Inventor 王玠许菲曹俊
Owner JIANGNAN UNIV
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