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Protein sericin gel based on transformation of human platelet derived growth factor gene silk and preparation method and application of protein sericin gel

A platelet-derived, growth factor technology, applied in the field of protein sericin gel preparation, to achieve the effects of promoting cell proliferation, good cell compatibility, and improving mechanical properties

Active Publication Date: 2019-10-22
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the extraction of active PDGF-BB protein from PDGF-BB silk and the preparation of gel biomaterials that can be used in tissue engineering.

Method used

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  • Protein sericin gel based on transformation of human platelet derived growth factor gene silk and preparation method and application of protein sericin gel
  • Protein sericin gel based on transformation of human platelet derived growth factor gene silk and preparation method and application of protein sericin gel
  • Protein sericin gel based on transformation of human platelet derived growth factor gene silk and preparation method and application of protein sericin gel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, the method for extracting sericin in PDGF-BB silk

[0037] The cocoon shells of the silkworms transformed with PDGF-BB ( figure 1 , A) ground into powder through liquid nitrogen ( figure 1 , B), standby. With a bath ratio of 50mg / ml, use extraction buffer (50mM Tris-HCl, 8M urea, pH 7.0) to extract in a water bath at 80°C for 45min ( figure 1 , C). After the extraction, filter with gauze to remove undissolved silk, then centrifuge at 18,000 rpm and 25° C. for 30 minutes, and take the supernatant to obtain a sericin solution. Utilize BCA method to measure total protein concentration, the result shows that total sericin concentration is 3.24mg / mL ( figure 2 ).

Embodiment 2

[0038] Embodiment 2, the preparation method of PDGF-BB sericin gel (SS-PDGF-BB)

[0039] plan 1:

[0040] The sericin solution prepared in Example 1 was added with a concentration equivalent to 1 / 4 of the volume of the sericin solution and was 1M Tris-HCl (pH 9.0), and 50 mL of the mixed sericin solution was packed into a dialysis bag (embranes , MWCO 3,500Da, Spectrum Laboratory, Inc, USA), and dialyzed in 3L of dialysis buffer (50mM Tris-HCl, 4M urea, pH 8.0) at 16°C for 12h ( figure 1 , D). After the dialysis, the sericin solution was introduced into abrasive tools of different shapes, and then placed at 4°C for 24-72h ( figure 1 , E), using low temperature to induce the gelation reaction of sericin. The solid sericin hydrogel was collected, put into a dialysis bag (embranes, MWCO 3,500Da, Spectrum Laboratory, Inc, USA) again, placed in PBS solution, and dialyzed at 4°C to geodesically remove the sericin hydrogel in the gel. Urea, replace the PBS solution once every 12h...

Embodiment 3

[0044] Embodiment 3, PDGF-BB sericin gel (SS-PDGF-BB) performance analysis

[0045] (1) SS-PDGF-BB electron microscope observation (SEM)

[0046] The SS-PDGF-BB with a length and width of 1 cm was freeze-dried, the cross-section of the sample was plated with gold, and observed and photographed with a scanning electron microscope (Supra 55sapphire, Zeiss). All experiments were performed at room temperature with a voltage of 3.0 kV. The number of samples in each experiment is 5 independent samples, and each sample is taken 3 times, and the results are as follows image 3 shown. The results showed that the hydrogel was a layered, porous and interconnected microporous structure. The pore size was measured. The pore size of PDGF-BB sericin hydrogel was 51.8±8.2 μm, and that of WT sericin hydrogel was 47.4±7.6 μm. μm. The porosity of the PDGF-BB sericin hydrogel was tested and analyzed, and the porosity of the PDGF-BB sericin hydrogel was 72.7%, and that of the WT sericin hydrog...

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Abstract

The invention discloses a protein sericin gel based on transformation of human platelet derived growth factor (PDGF-BB) gene silk and a preparation method and application of the protein sericin gel. By using the functional silk of the human-derived PDGF-BB, sericin with the active PDGF-BB protein is extracted, a gelling reaction of the sericin is induced at the low temperature, the prepared protein sericin gel is good in mechanical property, degradable and good in cell compatibility and does not have toxicity, the PDGF-BB can stably exist, apoptosis cannot be caused, the obvious cellular inflammatory response cannot be caused, and the sericin gel biological material with a function of improving the cell proliferation activity can be applied to the tissue engineering.

Description

technical field [0001] The invention relates to the field of biomedical materials, in particular to a protein sericin gel based on transhuman platelet-derived growth factor gene silk, and also to a preparation method and application of the protein sericin gel. Background technique [0002] Silk is mainly composed of silk fibroin in the inner layer and sericin in the outer layer, the content of which is 75% and 25% respectively. For a long time, a large amount of sericin protein produced by the silk reeling industry has been regarded as "waste" and discharged, resulting in serious environmental safety hazards. With the deepening of research, sericin has been revealed to have the functions of anti-oxidation, moisturizing, promoting cell proliferation and accelerating wound healing, and has become a potential raw material for biomaterials. Sericin can be prepared such as sericin ointment, sericin-gelatin film, sericin-carboxymethyl cellulose, sericin poly(ethylene) after chemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/22A61L27/52A61L27/54C08J3/075C08L89/00
CPCA61L27/227A61L27/52A61L27/54A61L2300/414C08J3/075C08J2389/00C08L89/00
Inventor 王峰侯凯王日远赵萍夏庆友
Owner SOUTHWEST UNIVERSITY
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