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Preparation and application of a kind of bionic medical silk-forming fibrin

A technology of silk protein and fiber, applied in the field of medical material preparation, can solve the problems of fast degradation, weak mechanical properties, and easy to cause immune reactions

Active Publication Date: 2021-06-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the natural collagen hydrogel has weak mechanical properties, rapid degradation and easy to cause immune reactions and other shortcomings; natural hyaluronic acid is sensitive to strong acid, strong alkali, heat, free radicals and hyaluronidase, has poor stability, and is prone to Degradation; Chitosan is insoluble in neutral solutions and most organic solvents due to its tight crystal structure, so its application is greatly limited

Method used

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  • Preparation and application of a kind of bionic medical silk-forming fibrin
  • Preparation and application of a kind of bionic medical silk-forming fibrin
  • Preparation and application of a kind of bionic medical silk-forming fibrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Screening of Sbp5-2 protein

[0024] 1. Cloning of the whole gene of Sbp5-2

[0025] 1.1 Combined with the sequence information of mass spectrometry identification results, design RACE primers

[0026] Using Primer Premier 5 software to design primers for RACE (Rapid Amplification of cDNA Ends) Sbp-5-2

[0027] 3' RACE primer: TCCCAACAATGGAGTATGTGAAGATGCTG,

[0028] 5' RACE primer: GCCTAGTGTAACAGTCATTCTCCAGCCAAG;

[0029] 1.2 RNA extraction

[0030] Three patellofrerio scallops in the adaptation period (0-2h), the growth period of silk secretion (2-16h) and the balance period of silk secretion (16-24h) were dissected and their feet were taken according to the RNA was extracted by the method of Hu et al. (2006) and mixed in equal amounts. Using SMARTer™ RACE cDNA Amplification Kit (Clontech, CA, USA), according to the instructions, construct 5'RACE and 3'RACE libraries.

[0031] 1.3 Cloning the 3' and 5' ends of the target gene respectively

[0032] Add...

Embodiment 2

[0041] Example 2: Preparation of silk-forming fibers

[0042] 1. RM 6 Clonal expression of CT

[0043] According to the amino acid sequence analysis of Sbp5-2, it was found that the part except the signal peptide sequence had multiple functional modules formed by paired cysteines, and a C-terminal (C Terminal) sequence different from the functional modules. The functional module contains a large number of hydrophilic amino acids, and the subclone RM of six functional modules connected after the C-terminal (C Terminal) sequence is selected by screening 6 CT (amino acid sequence number: 160-324) was purified and expressed. ( figure 1 ), the subclone RM 6 The sequence of CT is SEQ ID NO:2;

[0044]NKYYGCPNNGVCEDAEATRNNSCTSYMCVLSKRRTLMKWEILKTGCKTDEGCKYDTDEWTDPDASSCVTRRCDVTFNPTDKTYNSVNRVARHGCRASNGTCYYNSETWLENDCYTRRCDVTTTDKGESMAAIKIESGICKDADGSCKGYGEAMQYRSGAATLNCV;

[0045] 1.1RM 6 CT primer design

[0046] 5' Primer: GGGGTACCAACAGTTGCACCTCATATATGTGTA

[0047] 3' Primer: CCG...

Embodiment 3

[0070] Example 3: Properties of Filamented Fibers

[0071] 1. Characterization of the basic properties of silk-forming fibers:

[0072] 1) Material wet stability and water absorption detection

[0073] Soak the macroscopically formed wet fiber filaments (about 0.1g) in the PBS solution, take them out at 0, 12, 24, 36, 48, 60, 72, 84, 96, and 108 hours, and blot the surface moisture with filter paper. Call the mass M wet . After freezing at -80°C, freeze-drying is carried out, and its mass is called M after freeze-drying. dry , do three parallel sets.

[0074] Swelling rate = (M wet -M dry ) / M dry ×100%.

[0075] Water absorption = (M wet -M dry ) / M wet ×100%.

[0076] Figure 5 for RM 6 Test results of CT protein fiber swelling rate (left picture) and water absorption rate (right picture); the test results show that the swelling performance is relatively stable in a wet environment, and the swelling rate of the material is maintained at about 400% within 108 hour...

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Abstract

The present invention provides a fibroin, the sequence of which is SEQ ID NO:1. In one aspect, the present invention provides a method for preparing a protein fragment for preparing silk fibers, the protein fragment is the 160-324 amino group of scallop foot silk protein whose sequence is SEQ ID NO: 1; its sequence is SEQ ID NO: 1 ID NO: 2. The protein prepared into the silk fiber of the present invention adopts the genetic engineering method in the material source, has low cost, is environmentally friendly and can be produced on a large scale. The protein-based material of the present invention is derived from sea scallop silk, is polymerized and formed in a seawater-like environment, and has good adaptability and stability to a wet environment. It has a good development prospect in the field of medical materials. In terms of material biological safety, marine biological source materials avoid the risk of terrestrial biological disease transmission, and the tested materials have no cytotoxicity and good biocompatibility.

Description

technical field [0001] The invention belongs to the technical field of medical material preparation, and in particular relates to a silk-forming fiber and its application. Background technique [0002] Biomedical materials, also known as biomaterials, are special functional materials that can diagnose, treat, replace, repair, induce regeneration or enhance the functions of cells, tissues and organs in the body. The biological body contains a large amount of water-based extracellular fluid, and the body's cell tissue is always in a "wet" environment composed of interstitial fluid, plasma and lymph. Therefore, for in vivo repair and plugging, support materials must first have good underwater stability and swelling, and at a higher level, excellent biocompatibility and controllable biodegradation and absorption are also required. At present, the popular research materials mainly used in the in vivo environment mainly include some extracellular matrix (extracellular matrix, ECM...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435A61L27/22A61L27/50
CPCA61L27/227A61L27/50C07K14/43504C08L89/00
Inventor 刘伟治王硕硕徐平平
Owner OCEAN UNIV OF CHINA
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