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CN-121695839-B - Silk fibroin modified silica gel chromatographic packing and preparation method and application thereof

CN121695839BCN 121695839 BCN121695839 BCN 121695839BCN-121695839-B

Abstract

The invention discloses a silk fibroin modified silica gel chromatographic packing, a preparation method and application thereof, wherein the preparation method comprises the following steps of adding protease into regenerated silk fibroin solution, incubating and centrifuging, and dissolving collected silk fibroin precipitate into hydrogen bond inhibitor solution to obtain hydrophobic silk fibroin solution; dispersing inorganic silica gel powder into a hydrophobic silk fibroin solution, performing dialysis treatment, cleaning a solid product after the dialysis treatment, and performing vacuum freeze drying treatment to obtain the silk fibroin modified silica gel chromatographic packing. The preparation method provided by the invention is environment-friendly, has no toxic reagent and chemical crosslinking agent residues, and the prepared silk fibroin modified silica gel chromatographic packing has the advantages of regular surface structure, relatively uniform particle size and adjustable polarity, and can be applied to the chromatographic separation field of biological macromolecules.

Inventors

  • LIU YEZHUO
  • Request for anonymity
  • YANG WENHUA
  • ZHANG SHUAIXIN

Assignees

  • 复向丝泰医疗科技(苏州)有限公司

Dates

Publication Date
20260512
Application Date
20260224

Claims (8)

  1. 1. The preparation method of the silk fibroin modified silica gel chromatographic packing is characterized by comprising the following steps of: s1, adding protease into regenerated silk fibroin solution for incubation, and collecting silk fibroin precipitate through centrifugal treatment, dissolving the silk fibroin precipitate into hydrogen bond inhibitor solution to obtain hydrophobic silk fibroin solution, wherein the concentration of the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is 2-20 wt%, the protease is one or more selected from trypsin, alpha-chymotrypsin, pepsin, papain and proteinase K, and the hydrogen bond inhibitor solution is one or more selected from lithium bromide solution, sodium thiocyanate solution, zinc chloride solution and calcium chloride-ethanol-water ternary solution; s2, dispersing inorganic silica gel powder into the hydrophobic silk fibroin solution obtained in the step S1 under the stirring condition, and then performing dialysis treatment, wherein the stirring speed is 20-100 rpm; S3, adopting methanol and/or ethanol as a cleaning solvent, soaking the solid product subjected to dialysis treatment, cleaning, and performing vacuum freeze drying treatment to obtain the silk fibroin modified silica gel chromatographic packing.
  2. 2. The method according to claim 1, wherein the concentration of silk fibroin in the regenerated silk fibroin solution in S1 is 0.5-10 wt%.
  3. 3. The method according to claim 1, wherein in S1, the mass ratio of the protease to the regenerated silk fibroin in the silk fibroin solution is 1 (50-500).
  4. 4. The method according to claim 1, wherein in S2, the particle size of the inorganic silica gel powder corresponds to 100-1000 mesh; And/or the mass ratio of the inorganic silica gel powder to the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is (2-20): 1.
  5. 5. The method according to claim 1, wherein in S2, the dialysis treatment is performed for a period of 8 to 48 h and at a temperature of 2 to 40 ℃.
  6. 6. The method according to claim 1, wherein in S3, the vacuum degree of the vacuum freeze-drying treatment is 0-40 Pa, the temperature is-60 ℃ to-20 ℃ and the time is 6-24 h.
  7. 7. A silk fibroin modified silica gel chromatographic packing prepared by the method of any one of claims 1-6.
  8. 8. Use of a silk fibroin modified silica gel chromatographic packing according to claim 7 in biomacromolecule chromatographic separation.

Description

Silk fibroin modified silica gel chromatographic packing and preparation method and application thereof Technical Field The invention relates to the technical field of silica gel chromatographic packing, in particular to a silk fibroin modified silica gel chromatographic packing, and a preparation method and application thereof. Background The chromatographic separation technology is widely applied to the scenes of protein purification and the like in the biomedical field, and the traditional silica gel chromatographic packing is commonly used for separating small molecular compounds due to good mechanical stability and porous structure, but has the obvious defects of insufficient specific binding capacity, low separation efficiency and poor biocompatibility in the separation process of biological macromolecules (especially protein substances), is easy to cause denaturation of target proteins, and is difficult to meet the high-efficiency separation requirement of the biological macromolecules. Silk fibroin, which is a natural polymer compound extracted from silk, has excellent mechanical strength, controllable hydrophilicity and hydrophobicity and biodegradability, and has been widely used in the fields of tissue engineering, medical instruments, cosmetics and the like. The silk fibroin is composed of 18 amino acid residues, the molecular structure of the silk fibroin is composed of a highly ordered crystallization region and a loosely disordered amorphous region in an alternating mode, wherein the crystallization region is mainly formed by repeatedly arranging amino acids with smaller side groups such as glycine (G), alanine (A), serine (S) and the like, the silk fibroin has the characteristics of regular structure, hydrophobicity and weak polarity, and the amorphous region is rich in hydrophilic amino acids with larger side groups, and is easy to degrade and disordered in structure. Therefore, the silk fibroin with complete sequence has the problems of easy hydrolysis, poor mechanical property, unstable adsorption property and the like due to the structural characteristics, and cannot be directly used as chromatographic packing or coating. In the prior art, a part of patents relate to the preparation of a silk fibroin coating or a silica gel chromatographic packing, but have obvious limitations that Chinese patent CN110982429A discloses a preparation method of the silk fibroin coating for a medical catheter, is only used for optimizing the coating of the medical catheter, does not relate to the modification of the silk fibroin on inorganic silica gel and the application of biological macromolecule chromatographic separation, does not adopt an enzyme cutting technology to modify a silk fibroin structure, and is used for preparing a silica gel matrix chromatographic packing for strong polarity medicine separation, and a preparation method of a core-shell cholesterol-based silica gel chromatographic packing disclosed in Chinese patent CN118663236A are both modified in a chemical bonding mode, have complex reaction flow, introduce a large amount of toxic or non-environment-friendly organic reagents, have poor biocompatibility, are not related to the separation of silk fibroin, are only suitable for separating strong polarity medicines or tea saponin compounds, and cannot meet the separation requirements of nonpolar/weak polarity biological macromolecules. Disclosure of Invention The invention aims to solve the technical problems of insufficient specific binding capacity, low separation efficiency, poor biocompatibility, easy target protein denaturation and the like of the traditional silica gel chromatographic packing in the separation of biological macromolecules, and provides a silk fibroin modified silica gel chromatographic packing, a preparation method and application thereof, so as to realize the efficient and mild separation of biological macromolecules. The above object of the present invention is achieved by the following technical solutions: The first aspect of the invention provides a method for preparing a silk fibroin modified silica gel chromatographic packing, comprising the following steps: S1, adding protease into regenerated silk fibroin solution for incubation, and collecting silk fibroin precipitate through centrifugal treatment, dissolving the silk fibroin precipitate into hydrogen bond inhibitor solution to obtain hydrophobic silk fibroin solution, wherein the concentration of the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is 2-20 wt%; s2, dispersing inorganic silica gel powder into the hydrophobic silk fibroin solution obtained in the step S1 under the stirring condition, and then performing dialysis treatment, wherein the stirring speed is 20-100 rpm; S3, adopting methanol and/or ethanol as a cleaning solvent, soaking the solid product subjected to dialysis treatment, cleaning, and performing vacuum freeze drying treatment to obtain the silk fibroin modified sili