Preparation and application of bionic intelligent hydrogel
A hydrogel and protein technology, applied in the field of biomedical materials, can solve the problems of poor molecular weight uniformity of natural biopolysaccharide molecules, poor biocompatibility, and difficult to control product quality, and achieves controllable gelation time and mechanical properties. The effect of good compatibility and broad application prospects
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Embodiment 1
[0025] Example 1: Screening of protein fragments that can spontaneously form hydrogels
[0026] Chlamys farreri were kept in a laboratory environment (about 18°C). The roots of the silk were collected every 24 hours, frozen in liquid nitrogen, and ground in a mortar. Collect the ground silk, add protein extract, extract at 37°C for 1 hour, and centrifuge at 17,600g for 30 minutes. After centrifugation, the supernatant was dialyzed into pre-cooled deionized water (the molecular weight cut-off of the dialysis bag was 3.5 kDa) to remove guanidine hydrochloride and glacial acetic acid. Dialyzed samples were lyophilized. The extracted silk root protein was identified by Shotgun mass spectrometry, and the retrieved database was Chlamys farreri genome.
[0027] The following two conditions were used for screening: (1) specifically expressed genes; (2) the expression level of the protein and the content in the root of silk should be relatively high. Through the above conditions, t...
Embodiment 2
[0029] Example 2: Recombinant expression of protein and preparation of hydrogel
[0030] 1. Sample Preparation
[0031] The nucleotide encoding the protein whose amino acid sequence is SEQ ID NO: 1 was ligated to the pET32a vector. The ligated plasmid was transformed into the expression host Escherichia coli BL21(DE3). Single clones with correct sequencing were selected for recombinant expression.
[0032] The strains were cultured overnight in LB medium, and the overnight cultured Escherichia coli was expanded at a ratio of 1:100. OD 600 At about 0.6-0.8, induce with 0.2mM IPTG, induce overnight at 16°C, collect the bacteria, separate and purify or store at -80°C.
[0033] 2. Separation, purification and renaturation of recombinant protein
[0034] The cells were resuspended in PBS, placed in an ice-water mixture and sonicated for 30 minutes, with a power of 40%, and sonicated for 3 seconds with an interval of 6 seconds. Centrifuge at 17,600g for 10min and discard the s...
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