Collagen and collagen like peptide based hydrogels, corneal implants, filler glue and uses thereof
A hydrogel and corneal technology, which can be used in medical preparations without active ingredients, medical preparations containing active ingredients, and specific peptides, etc., and can solve problems such as unknown uses.
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Embodiment 1
[0133] Example 1: Collagen-like peptides
[0134] Using 36-amino acid peptide (Pro-Lys-Gly) 4 (Pro-Hyp-Gly) 4 (Asp-Hyp-Gly) 4 Collagen-like peptides were designed as a basis. Glycine and cysteine were added to this polypeptide sequence to allow covalent attachment to 8-arm polyethylene glycolmaleimide (PEG) and formation of robust hydrogels. Addition of additional peptide motifs thereto renders the polypeptide multifunctional.
[0135] A Pichia template vector (pPink-aMF-COL mimic-His-P4H-HC) comprising 10 repeats of the nucleic acid sequence encoding 38-amino acid CLP was used as the template vector. The nucleic acid sequence of the template vector is represented by SEQ ID NO: 1.
[0136] Using a forward primer comprising the nucleic acid sequence shown in SEQ ID NO: 2 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 3, one repeat of the nucleic acid encoding the 38-amino acid sequence was isolated. The nucleic acid sequence of the repeat sequen...
Embodiment 2
[0145] Example 2: Recombinant Production of Recombinant Collagen-like Peptides
[0146] The nucleic acid of Example 1 was cloned into pCOLDI expression vector (Takara Bio Inc). The vector map of the expression vector pCOLDI is as follows: figure 1 shown. The restriction sites used in this procedure were NdeI and XbaI. In addition, the forward primer and reverse primer used are represented by SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
[0147] For the post-translational modification of proline residues to hydroxyproline, a pET vector comprising a nucleic acid encoding prolyl 4-hydroxylase was used.
[0148] Both expression vectors (pCOLDI and pET) were transformed into Escherichia coli BL-21(DE3) strain. Escherichia coli BL-21 (DE3) electrocompetent strain with the trade name sell. BL21(DE3) bacterial cells are used for the production of endotoxin-free recombinant proteins.
[0149] For recombinant expression of CLP, inoculate 4-20 μL of pre-cultured bacteria of ampi...
Embodiment 3
[0153] Example 3: Purification of recombinant collagen-like peptides
[0154] Collagen-like polypeptides were purified using a Ni-NTI column that specifically binds a histidine tag attached to the N-terminus of CLP.
[0155] Equilibrate the Ni-NTA column with 10-15 CV of denaturing binding buffer and apply the supernatant of the protein sample. The column was washed with 3×2 mL of denaturing binding buffer, and the protein was eluted with 5CV of denaturing elution buffer (20 mM disodium hydrogen phosphate, 0.5 M sodium chloride, 0.5 M imidazole, 8 M urea, pH 8.0). Samples were collected and analyzed using SDS PAGE. SDS-PAGE was performed on recombinant CLP using standard protocols. The results of SDS-PAGE are as follows figure 2shown. E1-E5 of the SDS PAGE results show the eluate of CLP represented by SEQ ID NO:10. His-tagged polypeptides were visualized using 300 mM imidazole.
[0156] For dialysis, collect all protein-containing eluates and combine in one 50 ml tube. ...
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