Active peptide, recombinant carrier, recombinant cell, pharmaceutical composition and its preparation method and application
A technology of recombinant vectors and recombinant cells, applied in the biological field, can solve problems such as side effects and unsuitable for long-term use
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[0113] The natural active peptide can be obtained through the above preparation method of the active peptide, and the purity of the active peptide is relatively high.
[0114] A method for preparing an active peptide in one embodiment includes the following steps: synthesizing the active peptide by chemical synthesis.
[0115] In one of the embodiments, the chemical synthesis method is polypeptide solid-phase synthesis method. Further, the chemical synthesis method is Boc solid phase synthesis or Fmoc solid phase synthesis. Wherein, Boc is tert-butoxycarbonyl. In the Boc solid-phase synthesis method, the Boc group, which is easy to acidify, is used as the N-α-protecting group. Fmoc is 9-fluorenylmethoxycarbonyl. In the Fmoc solid-phase synthesis method, the Fmoc group, which is easy to acidify, is used as the N-α-protecting group.
[0116] The preparation method of the above-mentioned active peptide has simple process, convenient operation, and can prepare active peptide w...
Embodiment 1
[0121] The preparation process of the active peptide of the present embodiment is as follows:
[0122] (1) 2500 mL of ultrapure water was added to 200 g of minced meat of the stone turtle and mixed to obtain minced meat water. Add 700U / mL alkaline protease to the minced meat water and enzymolyze it at 45°C and pH 8.0 for 3 hours, put the enzymolyzed minced meat water at 85°C for 10 min to inactivate the enzyme, and centrifuge at 9000r / min for 10 min to collect supernatant; drying the supernatant to a water content of 0.1% to obtain an enzymatic hydrolyzate.
[0123] (2) Mix 500g of enzymatic hydrolyzate, 3500mL of ethyl acetate and 1000mL of ultrapure water, stand for extraction for 25min, collect the water phase layer, repeat the extraction four times, and mix the water phase layers collected from the four extractions Freeze-drying in vacuum at low temperature to obtain a dried aqueous phase layer.
[0124] (3) The dried aqueous phase layer is subjected to chromatographic s...
Embodiment 2
[0126] The preparation process of the active peptide of the present embodiment is as follows:
[0127] (1) Take 100 mg of Fmoc-Phe Wang Resin (Phenylalanine prepacked resin, purchased from Aladdin Company and the product number is 2018-01-225-NU) into a solid-phase synthesis tube, add 25 mL of N, N -Dimethylformamide (DMF) is allowed to stand still to fully swell the prepacked resin, the solvent is filtered off, and then 5 mL of DMF solution containing 20% piperidine by mass percentage is added, and the solvent is filtered off after shaking to obtain the treatment after the resin.
[0128] (2) 8 mg of Fmoc-His-OH (purchased from Aladdin Company and the product number is 2018-01-225-N22), 5 mg of 1-hydroxybenzotriazole, 3 mg of O-benzotriazole-tetra Methylurea hexafluorophosphate was dissolved in 20 mL of DMF, and after adding 30 mg of N,N-diisopropylethylamine and mixing well, it was protected from light to obtain activated Fmoc-His-OH. Add the activated Fmoc-His-OH to the...
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