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A kind of preparation method of sponge water-soluble peptide

A sponge-like, water-soluble technology, applied in the field of peptide preparation, can solve the problems of species, separation efficiency and scale limitation, and achieve the effect of less sample loading, convenient operation and easy scale-up

Inactive Publication Date: 2018-04-10
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the discovered sponge peptides are mainly obtained through organic extraction. Peptides are usually mixed with other types of compounds. At present, there is no method for systematic enrichment and separation of sponge peptides. The types of peptides, separation efficiency and scale severely restricted

Method used

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  • A kind of preparation method of sponge water-soluble peptide
  • A kind of preparation method of sponge water-soluble peptide
  • A kind of preparation method of sponge water-soluble peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Water-soluble peptides were secondary isolated from panting moss sponges (Tedania anhelans (Lieberkuhn, 1859)) with a wet weight of 1.3 kg.

[0030] The moss sponge obtained by sampling in the sea area was stored in a -80°C refrigerator, and the first-level crude extract of water-soluble peptides from the sponge was extracted according to the following method:

[0031] 1) Weigh about 1300g of frozen moss sponge and crush it to a size of no more than 3cm;

[0032] 2) Pour in an appropriate amount of liquid nitrogen, and the high-speed pulverizer pulverizes the frozen sponge block into powder, each pulverization time is <60s, after the powder is poured out, melt properly, then add liquid nitrogen to re-agglomerate, and pulverize again;

[0033] 3) Add 2L lysis buffer, stir magnetically for 45min, and sonicate for 30min;

[0034] 4) Centrifuge at 8000×g at 4°C for 30 minutes to obtain 2.4L of supernatant, 21.6g of total protein measured by Lowry method;

[0035] 5) PALL ...

Embodiment 2

[0046] Static adsorption and desorption rates of water-soluble peptides in moss sponges by four kinds of macroporous adsorption resins.

[0047] 1) Moss sponge with a wet weight of 165g According to the steps 1) to 5) of Example 1, 350 mL of the filtrate of the MWCO5K membrane bag was obtained, and the Lowry method measured 0.44 mg / mL;

[0048] 2) Treat four kinds of macroporous adsorption resins according to the instructions of the resins: DA201-C, D101, NKA-9, AB-8, mix 1mL resin with 5mL MWCO5K membrane bag filtrate, shake in a chromatographic cabinet at 4°C ;

[0049] 3) Take 50 μL supernatant at 2h, 4h, 6h, and 16h after mixing, and measure the concentration by Lowry method;

[0050] 4) After 16 hours, remove the supernatant, wash with pure water 5 times, blot the liquid, add 5 mL of 75% ethanol, shake for 1 hour, and measure the concentration of the supernatant by the Lowry method.

[0051] 5) The results are shown in Table 2. DA201-C has better adsorption and desorpti...

Embodiment 3

[0060] Liquid chromatography-tandem mass spectrometry analysis of primary crude extracts of water-soluble peptides from sponges.

[0061] The first-stage peptide crude extract obtained in step 8) of Example 1 was dissolved in 0.1% formic acid and 5% acetonitrile solution at a concentration of 0.3 μg / μl, and the liquid Phase chromatography tandem mass spectrometry analysis. Liquid phase parameters: 10μl on the machine, first enter the guard column (C18PepMap100, 300μm×1mm, 5μm, ), followed by an analytical column (Acclaim PepMap C18, 15cm×75μm, 2μm, ) was analyzed at a flow rate of 0.2 μl / min, and the mobile phase gradient was 5% to 45% mobile phase B (acetonitrile, 0.1% formic acid) for detection at 150 min. Mass spectrometry parameters: positive ion mode, CID collision mode, 120000 resolution, mass range 1060-2850, NCE 35%, top15 ion intensity for MS / MS analysis ( Figure 4 and 5 ). Searching the moss sponge transcriptome data determined in our laboratory, 262 peptides...

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Abstract

A method of preparing a water-soluble peptides sponges, a method for preparing a peptide.The sponges crushed, soaked with extraction buffer, sonicated, obtained after centrifugation soluble proteins / peptides aqueous solution, the adsorption / desorption After filtration, the peptides obtained after desalted crude extract of the first stage; the first peptide grade crude extract after the second stage rotary evaporation to afford a second stage 10 to 30 components, then lyophilized or powdered state in a low-temperature long-term storage in the dark, the specific method is the second stage purified acetonitrile - water gradient reverse phase HPLC method to obtain a 10 to 30 second stage component; the second stage component is a main component of the two components is less than 95% for the third stage purified by rotary evaporation to give sponges water-soluble peptides, then long-term storage or freeze-dried powder to a low temperature in the dark state.Peptide species can increase and improve the processing and separation efficiencies of scale, in order to facilitate the development of sponges active peptide drugs.

Description

technical field [0001] The invention relates to a method for preparing peptides, in particular to a method for preparing sponge water-soluble peptides. Background technique [0002] People have isolated more than 20,000 kinds of natural products from marine organisms, including peptides, proteins, polysaccharides, alkaloids, terpenes, macrocyclic polyesters, etc., among which a large number of compounds are peptides, Compared with large proteins, peptides have small molecular weight, no immunogenicity, simple structure, few side effects, and high biological activity. Due to the specific environment in which marine organisms live, the structure of marine peptides is very different from that of terrestrial animals and plants. The development of marine peptides has increasingly become a hot spot in marine drug research. [0003] Sponges belong to the phylum Porezoa, the lowest multicellular invertebrate among animals, and also the marine organism with the most potential for de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/36C07K1/34C07K1/22C07K1/20
Inventor 陈军丁少雄王德祥贺腾飞罗联忠赵晶
Owner XIAMEN UNIV
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