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Preparation method of spongia water-soluble peptides

A spongy, water-soluble technology, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of species, separation efficiency and scale limitations, and achieve a low sample load, convenient operation, and easy to scale up. Effect

Inactive Publication Date: 2015-04-29
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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  • Preparation method of spongia water-soluble peptides
  • Preparation method of spongia water-soluble peptides

Examples

Experimental program
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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

The invention discloses a preparation method of spongia water-soluble peptides and relates to a preparation method of peptides. The method comprises the following steps: crushing the spongia and soaking in an extraction buffer liquid; performing ultrasonic treatment; centrifuging to obtain an aqueous solution of soluble protein / peptides; filtering and performing adsorption / desorption to obtain a desalted first-level crude extract of peptides; performing rotary evaporation and concentration on the first-level crude extract of peptides and performing second-level purification to obtain 10-30 second-level components, or performing freeze drying and storing for long time without light and at a low temperature in a form of powder, wherein the second-level purification specifically refers to obtaining 10-30 second-level components by an acetonitrile-water gradient reversed phase HPLC method; and performing rotary evaporation and concentration on the second-level components with less than 95% of main ingredient and then performing third-level purification to obtain the spongia water-soluble peptides, or performing freeze drying and storing for long time without light and at a low temperature in a form of powder. The species of peptides can be increased, and the processing scale and separation efficiency are improved so as to facilitate the development of spongia 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 Applications(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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