A jellyfish hairpin polypeptide growth factor and its preparation method and application
A technology of peptide growth factor and jellyfish jellyfish, which is applied in the field of jellyfish jellyfish polypeptide growth factor and its preparation, can solve the problems of easy breakage, falling off of tentacles, consumption of nematocysts, and huge number of nematocysts, etc., to achieve Significant and highly conserved effect on promoting cell proliferation
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Embodiment 1
[0042] Embodiment 1: Preparation of jellyfish nematocystoxin
[0043] Take 200g of frozen jellyfish tentacles in a beaker to thaw, add 5 times the volume of pre-cooled distilled water, and stir continuously for 10 hours with a stirrer; the suspension is filtered with a 40-mesh sampling sieve with a pore size of 450 μm, and the filtrate is collected. Centrifuge at g for 5 min, remove the supernatant, and wash the precipitate with sterile artificial seawater 3 times to obtain nematocysts; add a small amount of 50 mmol / L, pH 3.0 acetic acid to the washed nematocysts, and transfer to a medium containing the nematocysts for crushing. Put the steel balls in the crushing tube, and then use the tissue grinder Mini-Beadbeater to crush at a speed of 4600rpm for 6 minutes. Every 30s of crushing, take out the crushing tube and place it in ice water to cool for 1 minute. After the crushing is completed, centrifuge at 10000×g for 10 minutes to collect the supernatant It is jellyfish nematoc...
Embodiment 2
[0044] Embodiment 2: Separation and purification of nematocystoxin from jellyfish
[0045] (1) Superdex30 column gel filtration chromatography
[0046] The jellyfish nematocystoxin was filtered with a microporous membrane with a pore size of 0.45 μm, and the filtrate was separated and purified on a Superdex30 column, and eluted with 50 mmol / L, pH 3.0 acetic acid at a flow rate of 1 mL / min, collected and eluted by a fixed volume Peak, MALDI-TOF mass spectrometry analysis shows that the components within the outflow volume of 110-115mL (see figure 1 Elution peak SE2) contains the polypeptide growth factor Cc-GRN-1.
[0047] (2) C8 column reversed phase high performance liquid chromatography (RP-HPLC)
[0048] The above effluent volume of 110-115mL components was eluted with a linear gradient with an elution system consisting of water containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid, and the HPLC reversed-phase C8 column chromatography ...
Embodiment 3
[0051] Example 3: Sequencing of Cc-GRN-1 Polypeptide Growth Factor of A.
[0052] The purity of Cc-GRN-1 was identified by high performance liquid chromatography (HPLC), and its molecular weight was determined by MALDI-TOF mass spectrometry (see Figure 4 ), and its amino acid sequence was determined with an automatic protein peptide sequencer.
[0053] The jellyfish jellyfish polypeptide growth factor Cc-GRN-1 prepared by the above method is a single-chain polypeptide isolated from nematocystoxin of jellyfish jellyfish jellyfish, with a molecular weight of 5782.9Da and the primary structure of the complete amino acid sequence of the polypeptide For: Asparagine - Valine - Isoleucine - Cysteine - Proline - Aspartic Acid - Glycine - Threonine - Serine - Phenylalanine - Cysteine - Alanine -serine-glycine-glutamine-threonine-cysteine-cysteine-lysine-leucine-serine-serine-glycine-serine-tyrosine-glycine-cysteine- cysteine-proline-leucine-proline-asparagine-alanine-valine-cyst...
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