Conotoxin polypeptide, preparation method and application thereof
A conotoxin and amino acid technology, applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve problems such as diplopia, unsuitable for wide use, unnatural expressions, etc., achieve inhibition of acetylcholine receptors, good economical and social effects, effects that apply to a wide range of
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[0036] The present invention also provides a preparation method of the conotoxin polypeptide, comprising the following steps:
[0037] 1) Solid phase synthesis of HPA-amino resin;
[0038] 2) Cleavage of HPA-amino resin to obtain HPA crude peptide;
[0039] 3) The crude HPA peptide was dissolved in 1% acetic acid solution, separated by gel column chromatography, and purified by reverse-phase high-performance liquid chromatography to obtain the refined HPA peptide.
[0040] Wherein, step 1) of the preparation method of the present invention adopts the Fmoc / tBu synthesis strategy, uses the amino resin with a suitable degree of substitution as a carrier, and couples amino acids with Fmoc protecting groups one by one in solid phase synthesis to obtain HPA-amino resin.
[0041] Preferably, the solid-phase synthesis is Fmoc-Arg (Pbf)-OH and amino resin to react to synthesize Fmoc-Arg (Pbf)-amino resin, and then adopt other amino acids of coupling Fmoc protecting group to obtain HPA...
Embodiment 1
[0065] Example 1: Peptide Screening
[0066] Using bioinformatics methods, select the sequence of small peptides in the cysteine ring of α3 / 5 conotoxin and perform homologous sequence alignment in blastp in NCBI (http: / / www.ncbi.nlm.nih.gov) , for one-to-one matching between proteins with the same sequence and the International Cosmetic Ingredient List (INCI), both of them can match the sequence as a candidate small peptide. Screen out NPA, HPA, WNPA, GKYYS, GKHFN.
Embodiment 2
[0067] Example 2: Synthesis of polypeptide HPA.
[0068] 1. Preparation of Fmoc-Arg(pbf)-amino resin
[0069] Add 10.03g of amino resin Fmoc-RinkAmide with a degree of substitution of 0.84mmol / g into a solid-phase reactor, add DMF to soak, make it fully swell for 30min, drain it, and add 20% piperidine / DMF solution to remove the Fmoc protecting group 2 The first reaction was 5 minutes, the second reaction was 15 minutes, and washed with DMF, DCM and methanol. Dissolve 11.1g of Fmoc-Arg(Pbf)-OH, 3.12g of HOBt, and 8.25g of HBTU in DMF and add to the reactor to balance for 5 minutes, add 7.0mL of DIPEA to react at room temperature for 1 hour, drain, and wash with DMF, DCM, and methanol for 3 times ,, 18.13g of Fmoc-Arg(pbf)-RinkAmide resin was obtained by dry weighing, and the detection degree of substitution was 0.68mmol / g.
[0070] 2. Preparation of HPA-amino resin
[0071] Put the Fmoc-Arg(pbf)-RinkAmide resin in the peptide bottle, add 20% piperidine / DMF solution to remov...
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