Alpha type conotoxin peptides and applications thereof
A technology of sequence and amino acid, applied in the field of α-type Cono snail polypeptide, can solve the problems of poor treatment effect, tolerance of toxic and side effects, etc.
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Embodiment 1
[0081] Embodiment 1, the discovery of α-type conus polypeptide
[0082] 1. Extraction of cone snail total RNA
[0083] Six kinds of live snails were collected from the Xisha Islands and Sanya, Hainan as sample materials, and the total RNA of the six kinds of snails was extracted by Trizol method. 6 kinds of cone snails: marble cone (Conus marmoreus), stately cone (Conus imperialis), signal cone (Conus litteratus), black heart cone (Conus emaciatus), maiden cone (Conus eberneus), calf cone (Conus Conus vitulinus).
[0084] The specific steps of Trizol method to extract total RNA:
[0085] ① Take the cone venom tube on ice first, freeze it in liquid nitrogen, grind it into powder with a grinder, and pour it into a micro homogenizer that has been ice-bathed in advance.
[0086] ②Pipe 1.2ml Trizol into the homogenizer and grind further to lyse the cone snail tissue to release RNA.
[0087] ③ Pour the ground slurry into a 1.5ml centrifuge tube and let stand at room temperature fo...
Embodiment 2
[0115] The synthesis of embodiment 2, alpha type conus polypeptide
[0116] The seven polypeptides shown in Table 3 were synthesized respectively.
[0117] Amino acid sequence of table 3α type conus polypeptide
[0118] name
sequence
T-1
NH 2 -GCCSNPACMLKNPNLC*
T-2
NH 2 -NCCTRSFCKRIYPDLC*
T-3
NH 2 -GCCDIPDCYNKNREQC*
T-4
NH 2 -NCCMFHTCPIDYSRFNC*
T-5
NH 2 -GNCCMFHTCPIDYSRFNC*
T-6
NH 2 -GNCCMFHTCPIDYSRFYC*
T-7
NH 2 -RKCCSNPACNRYNKLC
[0119] D-aspartic acid; E-glutamic acid; I-isoleucine; K-lysine; L-leucine; M-methionine; N-asparagine; Q-glutamine; R- Arginine; S-serine; T-threonine; G-glycine; F-phenylalanine; P-proline; H-histidine; A-alanine; Y-tyrosine. For T-1 and T-7, the disulfide linkages in each polypeptide are C1-C3, C2-C4. * indicates amidation (CONH 2 ).
[0120] 1. Peptides
[0121] (1) Synthesis of T-1 (artificially synthesized by Fmoc ...
Embodiment 3
[0173] Embodiment 3, the analgesic activity assay of α-type conus polypeptide
[0174] 1. Preparation of α-cono polypeptide Vc1.1
[0175] α-cono polypeptide Vc1.1: GCCSDPRCNYDHPEICONH 2 .
[0176] 1. Synthesis of peptide resin
[0177] using solid-phase synthesis techniques. It was carried out on a 433A polypeptide synthesizer (ABI Applied Systems Biological Instruments). The amino acid used in the synthesis is Fmoc protected amino acid (product of Advanced Chemtech Company in the United States), the resin used is Rink resin (product of Advanced Chemtech Company in the United States), and the reagents used include DCC (Acros Company), HOBt (product of Advanced Chemtech Company in the United States), NMP (PE company), piperidine (Shanghai Gil Biochemical), methanol (domestic analytical grade) and dichloromethane (domestic analytical grade).
[0178] The side chain protecting groups of Fmoc amino acids are as follows: the side chain protecting groups of His and Cys are tri...
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