South China Sea M superfamily conotoxin encoding sequence and applications thereof
A coding sequence, technology of conus, applied in the field of application in the development of ion channel drugs and analgesic drugs
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Embodiment 1
[0049] Example 1: High-throughput sequencing of cono venom tubes
[0050] Extraction of total RNA and synthesis of cDNA: The venomous tubes of Cono snails striped in the South China Sea were isolated, and the total RNA of the venomous tubes was extracted according to the instructions of TRIZOL LS reagent from Gibco BRL Company.
Embodiment 2
[0051] Example 2: Sequence Analysis of Conotoxin Gene Bt-S14
[0052] The plasmid and sequence of the neurotoxin gene Bt-S14 of Cono snail snails from the South China Sea were obtained from the venom c sequence of Cono snails from South China Sea. The precursor peptide of Cono snail neurotoxin gene Bt-S14 encodes 70 amino acid residues, and the mature peptide encodes 16 amino acid residues.
[0053] Using BLAST N and BLAST X (http: / / www.ncbi.nlm.nih.gov / BLAST) algorithms to search for homology of the tested nucleotide and derived protein sequences in existing public databases such as GenBank. Potential reading frames (ORFs) were mapped using SEQTOOLS 8.0. Sequence homology comparisons were performed using CLUSTAL X software. The protein streak peptide sequence prediction was performed through the website http: / / www.cbs.dtu.dk / services / SignalP. The molecular weight, theoretical isoelectric point, charge distribution and stability parameters of the protein were completed by P...
Embodiment 3
[0055] Embodiment 3: the synthesis of conotoxin mature peptide Bt-S14
[0056] (1) Weigh 1.5 g of chlorine resin (degree of substitution: 0.2 mmol / g) into a 150 ml reactor and soak with 50 ml of DCM.
[0057] (2) After 2 hours, wash the resin with DMF, then drain it, repeat this four times, and drain the resin for use.
[0058] (3) Weigh 192mg of threonine (Thr) in a 10ml centrifuge tube, add 5ml of DCM to dissolve it, then add 1ml of DIEA and shake it for 1min. After the solution is clarified, add it to the reactor, and then put the reaction The device was placed in a shaker at 30°C for reaction.
[0059] (4) After 2 hours, cap the head with a certain amount of methanol (methanol:DIEA:DCM=1:1:2) for half an hour, then wash it with DMF four times, and drain it for use.
[0060] (5) Add a certain amount of 20% piperidine (piperidine / DMF=1:4) into the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotect...
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