Pegylated thrombopoietin mimetic peptide homotetramer and use thereof
A technology of thrombopoietin and PEGylation, which is applied in the field of biotechnology and medicine, can solve the problems of low biological activity, short half-life, and difficult to meet clinical applications.
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example 1
[0034]Example 1, chemical solid-phase synthesis of thrombopoietin mimetic peptide dimer
[0035] The amino acid sequence of the NPC polypeptide is as follows:
[0036] SEQ ID NO: 1 (NPC):
[0037] Cys-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly- Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala
[0038] When the C-terminus is a carboxyl group, Wang resin is selected for the chemical solid-phase synthesis of the two polypeptides. After the synthesis is completed, the obtained polypeptide resin of the side chain protecting group is cleaved, and the C-terminus of the NPC polypeptide is cleaved from the resin to form a carboxyl group.
[0039] The above solid-phase synthesis was carried out on a peptide synthesizer of ABI Company. First connect the alanine at the C-terminal to the resin, and then carry out amino acids one by one from the C-terminal to the N-terminal. After the synthesis, deprotection group ...
example 2
[0040] Example 2, the recombinant preparation of NPC polypeptide in Escherichia coli
[0041] According to the amino acid sequence of NPC in Example 1, its cDNA sequence was designed using Escherichia coli preferred codons, and the whole gene was artificially synthesized by Dalian Baosheng Biological Company. The synthesized cDNA fragment (inserted into the pMB-19T plasmid) was digested with BamH2 and HindⅢ and recovered, and the recombinant fusion expression pMAL-c2X plasmid was also digested and the large fragment was recovered. Under the action of T4 ligase, the NPC gene fragment and pMAL-c2X were ligated and transformed into DH5α bacteria. The recombinant plasmid containing the inserted NPC gene was screened out and named pMAL-c2X-NPC. Reuse CaCl 2 Transformation expression host strain BL 21 In (DE3), a recombinant expression strain was obtained, which was induced to express the fusion protein of MBP-NPC by 1 mMol / LIPTG. After the fusion protein was purified by Ni-Seph...
example 3
[0046] Example 3, NPC-PEG 20K - Preparation of NPC homotetramer
[0047] Bismaleimide-activated polyethylene glycol (mal-PEG) with an average molecular weight of 20kD 20K -mal) at 20.0mg / ml dissolved in 100mMol / L sodium acetate buffer (pH6.5), under room temperature stirring conditions, gradually add NPC polypeptide until the molar concentration of the two reaches mal-PEG 20K -mal : NPC is 1:2~4. After reacting for 4-12 hours, the PEG-coupled dimer was detected by ELSD (evaporative light) by RP-HPLC to reach more than 85%. Add 1% TFA to terminate the reaction, and use a reverse C18 column to purify and separate unmodified NPC polypeptide and free mal-PEG 20K --mal and single modifier PEG 20K --NPC, purified NPC-PEG 20K --NPC homotetramer was lyophilized into dry powder for storage.
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