G-CSF (Granulocyte-Colony Stimulating Factor) fusion protein mutant, preparation method and application thereof

A technology of fusion proteins and mutants, applied in the fields of peptide/protein components, hybrid peptides, drug combinations, etc.

Active Publication Date: 2010-09-08
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] To sum up, the existing G-CSF preparations in the market still have considerable room for improvement

Method used

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  • G-CSF (Granulocyte-Colony Stimulating Factor) fusion protein mutant, preparation method and application thereof
  • G-CSF (Granulocyte-Colony Stimulating Factor) fusion protein mutant, preparation method and application thereof
  • G-CSF (Granulocyte-Colony Stimulating Factor) fusion protein mutant, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Screening of embodiment 1 mutation site

[0065] Multiple rHSA / G-CSF mutants were constructed by mutating multiple amino acid residues located at the G-CSF receptor binding interface and related ones. The construction method of the mutants was the same as that in Example 2 except for sequence differences. And it was expressed in Pichia pastoris (expression and purification method is as described in Example 3-5), and the mutants obtained were measured by SPR (surface plasmon resonance) technology, and the ligand-receptor affinity was measured, and the cell Level of biological activity test. The association rate constant and the dissociation rate constant can be obtained by the SPR technique and thus directly determine the ligand-receptor equilibrium dissociation constant (Zhou et al., Biochemistry, 32:8193-8198, 1993: Faegerstram and Osh annessy, In handbook of Affinity Chromatography, Marcel Dekker INc, NY, 1993), the larger the equilibrium dissociation constant, the l...

Embodiment 2

[0074] Example 2 Construction of rHSA / G-CSF and mutant rHSA / mG-CSF yeast expression strains

[0075] On the basis of the study in Example 1, the mutants were further optimized, and finally an optimized strain with low affinity to the receptor, long half-life, and high activity was screened out. This strain produced T1A, L3T, G4Y, Mutations at P5R, K34H, L35I, K40H, L41I. The construction method of the strain is as follows:

[0076] The DNA sequences encoding HSA / G-CSF (see SEQ NO: 5) and HSA / mG-CSF (see SEQ NO: 6) were synthesized by Shanghai Invitrogen Company and inserted into pMD18-T (TaKaRa) to construct plasmid HSA / G-CSF / pMD18-T and HSA / mG-CSF / pMD18-T. HSA has its natural signal peptide sequence, and a BamHI site is added before the signal peptide sequence, and an EcoRI site is added to the 3' end of G-SCF.

[0077] The HSA / G-CSF / pMD18-T plasmid was digested with BamHI / EcoRI, and the HSA / G-CSF and HSA / mG-CSF fragments were recovered, respectively, and connected to the...

Embodiment 3

[0079] Example 3 Expression screening of rHSA / G-CSF and mutant rHSA / mG-CSF

[0080] Inoculate the single colony of recombinant yeast transformed in Example 2 into 10 ml BMGY liquid medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH6.0, 1.34% YNB, 4×10-5% biotin, 1 % glycerol), 30 ℃, 250rpm after cultivating for 24 hours, let it stand overnight, discard the supernatant, add 10ml of BMMY liquid medium containing 1% methanol (1% yeast extract, 2% peptone, 100mM potassium phosphate, pH6. 0, 1.34% YNB, 4×10 -5 % biotin, 1% methanol), 30°C, 250rpm induction, supplemented with methanol every 24 hours, a total of 72 hours of induction. From 0 hour, every 10 hours, take 1ml of the induced bacteria solution into a 1.5ml centrifuge tube, and centrifuge at 5000g for 5 minutes. Take 40 μl supernatant and add 10 μl 5×Loadingbuffer, cook in boiling water bath for 5 minutes, perform electrophoresis on 12% SDS-PAGE, and analyze the expression status with GS115 empty bacteri...

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Abstract

The invention relates to a G-CSF (Granulocyte-Colony Stimulating Factor) fusion protein mutant, a preparation method and application thereof. The G-CSF fusion protein mutant is fusion protein having the effect of stimulating the proliferative activity of neutrophile granulocytes, and the structure of the G-CSF fusion protein mutant is G-CSF / carrier protein or carrier protein / G-CSF, wherein the G-CSF part contains multi-site mutations, which changes the activity and the receptor affinity. Compared with the traditional like products, the G-CSF fusion protein mutant has longer half-life period and higher biologic activity. Through injecting a proper dosage of the medicinal preparation, the neutrophilic granulocytopenia can be treated.

Description

technical field [0001] The invention relates to a new fusion protein with the function of accelerating granulocyte recovery, a method for preparing the fusion protein, a pharmaceutical preparation containing the fusion protein and in the field of medicine, especially for the treatment of neutropenia or leukopenia disease applications. Background technique [0002] Human granulocyte colony-stimulating factor (G-CSF), a long-chain polypeptide glycoprotein derived from monocytes and fibroblasts, can induce the proliferation and differentiation of hematopoietic stem cells, and promote the number of neutrophils in the blood In addition, it can stimulate the release of mature neutrophils from the bone marrow and activate the function of neutrophils. The main spatial structure of G-CSF is a helix, and 103 of the 174 residues form four α-helices (Hill CP et al., Proc Natl Acad Sci USA, 90:5167-5171, 1993), such as figure 1 shown. Since 1991, recombinant human granulocyte colony-s...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19C12N5/10C12P21/02A61K38/19A61K47/48A61P7/00C12R1/84C12R1/865A61K47/42
Inventor 温晓芳吴亦亮王叶飞杨志愉范敏王玉姣方晓春陆游
Owner JIANGSU T MAB BIOPHARMA
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