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Human granulocyte colony stimulating factor mutant recombinant fusion protein as well as preparation method and application thereof

A technology of colony stimulating factor and fusion protein, which is applied in the field of biological genetic engineering to achieve the effect of long half-life, prolonging half-life and improving biological activity

Active Publication Date: 2021-10-08
长春萤火虫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, human G-CSF mutants and ricin B-chain truncated peptides are used to form recombinant fusion proteins to improve the activity of recombinant fusion proteins in treating leukopenia and to prolong Studies on the half-life of recombinant fusion proteins have not been reported

Method used

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  • Human granulocyte colony stimulating factor mutant recombinant fusion protein as well as preparation method and application thereof
  • Human granulocyte colony stimulating factor mutant recombinant fusion protein as well as preparation method and application thereof
  • Human granulocyte colony stimulating factor mutant recombinant fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of recombinant hG-CSF-Mut / RTBD1 fusion protein

[0060] 1. Construction of human granulocyte colony-stimulating factor (hG-CSF) mutant expression vector

[0061] (1) Homologous modeling of human granulocyte colony-stimulating factor molecule and human granulocyte colony-stimulating factor receptor molecule was performed on the SWISS-MODEL online platform; human granulocyte colony-stimulating factor molecule and human granulocyte The colony-stimulating factor receptor molecule was docked to obtain the complex structure model; the full-length sequence of the complex structure model was mutated by alanine scanning to obtain potential mutation sites; the mutation was calculated for a single mutation site by virtual saturation mutation Changes in intermolecular binding force before and after.

[0062] (2) Preparation of recombinant plasmid pUC57-hG-CSF:

[0063] The human granulocyte colony-stimulating factor gene sequence was queried through the NCBI...

Embodiment 2

[0084] Example 2 Pharmacodynamics test of recombinant hG-CSF-Mut / RTBD1 fusion protein

[0085] 1. Construction of leukopenia mouse model

[0086] Select 18 mice and intraperitoneally inject cyclophosphamide 2 mg / mouse·day for three consecutive days. On the fourth day, the whole blood of the mice was taken for routine blood test, and the number of white blood cells (WBC) was detected. The success criteria of the model used in this experiment are: the number of white blood cells is lower than the lowest value of the reference range of 0.8×10 9 / L. Mice that meet the above conditions are deemed to have successfully prepared the mouse model of leukopenia. The normal group and the model control group were subcutaneously administered with normal saline the next day after modeling, and the administration group was subcutaneously administered 0.2 μgrhG-CSF-Mut / RTBD1 fusion protein to the neck and back of the mice on the next day after modeling.

[0087] Table 2 Grouping of animals...

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Abstract

The invention discloses a human granulocyte colony stimulating factor mutant recombinant fusion protein as well as a preparation method and application thereof, and relates to the field of biological genetic engineering, the recombinant fusion protein is formed by connecting a human granulocyte colony stimulating factor mutant and a ricin B chain truncated peptide through a flexible linker, and the flexible linker is (Gly4Ser) 3 connecting peptide. The preparation method of the human granulocyte colony stimulating factor mutant recombinant fusion protein comprises the following steps: constructing a human granulocyte colony stimulating factor mutant expression vector; constructing an escherichia coli recombinant expression vector; carrying out expression of the recombinant hG-CSF-Mut / RTBD1 fusion protein; and carrying out purification and renaturation on the recombinant hG-CSF-Mut / RTBD1 fusion protein. The recombinant fusion protein is long in half-life period and high in biological activity, the half-life period is prolonged, the biological activity is improved, the production cost of a granulocyte colony stimulating factor preparation is reduced, and the recombinant fusion protein can be applied to preparation of drugs for treating leucopenia.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a human granulocyte colony-stimulating factor mutant recombinant fusion protein and its preparation method and application. Background technique [0002] Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that mainly acts on the proliferation, differentiation and activation of neutrophil lineage hematopoietic cells. Recombinant human granulocyte colony stimulating factor (rhG-CSF-Mut / RTBD1) acts on hematopoietic progenitor cells to promote their proliferation and differentiation. And can promote the various functions of macrophages and eosinophils. G-CSF is mainly used clinically to prevent and treat leukopenia caused by tumor radiotherapy or chemotherapy, to treat bone marrow hematopoietic dysfunction and myelodysplastic syndrome, to prevent potential infectious complications of leukopenia, and to neutralize neutrophils caused by infection. Recovery...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/27C12N15/70C12N15/66C07K1/14C07K1/18C07K1/34C07K1/36C12N15/62C12P21/02A61K38/16A61K38/19A61K47/64A61K47/65A61P7/00
CPCC07K14/535C12N15/70A61K38/193A61K38/168A61K47/65A61K47/6415A61P7/00C07K2319/55C12N2800/22Y02A50/30
Inventor 许娜孙成彪石晶王燕
Owner 长春萤火虫生物科技有限公司
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