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Cat 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 effects of reducing preparation cost, improving protein stability and enhancing biological activity

Pending Publication Date: 2021-10-22
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, there is no report on the research of improving the activity of recombinant fusion protein in treating leukopenia and prolonging the half-life of recombinant fusion protein by combining cat G-CSF mutant and ricin B chain truncated peptide to form recombinant fusion protein

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1 Preparation of recombinant FeG-CSF-Mut / RTBD1 fusion protein

[0058] 1. Construction of expression vector for feline granulocyte colony-stimulating factor (FeG-CSF) mutation

[0059] (1) Carry out homologous modeling of feline granulocyte colony-stimulating factor molecule and feline granulocyte colony-stimulating factor receptor molecule through the SWISS-MODEL online platform; 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.

[0060] (2) Preparation of recombinant plasmid pUC57-FeG-CSF:

[0061] The feline granulocyte colony-stimulating factor gene sequence was queried through the NCBI Genbank database, and the obtained sequence was subjected...

Embodiment 2

[0082] Embodiment 2 Pharmacodynamics test of recombinant FeG-CSF-Mut / RTBD1 fusion protein

[0083] 1. Construction of leukopenia mouse model

[0084] 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 blood routine, and the number of white blood cells (WBC) was detected. The success standard of the model used in this experiment was: the number of white blood cells was lower than the lowest value of the reference range by 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 on the second day after modeling, and the administration group was subcutaneously administered 0.2 μgrFeG-CSF-Mut / RTBD1 fusion protein to the back of the neck of the mice on the second day after modeling.

[0085] Table 2 Grouping ...

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Abstract

The invention discloses a cat 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 cat granulocyte colony stimulating factor mutant and a ricin B chain truncated peptide through a flexible linker. The preparation method comprises the following steps: constructing a cat granulocyte colony stimulating factor mutation expression vector; constructing an escherichia coli recombinant expression vector; carrying out the expression of the recombinant FeG-CSF-Mut / RTBD1 fusion protein; and carrying out purification and renaturation on the recombinant FeG-CSF-Mut / RTBD1 fusion protein. The long-acting granulocyte colony-stimulating factor is obtained by connecting the cat G-CSF mutant and the ricin B chain truncated peptide, so that the biological activity of the granulocyte colony-stimulating factor can be enhanced, the protein stability of the granulocyte colony-stimulating factor in in-vivo blood is improved, and the half-life period of a drug in an organism is further prolonged. The recombinant fusion protein has the function of treating leucopenia.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to a cat 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 cat granulocyte colony-stimulating factor (rFeG-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. Rec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/36C07K1/34C07K1/22C07K1/18C12N15/62C12N15/70C12N15/66A61K38/19A61P7/06
CPCC07K14/535C12N15/70C12N15/66A61P7/06C07K2319/55C12N2800/22A61K38/00
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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