Transduced peptide-humanized granular leukocyte colony stimulating factor fusion protein and its medicinal composition

A technology of colony stimulating factor and fusion protein, applied in the field of fusion protein, can solve the problems of low incidence of allergic allergic reaction and respiratory distress, and achieve the effects of safe clinical medication, easy operation and simplified purification process.

Inactive Publication Date: 2007-11-21
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0032] 5. Aplastic anemia
The incidence of allergic allergic reaction caused by rhG-CSF is very low, and it occurs sooner or later, but respiratory distress syndrome may occur, which cannot be ignored

Method used

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  • Transduced peptide-humanized granular leukocyte colony stimulating factor fusion protein and its medicinal composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Extraction of human spleen mononuclear cell mRNA

[0059] Take fresh spleen that was ruptured and resected after trauma, prepare tissue suspension, separate mononuclear cells with Ficoll lymphocyte separation medium, culture for 2 hours to remove non-adherent cells, continue to culture in LPS (300ug / ml) medium for 2.5 hours, and then collect Cells, using Promega's RNAgentsRTotal RNA Isolation System to extract total RNA, and then purified by Oligo (dt) affinity chromatography column to obtain Poly (A) + mRNA.

Embodiment 2

[0060] Example 2 Cloning of hG-CSF cDNA

[0061] Primers were designed with reference to the hG-CSF cDNA sequence in Genebank: the second primer was used as a primer for reverse transcription.

[0062] Primer 1: 5'-GGAATTCACACCATTAGGCCCTGCCAGCTCCCTG-3' (SEQ ID NO: 7)

[0063] Primer 2: 5'-GGGATCCTCAGGGCTGGGCAAGGTGGCGTAGAAC-3' (SEQ ID NO: 8)

[0064] Using the mRNA obtained in Example 1 as a template, RT-PCR amplification was performed according to the protocol recommended by the kit manufacturer to obtain a full-length hG-CSF cDNA, which carried EcoRI and BamHI sites at its 5' and 3' ends, respectively.

Embodiment 3

[0065] Example 3 Construction and Identification of Recombinant hG-CSF Expression Plasmid

[0066] The above RT-PCR product of hG-CSF was double-digested with EcoRI and BamHI, then ligated with the expression vector pBV220 fragment recovered by the same digestion at a molar ratio of 4:1, transformed into competent Escherichia coli DH5α, and screened for positive recombinant pBV hG-CSF. Sent to Shanghai Shengong Company for sequencing.

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Abstract

A transduction peptide-humanized granulocyte colony stimulating factor fusion protein, nucleic acid molecule of nucleic acid sequence for encoding fusion protein, expression carrier containing the nucleic acid molecule and its usage of fusion protein in preparation of medicine are disclosed. It can be used for neutrophil reduction after tumor radiant chemo-radiotherapy, hematopoietic restoration after bone marrow transplantation, peripheral blood stem cells mobilization, anti-infective therapy, aregenerative anemia, systemic lupus erythematosus and immune response adjustment.

Description

Field of invention: [0001] The invention relates to a fusion protein with medicinal value, specifically a transduction peptide-human granulocyte colony-stimulating factor fusion protein, and a nucleic acid molecule encoding the fusion protein. The present invention also relates to pharmaceutical compositions containing the fusion protein. Background of the invention [0002] overview [0003] Granulocyte colony stimulating factor (G-CSF) is a polypeptide chain cell growth factor that stimulates bone marrow cells to form granulocyte colony forming units, increasing neutrophils (ANC), monocytes, T lymphocytes number of cells. G-CSF mainly acts on the proliferation and differentiation of granulocyte progenitor cells and granulocyte precursor cells, and enhances the functional activity of mature granulocytes; enhances the phagocytosis of ANC, improves the release function of ANC active oxygen, and strengthens its ability to kill pathogenic microorganisms , It is of great sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/435A61K38/19A61P35/00A61P37/02A61P31/00C12N15/62C12N15/63
Inventor 李前孙曼霁
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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