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Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof

A technology of colony-stimulating factor and granulocytes, applied in the field of long-acting recombinant human granulocyte colony-stimulating factor and its preparation, can solve the problems of low yield, increased protein molecular weight, high production cost, etc., and achieve the effect of simple preparation process

Inactive Publication Date: 2016-04-06
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the method of prolonging the half-life of protein is mainly to use PEG for chemical modification, which has the advantage of simple reaction and can significantly increase the in vivo half-life of the modified protein; the disadvantage is that the molecular weight of the modified protein increases sharply, which is not conducive to in vivo metabolism; there are many chemical modifications As a heterologous substance, frequent use may cause the body's immune response; and after chemical modification, the biological activity of the original protein will be significantly reduced, generally only 10% of that before modification; in addition, the final product of chemical modification needs to be purified once more , the yield is relatively low, and the modifier itself is expensive, and the production cost is relatively high

Method used

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  • Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof
  • Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof
  • Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of recombinant human granulocyte colony stimulating factor rhG-CSF and CTP fusion protein expression vector:

[0060] The recombinant human granulocyte colony stimulating factor rhG-CSF and CTP fusion protein coding gene sequence with restriction enzyme cut sites and signal peptides were artificially synthesized and cloned into pUC57simple plasmid vector to obtain a plasmid containing the fusion protein coding gene. (Completed by Nanjing KingScript Biotechnology Co., Ltd.). The 5'end of the plasmid fusion protein contains the XhoI restriction site and the signal peptide cleavage recognition site (i.e. 5’CTCGAGAAAAGA-); and the 3’ end contains the NotI restriction site (i.e. 3’GCGGCCGC-)

[0061] Take the pUC57simple-rhG-CSF-CTP3 plasmid and pPICZaA expression plasmid, and use XhoI and NotI restriction endonucleases for double digestion. The reaction system for double enzyme digestion is 20μL, in which plasmid 10μL, NotI1μL, XhoI1μL, 10XbufferH2μL, an...

Embodiment 2

[0064] Example 2 Expression of recombinant human granulocyte colony stimulating factor rhG-CSF and CTP fusion protein

[0065] First, prepare competent Pichia pastoris GS115 cells. The specific steps are as follows: first pick out the GS115 monoclonal colony in 3mL YPD medium, and incubate for 48h at a constant temperature of 30°C at a rotation speed of 180rpm. Then, the cultured Pichia pastoris GS115 cells were transferred into 100 mL of YPD medium with an inoculum of 1:200, and cultured to O.D.600=1.2. Centrifuge at 4000 rpm / min 4°C for 5 minutes, discard the supernatant, collect the precipitate and resuspend it twice with 1mol / L sorbitol solution, centrifuge at 4000 rpm / min 4°C for 10 minutes, and resuspend the precipitate with 100 μL ice-cold sorbitol solution. Prepare competent Pichia pastoris GS115 cells.

[0066] Secondly, the recombinant human granulocyte colony stimulating factor rhG-CSF and CTP fusion protein expression vector pPICZaA-rhG-CSF-CTP3 was sequenced and linea...

Embodiment 3

[0069] Example 3 In vitro activity determination of recombinant human granulocyte colony stimulating factor rhG-CSF and CTP fusion protein

[0070] In this example, in accordance with the Pharmacopoeia of the People's Republic of China (2010 Edition, Part III), the G-CSF-dependent cell line NFS60 was selected, and the biological activity was measured by the MTT method. The specific steps are as follows: FS60 cell line with complete culture medium at 37℃, 5% CO 2 Culture, control the cell concentration to 1*10 per milliliter 5 Cells are used for the assay. Take a sufficient amount of NFS60 cell culture, collect the cells by centrifugation, wash twice with RPMI1640 culture medium, and then resuspend in the basic culture medium (RPMI1640900ml + newborn calf serum 100ml) to make 2*10 per ml 5 Cell suspension of two cells. Add 50μL of cell suspension to each well of a 96-well plate with standard and test products, at 37℃, 5% CO 2 Cultivate for 72 hours. Add 20μL of MTT solution to ea...

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Abstract

The invention belongs to the technical field of biological pharmacy and relates to a recombinant fusion protein of a human granulocyte colony-stimulating factor, particularly a long-acting recombinant human granulocyte colony-stimulating factor and a preparation method therefor and use thereof. A recombinant human granulocyte colony-stimulating factor and a short peptide (CTP) formed by amino acids from site 118 to site 145 of beta subunit end of one or more human chorionic gonadotropin (hCG) are expressed in a fusion manner to prepare a long-acting fusion protein; on the premise of maintaining the original activity of the human granulocyte colony-stimulating factor, the half-life of the protein in vivo is prolonged, so that the protein is further used for preparing drugs for resisting chemoradiotherapy or treating neutrophilic granulocytopenia and myelosuppression caused by other reasons. The fusion protein provided by the invention can be efficiently expressed in pichia pastoris, is simple in preparation process, and can be used for production and preparation of drug-level fusion proteins on a large scale.

Description

Technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a recombinant fusion protein of human granulocyte colony stimulating factor, and specifically relates to a long-acting recombinant human granulocyte colony stimulating factor and a preparation method and application thereof. The invention prolongs the half-life of the protein in the body under the premise of maintaining the original activity of the human granulocyte colony stimulating factor, and is further used to prepare drugs against neutropenia and bone marrow suppression caused by radiochemotherapy or other reasons. Background technique [0002] According to reports, malignant tumors are currently one of the major diseases that seriously affect human health and threaten human lives. According to statistics, in 2007, 7.6 million people died of cancer globally, of which about 24% occurred in China, showing a gradual younger trend. Surgery, radiotherapy, and chemotherapy a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/19A61K47/48A61P7/00A61P37/04A61K47/64
Inventor 鞠佃文范佳君李玉彬王子玉王绍飞宋平陈其成
Owner FUDAN UNIV
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