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Interfusion protein between diphtheria toxin and GM CSF mutant, coded gene and application

A technology of GM-CSF and diphtheria toxin, which is applied to the fusion protein of diphtheria toxin and GM-CSF mutant and its encoding gene and application field, can solve the problems of low expression amount, unfavorable production, large resistance to research and development, etc. The effect of increasing the amount of

Inactive Publication Date: 2007-10-10
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although clinical trials have shown that DT 388 -GMCSF has 10% effectiveness, but it was found that the immunotoxin in the form of DT-GMCSF has 2 important disadvantages: First, its expression in E. coli is very low, which is not conducive to mass production (Williams MD, RostovtsevA, Narla RK , Uckun FM. Production of recombinant DT ct GMCSF fusion toxin ina baculovirus expression vector system for biotherapy of GMCSF-receptorpositive hematologic malignancies. Protein expr purif, 1998.13: 210-221)
Although it has been found that high expression can be obtained in the baculovirus expression system, since no baculovirus-expressed protein drug has been approved by the FDA so far, the resistance to further development may be relatively large

Method used

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  • Interfusion protein between diphtheria toxin and GM CSF mutant, coded gene and application
  • Interfusion protein between diphtheria toxin and GM CSF mutant, coded gene and application
  • Interfusion protein between diphtheria toxin and GM CSF mutant, coded gene and application

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Embodiment 1

[0045] Embodiment 1, fusion protein DT of diphtheria toxin and GM-CSF mutant 386 -Expression of GMCSF 123GVT

[0046] 1. The fusion protein DT of diphtheria toxin and GM-CSF 386 -Expression of GMCSF

[0047] 1. DT 386 - Construction of GMCSF expression plasmid

[0048] The ORF of the human GM-CSF gene composed of E. coli preferred codons (the gene is called GM-CSFm) was artificially synthesized and inserted into the EcoR V site of pcDNAII (Invitrogen, V40020) to obtain the recombinant vector pcDNAII-GMCSFm. Wherein, the nucleotide sequence of the ORF of GM-CSFm is sequence 3 in the sequence listing (sequence 3 consists of 390 nucleotides).

[0049] Using pcDNAII-GMCSFm as a template, primer GFS with SphI restriction site and primer GFP14 with BamH I restriction site were used for PCR amplification. The length of the PCR product is about 400bp, and the blunt end connection is inserted into the EcoR V site of the pcDNAII plasmid. The results of Sph I and Bam HI double enzy...

Embodiment 2

[0102] Example 2, DT 386 - Activity assay of GMCSF 123GVT

[0103] In order to observe the effect of immunotoxin on killing tumor cells in vivo, HL60 cells were inoculated into mice to grow into tumor masses, and then tumor single cells were prepared for the determination of cytotoxic activity. NOD / SCID mice aged 6-8 weeks (purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences) were injected with HL60 cells (5×10 6each mouse), about 4 weeks later a human leukemia model was formed, and some mice developed tumors. Tumor-bearing NOD / SCID mice were taken to peel off the tumor body, placed in 10% FCS RPMI 1640 culture medium, shredded fully, and passed through a 400-mesh sieve. Centrifuge the cell suspension to 4,000rpm to stop, discard the supernatant, and wash 3 times with RPMI 1640 culture medium. The cell pellet was suspended in RPMI 1640 medium with 10% FCS. Take part of the tumor single cell suspension or HL60 cells, and incubate with m...

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Abstract

This invention discloses fusion protein of diphtherin and GM-CSF mutant, its coding gene, and its application. The fusion protein is selected from: (a) the protein shown in SEQ ID No.2; (b) the protein derived from SEQ ID No.2 by substituting, deleting and / or adding one or more amino acid residues, which can kill acute myeloid leukemia cells. The fusion protein can kill target cells, and has a high expression level.

Description

technical field [0001] The invention relates to the fusion protein of diphtheria toxin and GM-CSF mutant, its encoding gene and application. Background technique [0002] Acute Myeloid Leukemia (AML) is the leukemia with the highest incidence rate among adults and the second highest incidence rate among children. The number of new cases in the United States in 2006 is expected to be 11,930. At present, chemotherapy drugs such as cytarabine are effective in treating AML, and the complete remission rate can reach 70%. However, most patients develop drug resistance after long-term medication, and eventually die of relapse or complication of treatment. The median survival of patients with relapsed and refractory AML is weeks or months, so there is an urgent need for novel drugs with unique therapeutic mechanisms that can avoid the multidrug resistant phenotype. It is currently known that 88% of AML patients overexpress granulocyte-macrophage colony stimulating factor (Granuloc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/16A61P35/02C12N1/21C12P21/02
Inventor 欧阳晶王健伟洪涛
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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