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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 in the fusion protein of diphtheria toxin and GM-CSF and its coding gene and application field, can solve the problems of high resistance to development, low expression level, unfavorable mass industrial production, etc., and achieve survival The effect of time improvement and high expression volume

Inactive Publication Date: 2007-10-10
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 level in Escherichia coli is very low, which is not conducive to large-scale industrial production (Williams MD, Rostovtsev A, Narla RK, Uckun FM. Production of recombinant DT ct GMCSF fusiontoxin in a baculovirus expression vector system for biotherapy of GMCSF-receptor positive 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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Effect test

Embodiment 1

[0054] Embodiment 1, diphtheria toxin and GM-CSF fusion protein DT 386 -Expression of GMCSF

[0055] 1. The fusion protein DT of diphtheria toxin and GM-CSF 386 -Expression of GMCSF

[0056] 1. DT 386 - Construction of GMCSF expression plasmid

[0057] 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).

[0058] 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 enzyme digestion and ...

Embodiment 2

[0085] Embodiment 2, GMCSF-DT 386 activity assay

[0086] 1. GMCSF-DT 386 Specific cytotoxic effect on HL60

[0087] The HL60 cell line belongs to promyelocytic leukemia cells and is a commonly used cell line for leukemia research. Because there are a large number of GM-CSF receptors on the cell surface, it can be used as the target cell of the immunotoxin in this study to detect the cytotoxic activity of the toxin. Determination of immunotoxin GMCSF-DT by MTS method 386 Cytotoxicity of purified fractions of P1 and P2 against HL60.

[0088] The MTS method was operated according to the instructions of the MTS detection kit of Promega Company. Collect HL60 cells in the logarithmic growth phase and adjust to 5×10 with RPMI 1640 culture medium with 10% FCS 5 cells / ml, insert 0.1ml / well into 96-well cell culture plate, culture for 24h, add 0.1ml of GMCSF-DT 386 Purified fraction of P1 or 0.1 ml of GMCSF-DT 386 P2 purified fraction or 0.1ml of DT 386 -GMCSF or 0.1ml of DT ...

Embodiment 3

[0104] Embodiment 3, GMCSF-DT 386 Mechanism of Cytotoxicity

[0105] 1. Morphological study of apoptotic cells

[0106] HL60 cells were treated with 10 5 A / ml concentration into 25cm 2 Culture flasks or 6-well plates for 24 hours. Join GMCSF-DT 386 Purified fraction of P1 or GMCSF-DT 386 The P2 purified fraction to a final concentration of 0; 1×10 -9 mol / L; 1×10 -8 mol / L; 1×10 -7 After mol / L treatment for 48 hours, cells were collected by centrifugation at 1,000 rpm for 10 minutes, washed once with PBS, centrifuged at 1,000 rpm for 10 minutes, resuspended in serum-free medium, and stained with 10 μg / ml Hoechst 33342. Unstained or stained cell drops were observed and photographed under a fluorescent microscope.

[0107] Diphtheria toxin kills cells by inactivating ADP-ribosylated EF-2 (elongation factor), thereby inhibiting cell protein synthesis and eventually causing cell death. This experiment first observed GMCSF-DT morphologically 386 Specific cytotoxicity again...

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Abstract

This invention discloses fusion protein of diphtherin and GM-CSF, 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, its coding 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 (Granulocyte Macro...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/16A61P35/02C12N1/21C12P21/02
Inventor 欧阳晶王健伟洪涛
Owner 中国疾病预防控制中心病毒病预防控制所
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