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Acute promyelocytic leukemia DNA vaccine, preparing method thereof and applications

A promyelocytic, DNA vaccine technology, applied in the field of hematological tumor immunotherapy, can solve the problem of fusion gene construction and other problems

Inactive Publication Date: 2008-07-30
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no construction of the fusion gene pIRES-PML-RARα(245bp)-hGM-CSF

Method used

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  • Acute promyelocytic leukemia DNA vaccine, preparing method thereof and applications
  • Acute promyelocytic leukemia DNA vaccine, preparing method thereof and applications
  • Acute promyelocytic leukemia DNA vaccine, preparing method thereof and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Construction of Acute Promyelocytic Leukemia DNA Vaccine (pIRES-PML-RARα(245bp)-hGM-CSF Recombinant Plasmid)

[0067] The construction flow chart is shown in Figure 1, using RT-PCR to amplify the PML-RARα gene, and inserting the gene into the multiple cloning site A of the pIRES plasmid (shown in Figure 2) to construct pIRES-PML-RARα (245bp) , and then amplify the hGM-CSF gene from the pORF-hGM-CSF plasmid by PCR, and insert the gene into the multiple cloning site B of pIRES-PML-RARα (245bp) to construct pIRES-PML-RARα (245bp)- After hGM-CSF was sequenced correctly, the pIRES-PML-RARα(245bp)-hGM-CSF plasmid was successfully constructed.

[0068] Specific steps are as follows:

[0069] 1 Construction of pIRES-PML-RARα recombinant plasmid

[0070] 1.1 Amplification of PML-RARα gene

[0071] Extract RNA from NB4 cell line and synthesize cDNA by reverse transcription as a template (the RNA extraction and cDNA synthesis of NB4 cells are carried out according to ...

Embodiment 2

[0108] Example 2 Study on gene and protein expression of the constructed acute promyelocytic leukemia DNA vaccine (i.e. pIRES-PML-RARα(245bp)-hGM-CSF recombinant plasmid) transfected into eukaryotic cells

[0109] 1 Screen suitable cells to be transfected, using Lipofectamine TM 2000Kit transfected K562 cells

[0110] Nested PCR proved that the total RNA of K562 cells does not contain hGM-CSF gene and PML-RARα gene, so the pIRES-PML-RARα(245bp)-hGM-CSF recombinant plasmid constructed by inserting can be transfected into K562 cell line. Using Lipofectamine TM 2000Kit transfected K562 cells, put the culture plate into 5% CO 2 After culturing in an incubator (37° C.), the medium was replaced with a medium containing fetal calf serum and antibiotics after 4 to 6 hours, and the supernatant and cells were collected after 48 hours.

[0111] 2RT-PCR detection

[0112] K562 cells cultured for 48 hours after transfection were collected to extract total RNA, and cDNA was synthesize...

Embodiment 3

[0116] Example 3 Study of gene and protein expression, antibody production and specific CTL effect at different stages after injection of acute promyelocytic leukemia DNA vaccine (pIRES-PML-RARα(245bp)-hGM-CSF recombinant plasmid) in mice

[0117] Thirty healthy pure-line male BALB / c mice of 6-8 weeks were randomly divided into 5 groups (6 mice in each group). Second-rate. Group A: intramuscular injection of pIRES 200 μg each time into bilateral quadriceps; group B: intramuscular injection of PML-RARα-pIRES 200 μg each time into bilateral quadriceps; PML-RARα-hGM-CSF-pIRES 200 μg were injected intramuscularly into the quadriceps femoris respectively; Group D: 200 μg of hGM-CSF-pIRES were injected into the quadriceps femoris each time; Group E: each time Intramuscularly inject 200 μg of normal saline into the quadriceps femoris. On the 7th day after the second immunization and the last immunization, 3 mice in each group were randomly selected and sacrificed, and the tibialis ...

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Abstract

The invention discloses DNA bacterin of acute promyeloblastic leukemia, i.e., fusion gene pIRES-PML-RAR Alpha (245bp)-hGM-CSF. The invention also discloses the medicine for treating acute promyeloblastic leukemia which is prepared by the DNA bacterin. The fusion gene PML-RARAlpha is used for constructing the DNA bacterin for eradicate MRD of APL. The invention provides a PML-RAR Alpha (245bp)-hGM-CSF dihybrid DNA bacterin that can induce to generate specific APL immunological effect so as to provide important data and information for the research and development of the DNA bacterin for treating APL; the method provided by the invention can also be used as reference for the research of other curative tumor DNA bacterins. The DNA bacterin of the invention can eradicate tiny residual pathologic change in APL sufferers, and realize the object of curing APL finally.

Description

technical field [0001] The invention belongs to the technical field of hematological tumor immunotherapy, and in particular relates to an acute promyelocytic leukemia (ie, pIRES-PML-RARα(245bp)-hGM-CSF) DNA vaccine and its preparation method and application. Background technique [0002] Immunotherapy is a current trend in the treatment of tumors and leukemia. DNA vaccines have many advantages that traditional vaccines cannot match, and can induce specific humoral immunity and cellular immunity. Acute promyelocytic leukemia (APL) is currently the most effective type of leukemia, but the minimal residual disease (MRD) of the tumor is difficult to be eradicated by the current treatment plan. Therapeutic options to eradicate MRD in APL are urgently needed. [0003] Although the combined chemotherapy regimen for acute promyelocytic leukemia (APL) has made great progress, and the prognosis of patients has also been greatly improved, the minimal residual disease (MRD) of the tumo...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/00C12N15/85C12N15/62C12N15/66A61P35/02
Inventor 李扬秋胡刚陈少华杨力建周羽竝岑东芝
Owner JINAN UNIVERSITY
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