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Acute progranulocyte leukemia DNA vaccine PML-RAR alpha 384-hIL-2 and preparing method and application thereof

A 384-hil-2, promyelocytic technology, applied in the field of hematological tumor immunotherapy, can solve the problem of fusion gene construction and other problems

Inactive Publication Date: 2010-09-29
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(384bp)-hIL-2 (human interleukin 2)

Method used

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  • Acute progranulocyte leukemia DNA vaccine PML-RAR alpha 384-hIL-2 and preparing method and application thereof
  • Acute progranulocyte leukemia DNA vaccine PML-RAR alpha 384-hIL-2 and preparing method and application thereof
  • Acute progranulocyte leukemia DNA vaccine PML-RAR alpha 384-hIL-2 and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Construction of Acute Promyelocytic Leukemia DNA Vaccine (pIRES-PML-RARα(384bp)-hIL-2 Recombinant Plasmid)

[0086] Build a flowchart such as figure 1 As shown, use RT-PCR to amplify the PML-RARα gene, insert the gene into the multiple cloning site A of the pIRES plasmid to construct pIRES-PML-RARα (384bp), and then amplify it from Jurkat cell cDNA by PCR The hIL-2 gene was inserted into the multiple cloning site B of pIRES-PML-RARα (384bp) to construct pIRES-PML-RARα(384bp)-hIL-2, and after the sequence was correct, PML-RARα384 was successfully constructed -hIL-2 is pIRES-PML-RARα(384bp)-hIL-2 plasmid.

[0087] Specific steps are as follows:

[0088] 1 Construction of pIRES-PML-RARα recombinant plasmid

[0089] 1.1 Amplification of PML-RARα gene

[0090] Extract RNA from NB4 cell line and synthesize cDNA by reverse transcription as a template (RNA extraction and cDNA synthesis of NB4 cells are carried out according to conventional methods), and PCR amplif...

Embodiment 2

[0124] Example 2 Study on gene and protein expression after transfection of eukaryotic cells by the constructed acute promyelocytic leukemia DNA vaccine (i.e. pIRES-PML-RARα(384bp)-hIL-2 recombinant plasmid)

[0125] 1. Select suitable cells to be transfected. Since the transfection efficiency of suspension cells is low, in order to further improve the transfection efficiency, we use adherent cells for transfection. Using Lipofectamine TM 2000 Kit was transfected into A549 cells. It was proved by nested PCR that the total RNA of A549 cells did not contain hIL-2 gene and PML-RARα gene. A549 cell line was infected. Using Lipofectamine TM 2000Kit transfected A549 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.

[0126] 2 RT-PCR detection

[0127] A549 cells cultured for 48 hours...

Embodiment 3

[0141] 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(384bp)-hIL-2 recombinant plasmid) in mice

[0142] 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 in bilateral quadriceps; group B: intramuscular injection of PML-RARα-pIRES 200 μg each time in bilateral quadriceps; PML-RARα-hIL-2-pIRES 200 μg was injected intramuscularly into the quadriceps femoris respectively; Group D: 200 μg of hIL-2-pIRES was 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 anterior mus...

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Abstract

The invention discloses acute promyelocytic leukemia DNA vaccine PML-RAR Alpha 384-hIL-2 which provides the fusion gene pIRES-PML-RAR Alpha-hIL-2 and also the medicine for the application of the DNA vaccine to prepare acute promyelocytic leukemia. The invention constructs DNA vaccine by using the PML-RAR Alpha fusion gene to eradicate the MRD of the APL provides PML-RAR Alpha-hIL-2 double-gene DNA vaccine which can induce and produce the PML-RAR Alpha-hIL-2 with specific anti APL immune effect and provides important data and information for the research of DNA vaccine that cures the APL. The method of the invention can also be referred by other research institutes on curing tumor DNA vaccine; the DNA vaccine of the invention can clear the minimal residual diseases of the bodies of APL patients, so as to finally achieve the aim of curing the APL.

Description

technical field [0001] The invention belongs to the technical field of hematological tumor immunotherapy, in particular to a DNA vaccine PML-RARα384-hIL-2 for acute promyelocytic leukemia (i.e. pIRES-PML-RARα(384bp)-hIL-2 plasmid) 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/62C12N15/63A61P35/02
Inventor 李扬秋杨力建陈少华胡刚岑东芝周羽竝
Owner JINAN UNIVERSITY
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