Acute promyelocytic leukemia DNA vaccine PML-RAR alpha-hGM-CSF and preparation and application thereof

A technology of promyelocytes and DNA vaccines, applied in the field of hematological tumor immunotherapy, can solve problems such as the construction of fusion genes that have not yet been established

Inactive Publication Date: 2008-07-09
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)-hGM-CSF

Method used

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  • Acute promyelocytic leukemia DNA vaccine PML-RAR alpha-hGM-CSF and preparation and application thereof
  • Acute promyelocytic leukemia DNA vaccine PML-RAR alpha-hGM-CSF and preparation and application thereof
  • Acute promyelocytic leukemia DNA vaccine PML-RAR alpha-hGM-CSF and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Construction of Acute Promyelocytic Leukemia DNA Vaccine PML-RARα-hGM-CSF (pIRES-PML-RARα(384bp)-hGM-CSF Recombinant Plasmid)

[0064] The construction flow chart is shown in Figure 1. Use RT-PCR to amplify the PML-RARα gene, insert the gene into the multi-cloning site A of the pIRES plasmid to construct pIRES-PML-RARα (384bp), and then pass PCR from pORF - The hGM-CSF gene was amplified from the hGM-CSF plasmid, and the gene was inserted into the multiple cloning site B of pIRES-PML-RARα (384bp) to construct pIRES-PML-RARα (384bp)-hGM-CSF, which was sequenced After being correct, the pIRES-PML-RARα(384bp)-hGM-CSF plasmid was successfully constructed.

[0065] Specific steps are as follows:

[0066] 1 Construction of pIRES-PML-RARα recombinant plasmid

[0067] 1.1 Amplification of PML-RARα gene

[0068] 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 ou...

Embodiment 2

[0101] Example 2 Study on the gene and protein expression after transfection of the constructed acute promyelocytic leukemia DNA vaccine PML-RARα-hGM-CSF (i.e. pIRES-PML-RARα(384bp)-hGM-CSF recombinant plasmid)

[0102] 1 Screen suitable cells to be transfected, using Lipofectamine TM 2000Kit transfected K562 cells and nested PCR proved that the total RNA of K562 cells did not contain hGM-CSF gene and PML-RARα gene, so the pIRES-PML-RARα(384bp)-hGM-CSF recombinant plasmid constructed by inserting can be transfected into K562 cell line. Using Lipofectamine TM 2000 Kit 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.

[0103] 2 RT-PCR detection

[0104] K562 cells cultured for 48 hours after transfection were collected to extract total RNA, and cDNA was synthesi...

Embodiment 3

[0108] Example 3 Study of gene and protein expression, antibody Production and specific CTL effects, etc.

[0109] 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 anterior muscle was isolated. RT-PCR and immunohistochemical staining were used to detect the transcription and expression of the t...

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Abstract

The invention discloses an acute promyelocytic leukemia cell leukemia DNA vaccine PML-RAP Alpha-hGM-CSF, i.e. the fusion gene pIRES-PMI-RAR Alpha- hGM-CSF. The invention also discloses that the DNA vaccine is applicable in preparing medicine for curing acute promyelocytic leukemia cell leukemia. The invention uses the PML-RAP Alpha fusion gene to construct the DNA vaccine for eradicating the MRD of APL. The invention provides a PML-RAP Alpha-hGM-CSF double-gene DNA vaccine capable of inducing to produce specific anti-APL immune response, which provides important data for research and development of the DNA vaccine for curing APL. The method of the invention can also provide reference for research on other curative tumor DNA vaccines. The DNA vaccine of the invention can clear the minimal residual diseases in the body of APL patients, and finally cure the APL.

Description

technical field [0001] The invention belongs to the technical field of hematological tumor immunotherapy, in particular to a DNA vaccine PML-RARα-hGM-CSF for acute promyelocytic leukemia (i.e. pIRES-PML-RARα(384bp)-hGM-CSF) 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 combination chemotherapy for APL has made great progress and the prognosis of patients has been greatly improved, it is difficult for the tumor MRD to be completely eliminated by the current ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/62C12N15/63A61P35/02
Inventor 李扬秋岑东芝胡刚周羽竝陈少华杨力建
Owner JINAN UNIVERSITY
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