Chronic myeloid leukemia DNA vaccine BCR/ABL-pIRES-SEA and preparation method and application thereof

A chronic granulocyte and DNA vaccine technology, applied in the field of hematological tumor immunotherapy

Inactive Publication Date: 2010-09-22
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no conclusion on the selection of specific target genes for the preparation of BCR/ABL gene vaccine...

Method used

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  • Chronic myeloid leukemia DNA vaccine BCR/ABL-pIRES-SEA and preparation method and application thereof
  • Chronic myeloid leukemia DNA vaccine BCR/ABL-pIRES-SEA and preparation method and application thereof
  • Chronic myeloid leukemia DNA vaccine BCR/ABL-pIRES-SEA and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] The construction of chronic myelogenous leukemia DNA vaccine BCR / ABL-pIRES-SEA recombinant plasmid, such as figure 1 Shown:

[0130] Utilize RT-PCR to amplify the BCR / ABL fragment, insert the fragment into the multiple cloning site A of the pIRES plasmid to construct the BCR / ABL-pIRES, and then amplify the SEA gene from the Staphylococcus aureus genomic DNA by PCR, The gene was inserted into the multiple cloning site B of BCR / ABL-pIRES to construct BCR / ABL-pIRES-SEA. After the sequencing was correct, the BCR / ABL-pIRES-SEA plasmid was successfully constructed.

[0131] Specific steps are as follows:

[0132] 1 Construction of BCR / ABL-pIRES recombinant plasmid

[0133] 1.1 Amplification of BCR / ABL fragment

[0134] RNA was extracted from the leukemia cell line K562 cells (purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai), and the cDNA obtained by reverse transcription was used as a template (the RNA extraction and cDNA synthesis of K562 cells...

Embodiment 2

[0178] Study on the gene and protein expression of the constructed chronic myelogenous leukemia DNA vaccine (namely BCR / ABL-pIRES-SEA recombinant plasmid) transfected into eukaryotic cells

[0179] 1 Screen suitable cells to be transfected, using Lipofectamine TM 2000 Kit (Invitrogen Company) was used to transfect human embryonic kidney epithelial K293 cells (Shanghai Cell Institute, Chinese Academy of Sciences).

[0180] Using Lipofectamine TM 2000 Kit transfected K293 cells, put the culture plate into 5% CO 2 Cultivate in an incubator (37°C), and after 4 to 6 hours, transfer the Opti-MEM containing transfection reagent The I medium was replaced with DMEM medium containing 10% fetal bovine serum by volume, and the supernatant and cells were collected after 48 hours.

[0181] 2 RT-PCR detection

[0182] K293 cells cultured for 48 hours after transfection were collected to extract total RNA, and cDNA was synthesized by reverse transcription as a template. Primers B1, B2 ...

Embodiment 3

[0185] Example 3 Study of gene and protein expression and specific CTL effect at different stages after injection of chronic myelogenous leukemia DNA vaccine into mouse muscle (BCR / ABL-pIRES-SEA recombinant plasmid)

[0186] Thirty 6- to 8-week-old healthy pure-line male BALB / c mice (SCXK Guangdong 20040011, purchased from the Experimental Animal Center of Sun Yat-sen University) were randomly divided into 5 groups (6 mice in each group), and each mouse was treated at the first week. , 2 weeks, 4 weeks each immunization once, a total of 3 times. Group A (Group P): Inject 200 μg of pIRES into each quadriceps muscle; Group B (Group B-P): inject BCR / ABL-pIRES 200 μg into each quadriceps muscle; Group C (Group B-P-S): BCR / ABL-pIRES-SEA 200 μg was injected intramuscularly into the bilateral quadriceps each time; Group D (Group S-P): SEA was injected intramuscularly into the quadriceps femoris each time - pIRES 200 μg; Group E (Group N): 200 μg of normal saline was injected intramu...

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Abstract

The invention discloses a chronic myeloid leukemia DNA vaccine BCR/ABL-pIRES-SEA and a preparation method and an application thereof, the DNA vaccine contains a pIRES plasmid, a BCR/ABL gene and an SEA gene, wherein, the BCR/ABL gene and the SEA gene are respectively positioned on multi-clone sites at two sides of an IRES element on the pIRES plasmid; nucleotide sequences of the BCR/ABL gene and the SEA gene are shown in SEQ ID No.1 and SEQ ID No.2; the DNA vaccine can express BCR/ABL protein and SEA protein; the DNA vaccine can express BCR/ABL protein and SEA protein in eukaryocyte; when being injected into mice, the DNA vaccine can stimulate organism to generate specificity CD8+T cells aiming at CML cells, accompanying increased secretion of a cell factor, thus eradicating minimal residual disease in the bodies of patients, therefore the invention can be applied to prepare drugs for treating chronic myeloid leukemia.

Description

technical field [0001] The invention belongs to the technical field of hematological tumor immunotherapy, and in particular relates to a chronic myelogenous leukemia DNA vaccine BCR / ABL-pIRES-SEA and a preparation method and application thereof. Background technique [0002] At present, the treatment of chronic myelogenous leukemia (CML) mainly includes traditional chemotherapy, IFN-α and targeted therapy of tyrosine kinase inhibitors. Although these treatment methods have been able to improve the quality of life of patients to a large extent, and the prognosis of patients has also been greatly improved, but the minimal residual disease (MRD) of the tumor is difficult to be eradicated by the current treatment options. Although hematopoietic stem cell transplantation can eradicate MRD, few bone marrow sources, low matching success rate, high transplantation costs and serious transplantation-related complications limit the promotion of this therapy. Since many leukemia patien...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/00A61P35/02C12N15/12C12N15/85
Inventor 林晨田红霞高永鹏陈少华杨力建周羽竝李扬秋
Owner JINAN UNIVERSITY
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