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Recombinant retroviral vector

A technology of retroviruses and vectors, applied in the field of medical biology, can solve the problems of insufficient number of T lymphocytes, immune activity, low tumor specificity, slow progress of T cell adoptive therapy, etc., to avoid autoimmune diseases and T Lymphocytoma, maintenance of antitumor activity, effect of ensuring safety

Inactive Publication Date: 2010-06-30
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for the treatment of solid tumors, T cell adoptive therapy has made slow progress in recent years, and there are only a few reports of using this therapy to treat melanoma patients (3)
At present, the limitations of T cell adoptive transfer therapy are: the number of T lymphocytes isolated from tumor patients is insufficient; their immune activity and tumor specificity are not high (4)
This risk is increased after transfection of the interleukin 2 gene or the interleukin 15 gene into T cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of Recombinant Retroviral Vectors

[0035] Schematic reference for recombinant retroviral vectors IL15-TK and IL2-TK figure 1 A and 1B.

[0036] The construction of the recombinant retroviral vector is based on the retroviral vector tgls(+)HyTKEF1a (abbreviated as TK vector) carrying the TK suicide gene. tgls(+)HyTKEF1a was transformed by introducing eukaryotic promoter EF1a from tgLS(+)HyTK plasmid (14). The TyTK fusion gene carried by tgls(+)HyTKEF1a encodes hph and HSV-1 TK enzyme activities.

[0037] Construction of IL2-TK recombinant retroviral vector:

[0038] a) Obtain hIL2 gene fragment:

[0039] The plasmid vector pcDNA3-hIL2 carrying the human interleukin-2 gene (see figure 1 D, its sequence is SEQ.ID.No.6, wherein the human interleukin 2 gene is located at base 913-1374) (this plasmid was donated by Wang Yongxiang’s laboratory of Hebei Medical University, see reference 15) as a template to design human interleukin 2 Gene primers,...

Embodiment 2

[0079] Example 2: Construction of retroviral packaging vector cells

[0080] 1. Put 5*10 5 The human embryonic kidney cell line 293T was spread in a six-well plate, and 1ml of serum-free and antibiotic-free 1640 medium was added to each well, and incubated at pH 7.2-7.4, temperature 37°C, relative humidity 95%, and 5% CO2 culture conditions for 12 Hour.

[0081] 2. Add 3ul lipofectamine2000 transfection reagent to 100ul serum-free, antibiotic-free 1640 medium, and add 0.2ug recombinant retroviral vector (IL2-TK, IL15-TK) to 100ul serum-free, antibiotic-free 1640 medium or TK) and 1ug of virus packaging vector 10A1, placed at room temperature for 5 minutes.

[0082] 3. Mix the transfection plasmid and transfection reagent dissolved in the culture medium, and place at room temperature for 15 minutes.

[0083] 4. Replace the original 293T cell culture medium with 800ul serum-free, antibiotic-free 1640 medium.

[0084] 5. Add the mixture of transfection plasmid and transfectio...

Embodiment 3

[0089] Embodiment 3: Recombinant retroviral vector infects human Jurkat cell line

[0090] Infection experiments were performed according to conventional methods known to those skilled in the art. A brief description of the infection steps is as follows:

[0091] 1. Spread 5*10 cells in a 8cm diameter cell culture dish 6 The virus packaging cells obtained in Example 2. Add 5ml of 1640 medium. Incubate for 48 hours under the conditions of pH value 7.2-7.4, temperature 37° C., relative humidity 95%, and 5% CO2.

[0092] 2. Collect the culture supernatant of packaging cells containing the retrovirus, and filter through a 0.45 μm filter to eliminate cell debris.

[0093] 3. Adjust the virus titer collected in step 2 to 2*10 6 / ml, add polybrene (polybrene) to a final concentration of 6ng / ml.

[0094] 4. Put 2*10 5 Jurkat cells were resuspended in 1 ml of the virus supernatant from step 3 (virus:target cells (number ratio)=10:1).

[0095] 5. Centrifuge at 1500g, 30°C for 90...

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Abstract

The invention provides a novel recombinant retroviral vector which comprises a human interleukin 15 gene and a thymidine kinase gene (HSV TK). The recombinant retroviral vector can greatly amplifies T lymphocytes in vitro and maintains the tumor antigen specificity thereof. The vector can introduce the human interleukin 15 gene into a T lymphocyte so that the T lymphocyte can secrete human interleukin 15 per se to stimulate the multiplication of the T lymphocyte, and meanwhile, the tumor antigen specificity of the T lymphocyte is also maintained. The invention also relates to the application of the recombinant retroviral vector.

Description

technical field [0001] The invention relates to a novel recombinant retroviral vector, which can be applied to clinical tumor immunotherapy and belongs to the field of medical biotechnology. Background technique [0002] Malignant tumor is a disease that seriously threatens human health. Because traditional chemotherapy, radiotherapy and surgery are difficult to treat tumor patients with metastatic tumors, tumor immunology therapy has attracted extensive attention and research from immunologists and medical workers. In the field of clinical treatment, T cell adoptive transfer therapy as an important passive immunotherapy has been successfully applied to tumor diseases of hematological origin (1, 2). However, for the treatment of solid tumors, T cell adoptive therapy has made slow progress in recent years, and there are only a few reports of using this therapy to treat melanoma patients (3). At present, the limitations of T cell adoptive transfer therapy are: the number of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/24C12N7/01A61K48/00A61P35/00A61P35/04
Inventor 秦志海吕继洲徐迎新
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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