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Recombinant expression plasmid and application thereof in preparing anti-tumor immunogene therapeutic medicament

A technology of anti-tumor immunity and expression plasmid, applied in the field of biopharmaceuticals

Inactive Publication Date: 2013-07-17
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the literature has confirmed that IL-12, GM-CSF and B7.1 can be used in combination with each other, it is obvious that the combined application of more cytokines still has limitations, and it is necessary to find more factors for combined application and combined application. method is a new topic for researchers

Method used

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  • Recombinant expression plasmid and application thereof in preparing anti-tumor immunogene therapeutic medicament
  • Recombinant expression plasmid and application thereof in preparing anti-tumor immunogene therapeutic medicament
  • Recombinant expression plasmid and application thereof in preparing anti-tumor immunogene therapeutic medicament

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, preparation of recombinant expression plasmid pVAX-IL-12-GB

[0024] The active ingredient of the anti-tumor immune gene therapy drug of the present invention is the recombinant expression plasmid pVAX-IL-12-GB carrying the human immunoregulatory factor IL-12 gene, GM-CSF gene and B7.1 gene. In this plasmid, human immune The positions of the regulatory factors IL-12 gene, GM-CSF gene and B7.1 gene in the carrier are human immune regulatory factor IL-12 gene, GM-CSF gene and B7.1 gene from upstream to downstream, and the GM- The CSF gene and the B7.1 gene are connected through a linker to form a fusion gene (GM-CSF-B7.1), and connected to the IL-12 gene through the IRES sequence, that is, the IL-12 gene from the upstream of the IRES, and the fusion gene from the downstream of the IRES GM-CSF-B7.1.

[0025] The plasmid construction method is as follows:

[0026] The pCI-IL-12 plasmid containing the human IL-12 gene (see the literature for the construction ...

Embodiment 2

[0030] Example 2, detection of expression levels of IL-12 gene and GM-CSF-B7.1 fusion gene in cells transfected with plasmid pVAX-IL-12-GB

[0031] 1. Transfection of pVAX-IL-12-GB recombinant expression plasmid into 293T cells

[0032] Inoculate the positive clones transfected with the pVAX-IL-12-GB recombinant expression plasmid in 200mL LB liquid medium, culture at 37°C and shake at 200rpm for 12 hours, and extract a large amount of pVAX-IL-12-GB ultrapure plasmid (Use Tiangen Biochemical Technology Company’s endotoxin-free plasmid mass extraction kit), take 293T cells in the logarithmic growth phase and divide them into six-well plates one day in advance, and when they grow to 60%-70% on the second day, transfect Reagent Lipofectamine 2000 (Invitrogen Company) transfected 293T cells with pVAX-IL-12-GB ultrapure plasmid, and replaced 1640 complete medium (product of Gbico Company) with 10% fetal bovine serum after 6 hours to transfect pVAX1 empty vector Plasmids (products ...

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Abstract

The invention discloses an anti-tumor immunogene therapeutic medicament capable of activating the immunoreaction of the organism and having anti-tumor effect. The active ingredient of the anti-tumor immunogene therapeutic medicament is pVAX-IL-12-GB; three immunoregulation factor genes of human IL-12, GM-CSF and B7.1 are cloned to the same plasmid DNA vector pVAX1, and co-expression of upstream and downstream genes is realized through an internal ribosome entry site (IRES); and the vector is imported into a host so that the human immunoregulation factors IL-12, GM-CSF and B7.1 can be expressed simultaneously. The invention provides a novel anti-tumor immunogene therapeutic agent, wherein a plurality of anti-tumor immunoregulation factors are combined for use so that mutual costimulation of the factors can be developed and the organism can be better induced to produce anti-tumor immunoreaction; and therefore, the anti-tumor immunogene therapeutic medicament lays a firm foundation for further developing tumor immunogene treatment study and is wide in application prospect.

Description

technical field [0001] The invention belongs to the gene medicine in the field of biopharmaceuticals, in particular to an anti-tumor immunity gene therapy medicine capable of activating the body's immune response and having an anti-tumor effect. Background technique [0002] In addition to the characteristic low expression of major histocompatibility antigen and co-stimulatory molecules such as B7.1 and CD86, the immune tolerance of malignant tumors is also closely related to the microenvironment of the tumor. There are a large number of immunosuppressive cytokines such as IL-10 and tumor growth factor β in the tumor microenvironment. Tumor cells escape anti-tumor immune response through the regulation of the cytokine network in the microenvironment (Coussens LM, Werb Z. Inflammation and Cancer [J]. Nature, 2002, 420 (6917): 860-7; 6abrilovich D. Mechanisms and functional significance of tumor-induced dendritic-cell defects [J]. Nat Rev Immunol, 2004, 4 (12): 941 -52.). Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A61K48/00A61P35/00
Inventor 于继云徐元基阎瑾琦董金凯王伟张亮
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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