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

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...

Examples

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 ...

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...

Examples

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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