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

A technology of anti-tumor immunity and expressing plasmids, applied in the field of biopharmaceuticals

Inactive Publication Date: 2012-11-14
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 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 a recombinant expression plasmid pVAX-IL-12-GB carrying human immune regulatory factors 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 vector are the human immunoregulatory factor IL-12 gene, GM-CSF gene and B7.1 gene in order from upstream to downstream. CSF gene and B7.1 gene are linked by linker to form a fusion gene (GM-CSF-B7.1), and are linked to IL-12 gene by IRES sequence, that is, IL-12 gene from upstream of IRES and fusion gene from downstream of 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 method: Wang Wei, Gao Jiangping, Yu Jiyun, etc.) The construction of t...

Embodiment 2

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

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

[0032] The positive clones transfected with pVAX-IL-12-GB recombinant expression plasmid were inoculated into 200mL LB liquid medium, cultured at 37℃ at 200rpm and shaken for 12 hours to extract a large amount of pVAX-IL-12-GB ultrapure plasmid (Using the endotoxin-free plasmid mass extraction kit of Tiangen Biochemical Technology Company), take the 293T cells in the logarithmic growth phase into a six-well plate 1 day in advance, and when the growth reaches 60%-70% on the second day, use transfection Reagent Lipofectamine 2000 (Invitrogen) transfected pVAX-IL-12-GB ultra-pure plasmid into 293T cells, 6 hours later, replaced with 10% fetal calf serum 1640 complete medium (product of Gbico) to transfect pVAX1 empty vector Plasmid (product of Invitrogen) is a blank ...

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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 a gene medicine in the field of biopharmaceuticals, and particularly relates to an anti-tumor immune gene therapy drug that can activate the body's immune response and has an anti-tumor effect. Background technique [0002] In addition to the characteristic low expression of major histocompatibility antigens and costimulatory 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. It is through the regulation of the cytokine network in the microenvironment that tumor cells escape the anti-tumor immune response (Coussens LM, Werb Z. and Cancer[J].Nature,2002,420(6917):860-7;6abrilovich D.Mechanisms and functional significance of tumour-induced dendritic-cell defects[J].Nat Rev Immunol,2004,4(12):941 -52.). Therefore, the cytokin...

Claims

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Application Information

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Patent Type & Authority Applications(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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