Livin-based immune-stimulating complex, preparation method thereof and application thereof

An immunostimulatory and complex technology, applied in the field of genetic engineering, to achieve the effect of improving immunogenicity, efficiently inducing cytotoxic killing effect, and inhibiting growth

Inactive Publication Date: 2013-06-12
FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] But so far, there are no relevant reports on the immunostimulatory complex involving Livin gene, IgG Fc segment gene and CpG motif at home and abroad

Method used

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  • Livin-based immune-stimulating complex, preparation method thereof and application thereof
  • Livin-based immune-stimulating complex, preparation method thereof and application thereof
  • Livin-based immune-stimulating complex, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the preparation of the immunostimulatory compound based on Livin

[0037] 1. Construction of recombinant eukaryotic vector for co-expression of Livin and IgG Fc segment double genes

[0038] 1. Cloning of Livin full-length cDNA

[0039] According to the Livin gene sequence registered in GenBank (accession number is NM_001163247), the following primers were designed and synthesized: Upstream primer F1: 5’-aat ctgcag atgttctcacctgcagacttatt-3' (SEQ ID No.3), the underlined part is the PstⅠ restriction site; downstream primer R1: 5'-tac gaattc ggcttcacttccgtgtatgac-3' (SEQ ID No.4), the underlined part is the EcoRI restriction site. Glioma cells G422 were routinely cultured in RPMI 1640 medium, and the cell density was adjusted to 2×10 7 Cells / mL were collected, washed 3 times with PBS, and total RNA was extracted with a total RNA extraction kit, and operated according to the kit instructions. The obtained total cellular RNA was reverse-transcribed in...

Embodiment 2

[0047] Embodiment 2, the preparation of dendritic cell vaccine

[0048] Mouse femoral bone marrow cells were extracted under sterile conditions, mixed with RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 U / mL streptomycin, and removed after 3 hours. For adherent cells, add interleukin-4 (IL-4) at a final concentration of 500 U / mL and granulocyte-macrophage colony-stimulating factor (GM-CSF) at a final concentration of 1000 U / mL, and set the temperature at 37°C, CO 2 They were cultured in an incubator with a gas volume fraction of 5%, and half of the culture medium was replaced every 3 days, while IL-4 and GM-CSF were added to induce the generation of dendritic cells. Mix 0.1mg Livin-based immunostimulatory complex with 1×10 6 Dendritic cells were co-incubated at 37°C for 24 hours, activated dendritic cells ( Figure 4 ) is the dendritic cell vaccine.

Embodiment 3

[0049] Example 3, Detection of Anti-glioma Activity of Dendritic Cell Vaccine

[0050] 1. Anti-glioma activity detection in vitro

[0051] Thirty 6- to 8-week-old female Kunming mice were randomly divided into 3 groups: experimental group, control group and blank group, with 10 mice in each group. In each group, 1×10 dendritic cells were subcutaneously injected at the base of the dorsal tail of each mouse. 6 (experimental group is the dendritic cell vaccine that the embodiment 2 prepares namely the dendritic cell stimulated by the immunostimulatory complex of the present invention, the control group is the dendritic cell stimulated by the blank immunostimulatory complex, and the blank group is the dendritic cell stimulated by the immunostimulatory compound of the present invention, and the blank group is the dendritic cell stimulated by the immunostimulatory compound of the present invention, Stimulated dendritic cells), followed by a booster immunization with the same meth...

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Abstract

The invention discloses a Livin-based immunostimulatory complex, which is composed of a recombinant eukaryotic vector co-expressed with two genes of Livin and IgG Fc segment, a CpG motif, saponin QuilA, cholesterol and lecithin; the preparation method is at a pH of 7.2~ In the phosphate buffer solution of 7.4, add the recombinant eukaryotic vector with the final concentration of 0.5mg / mL of Livin and IgGFc double gene co-expression, 0.1mg / mL of CpG motif, 1mg / mL of saponin QuilA, 0.1mg / mL of Cholesterol and 0.1mg / mL lecithin, ultrasonic treatment in an ice bath to fully mix and emulsify the solution, and then dialyze with PBS, the obtained microfloccules are the immunostimulatory complex based on the Livin gene; the immunostimulatory complex can be large Significantly improve the immunogenicity of Livin, and co-incubating it with dendritic cells can stimulate the maturation and activation of dendritic cells and exert an efficient antigen presentation ability, thereby efficiently inducing the cytotoxic killing effect of CTL and effectively inhibiting glial cells The growth of the tumor, prolonging the average survival period of tumor-bearing nude mice and improving the average survival rate can be used to prepare dendritic cell therapeutic vaccine against glioma.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an immunostimulating complex (immunostimulating complex, ISCOM), in particular to a Livin-based immunostimulating complex, and also relates to a preparation method of the immunostimulating complex and its application in the pharmaceutical field. application. Background technique [0002] Glioma is the most common malignant tumor of the human central nervous system. Because of its infiltrating unlimited proliferation, no obvious boundary with normal tissue and rich blood vessels, it has a high recurrence rate and low cure rate, mortality and disability. Rates are high. Finding new effective therapeutic drugs and methods to completely conquer glioma has always been one of the main topics in the world medical field. In recent years, the continuous development of immunology has brought new means and methods to the treatment of glioma. Immunotherapy has been regarded as th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/39A61K39/00C12N15/79A61P35/00
Inventor 陈宏颉王守森郑兆聪袁邦青王如密高进喜赵琳
Owner FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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