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DC cell for overexpression of TRAF6, DC cell vaccine, construction method and application

An overexpression, cell technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., to achieve good tumor killing activity, enhance anti-tumor immune response, and improve the effect of tumor inhibition rate

Pending Publication Date: 2021-03-09
康九生物科技(长春)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a lack of a DC cell capable of overexpressing TRAF6 and its application in the prior art

Method used

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  • DC cell for overexpression of TRAF6, DC cell vaccine, construction method and application
  • DC cell for overexpression of TRAF6, DC cell vaccine, construction method and application
  • DC cell for overexpression of TRAF6, DC cell vaccine, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0051] 1. Materials

[0052] Experimental reagents: GM-CSF and IL-4 cytokines were purchased from PeproTech Company, LPS was purchased from Sigma Company, endotoxin-free plasmid extraction kit was purchased from Tiangen Company, GAPDH, TRAF6, JNK, p-JNK, ERK, p- ERK, p38, p-p38, IκB, and p-IκB antibodies were purchased from CST Company, goat anti-mouse IgG (H+L), goat anti-rabbit IgG (H+L) were purchased from Proteintech Company, and ECL luminescent liquid was purchased from Thermo company. TRAF6 knockout and overexpression lentiviral vectors were synthesized by Jiangsu Nanjing PPL Company. Experimental animals and cell lines: C57 BL / 6 mice (SPF grade, female, 6-8 weeks old, weighing 18-22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. 293T cells are owned by our laboratory.

[0053] 2. Method

[0054] 2.1 Construction of lentiviral vector inserted into TRAF6 gene fragment

[0055] The lentiviral vector for inserting the TRAF6 gene fragment is to insert...

experiment example 2

[0069] 1. Materials

[0070] Experimental reagents: GM-CSF and IL-4 cytokines were purchased from PeproTech Company, LPS was purchased from Sigma Company, CD16 / 32 antibodies were purchased from eBioscience Company, CD11c, CD40, CD80, CD86, MHCⅠ, MHCⅡ antibodies were purchased from Sanjian Company, The endotoxin-free plasmid extraction kit was purchased from Tiangen Company, the IL-12p70 detection kit was purchased from Invitrogen Company, and the TRAF6 overexpression lentiviral vector was synthesized by Jiangsu Nanjing PPL Company. Experimental animals and cell lines: C57 BL / 6 mice (SPF grade, female, 6-8 weeks old, weighing 18-22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0071] 2. Method

[0072] 2.1 The effect of overexpression of TRAF6 on the expression of mouse DC cell surface molecules

[0073] Recount the successfully induced DC cells above, and spread 6-well plates with RPMI-1640 complete medium containing 20ng / ml GM-CSF and 20ng / ml IL-4 cyt...

experiment example 3

[0081] 1. Materials

[0082] Experimental reagents: GM-CSF and IL-4 cytokines were purchased from PeproTech Company, LPS was purchased from Sigma Company, CD16 / 32 antibody, FITC-CD3 and APC-CD8 antibody were purchased from eBioscience Company, endotoxin-free plasmid extraction kit was purchased from Tiangen Company, IFN-γ detection kit and IL-4 detection kit were purchased from Invitrogen Company, and the TRAF6 overexpression lentiviral vector was synthesized by Jiangsu Nanjing PPL Company. Experimental animals and cell lines: C57 BL / 6 mice (SPF grade, female, 6-8 weeks old, weighing 18-22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0083] 2. Method

[0084] 2.1 Isolation of mouse mononuclear cells and sorting CD4 + T cell

[0085] The mouse was euthanized, and the spleen was taken out aseptically. The spleen was placed on gauze poured with 5 ml of lymphocyte separation solution, and ground to prepare a spleen cell suspension. Cover the cell suspen...

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Abstract

The invention provides DC cell for overexpression of TRAF6, a DC cell vaccine, a construction method and application, and belongs to the technical field of DC vaccines. According to the invention, theDC cell for overexpression of the TRAF6 is successfully constructed. The DC cell for overexpression of the TRAF6 is easier to maturate, can better present an antigen, stimulate immune response of T cell with stronger antigen specificity, enhance proliferation of CD4+T cells and induce Th1 type immune response, and can influence molecular expression of the downstream part of a TRAF6 signal path. The DC cell for overexpression of the TRAF6 can load a tumor antigen to obtain the DC cell vaccine. The DC cell vaccine provided by the invention has better tumor killing activity, can significantly improve the tumor inhibition rate, and can enhance the anti-tumor immune response, so that the DC cell vaccine can be used for preventing or treating adenocarcinoma, liver cancer, hematologic tumor andthe like, and brings good news to the majority of cancer patients.

Description

technical field [0001] The invention relates to the technical field of DC vaccines, in particular to a DC cell overexpressing TRAF6, a DC cell vaccine, a construction method and application. Background technique [0002] DC cells are important antigen-presenting cells, which can absorb and process antigens, present antigens to T lymphocytes, and initiate patients' own specific anti-tumor immune responses. [0003] At present, a variety of DC cell immunotherapies have been developed, and DC cell vaccines based on loading one or more specific tumor antigens have been proved to be feasible and safe in many clinical studies. DC cells pulsed with glioma-associated peptides can improve the survival rate of glioma model rats, and can induce specific anti-tumor immune responses and enhance CD4 + and CD8 + Infiltration of T lymphocytes. The first FDA-approved use of ex vivo peptide-loaded autologous DC cells (Sipuleucel-T) against prostate cancer. Due to the limited ability of DC...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/52C12N15/867A61K39/00A61P35/00
CPCC12N9/93C12N5/0639C12Y603/02019C12N15/86A61K39/001154A61P35/00C12N2740/15043A61K2039/5154C12N2510/02Y02A50/30
Inventor 台桂香王晶晶
Owner 康九生物科技(长春)有限公司
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