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Recombinant tumor antigen and its preparation method, encoding nucleic acid and use

A tumor antigen and encoding nucleic acid technology, applied in the field of recombinant tumor antigen preparation, can solve the problems of easy damage to normal cells, poor targeting, and adverse reactions.

Inactive Publication Date: 2018-03-13
BEIJING TRICISIONBIO THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Surgical treatment is only suitable for early-stage tumors; radiotherapy and chemotherapy are prone to damage normal cells and cause adverse reactions due to their poor targeting

Method used

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  • Recombinant tumor antigen and its preparation method, encoding nucleic acid and use
  • Recombinant tumor antigen and its preparation method, encoding nucleic acid and use
  • Recombinant tumor antigen and its preparation method, encoding nucleic acid and use

Examples

Experimental program
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Embodiment 1

[0015] Example 1: Synthesis and preparation of recombinant antigen gene

[0016] 1. According to the above-mentioned rules, codon optimization was performed on the human AKR1B10 gene, gp96 gene and LAMP1 gene, the optimized genes were synthesized, and the synthesized recombinant genes were cloned into the vector psp73.

[0017] 2. The synthetic vector psp73-AKR1B10 was transformed into Escherichia coli competent cell stb13, and after overnight culture and amplification, the recombinant plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0018] 3. Digest the extracted recombinant plasmid according to the following system (take 100ul system as an example): 10x cutsmartbuffer, 10ul; recombinant plasmid DNA (1000ng / ul), 50ul; ultrapure water, 35ul; SpeI-HF restriction endonuclease Enzyme, 5ul. Mix well and react at 37°C for 2 hours.

[0019] 4. Add 70ul of isopropanol to the digested product, mix well and put it at -20°C for precipitation for 30 minutes.

[...

Embodiment 2

[0027] Example 2: Preparation and phenotypic identification of DC-AKR1B10 tumor vaccine

[0028] 1. Induction culture of immature DC cells in vitro

[0029] Aseptically extract 50ml of venous blood from a healthy person, separate the peripheral blood mononuclear cells with lymphocyte separation medium in the ultra-clean workbench, add the mononuclear cells to the AIM-V medium, and put them in a 37°C, 5% CO2 incubator. to allow monocytes to adhere to the wall. After 1-2 hours, remove the non-adherent cells, add iDC medium (the final concentration of 800U / mL of GM-CSF and 500U / mL of IL-4 to the AIM-V medium) for the adherent cells, and put them in 37℃ , cultured in a 5% CO2 incubator for 7 days, and half of the medium was changed every 2 to 3 days.

[0030] 2. Recombinant tumor antigen transfection of immature DC cells

[0031] On the day of transfection, digest the immature DC cells into a cell suspension with a non-enzymatic cell digestion reagent, resuspend the cells in op...

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Abstract

The invention relates to a recombinant tumor antigen and its preparation method, encoding nucleic acid and use in tumor treatment. The recombinant tumor antigen comprises a human GP96 gene signal peptide modified by a codon, a coding sequence of a human AKR1B10 gene, a human LAMP1 gene signal sequence and a ployA structural region of A64. The modified AKR1B10 recombinant antigen mRNA is transfected into immature DC cells and then is subjected to cell factor-induced maturation so that a mature dendritic cell tumor vaccine DC-AKR1B10 is obtained. The tumor vaccine stimulates the human body so that cytotoxic T cells are produced and the effective anti-tumor immune response is caused without causing autoimmunity.

Description

technical field [0001] The present invention relates to the field of pharmaceutical products containing antigens, specifically, a recombinant tumor antigen, its preparation method, encoding nucleic acid and its use. Background technique [0002] Traditional cancer treatment methods, such as surgery, radiation therapy and chemotherapy, all have certain limitations. Surgical treatment is only suitable for early-stage tumors; radiotherapy and chemotherapy are prone to damage normal cells and cause adverse reactions due to their poor targeting. Malignant tumors have biological characteristics of easy invasion and recurrence, so more targeted and less toxic treatment options are needed. With the development of oncology, biological immunotherapy has become the fourth means of tumor treatment. Studies have shown that tumor cells have specific tumor antigens, and the immune system can distinguish tumor cells from normal cells by recognizing tumor antigens. In cancer patients, the...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/00A61P35/00
CPCA61K39/0011C07K2319/02C07K2319/20C12N9/0006C12Y101/01021
Inventor 不公告发明人
Owner BEIJING TRICISIONBIO THERAPEUTICS INC