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Artificial antigen submit cell and preparation method thereof

An artificial antigen and cell technology, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of increasing treatment costs and achieve the goal of prolonging survival and strengthening interactions Effect

Inactive Publication Date: 2007-09-26
YANGZHOU UNIV
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AI Technical Summary

Problems solved by technology

However, this artificial antigen-presenting cell mainly stimulates T cells broad-spectrum and non-specifically, and there is a risk of causing autoimmune diseases when reinfused into the body; on the other hand, each time the cells are prepared, commercial CD3, CD28 Monoclonal antibodies, greatly increasing the cost of treatment

Method used

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  • Artificial antigen submit cell and preparation method thereof
  • Artificial antigen submit cell and preparation method thereof
  • Artificial antigen submit cell and preparation method thereof

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Experimental program
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Embodiment Construction

[0045] 1. Construct the cDNA expression vector of CD32, transfect the CD32 recombinant plasmid into K562 cells, screen with G418, identify and sort by FACS, and obtain K32 cells.

[0046] 1. Acquisition of CD32a cDNA: U937 cells in logarithmic phase were taken, RNA was routinely extracted with trizol, and mRNA was reverse transcribed to generate cDNA with Oligod(T)n as a primer. Primers for amplifying CD32a were designed with primer5.0 software, and HindIII and EcoRI restriction sites and protective bases were added to both ends.

[0047] The upstream primer sequence is cgcaagcttg atggctatgg agacccaaat g

[0048] The downstream primer sequence is ccggaattca gcaagctgag agtatgacca c

[0049] The PCR amplification program was pre-denaturation at 94°C for 5 minutes, followed by 30 seconds at 94°C, 1 minute at 55°C, and 90 seconds at 72°C for a total of 33 cycles, and a final extension for half an hour. The product of CD32a cDNA was obtained, and the fragment size was about 1000b...

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Abstract

The invention discloses an artificial antigen submitting cell and preparing method, which comprises the following steps: choosing human chronic granular leukocyte leukemia bacterial strain K562 cell as carrier; transfer-dying and expressing eucaryon expressing carrier of CD32a; expressing CD32 molecular on the surface of K562 cell stably through G418 screening and flowed cell sorting device sorting; forming K32 cell; producing eucaryon expressing carrier of double expressing CD86 and 4-1BBL co-simulating signal; proceeding homomycin screen and sort with flowed cell device for the K32 cell; getting stable expression K32 cell of CD86 and 4-1BBL molecule; getting the end product. This invention possesses low cost and good repeatability, which can inhibit die effectively.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of a novel antigen-presenting cell. Background technique [0002] Tumor adoptive immunotherapy has always been one of the most active research fields in tumor biotherapy. The infusion of autologous antigen-specific T cells (CTL) can not only specifically and effectively kill target cells, but also overcome the "incompetence" of immune cells in the body. The state leads to the low problem of the therapeutic effect of the vaccine. Therefore, fully and effectively activate CTL cells in vitro, so that they can reproduce and survive without apoptosis after being infused into the body, chemotaxis to the tumor site, specifically recognize and kill target cells, and have no toxicity to normal cells. Reference indicators for people to choose suitable adoptive immunotherapy. [0003] To fully and effectively activate CTL cells in vitro, three conditions for T cell activati...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/09C12N15/85
Inventor 龚卫娟季明春万兵钱莉
Owner YANGZHOU UNIV
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