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Interleukin-15 gene modified natural killing cell strain and its preparation method

A technology of natural killer cells and interleukins, which is applied to cells modified by the introduction of foreign genetic material, biochemical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., and can solve problems such as failure to achieve

Active Publication Date: 2004-05-05
UNIV OF SCI & TECH OF CHINA +1
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Problems solved by technology

Although the IL-2 gene-modified NK cell lines obtained by Nagashima S and Tam YK et al. overcome the dependence on IL-2 and do not need to inject IL-2 in clinical application, their biological characteristics are similar to those of unmodified cells. NK cells are similar, and they can secrete a certain dose of IL-2 by themselves, but there are still potential disadvantages, which do not meet the ideal requirements

Method used

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  • Interleukin-15 gene modified natural killing cell strain and its preparation method
  • Interleukin-15 gene modified natural killing cell strain and its preparation method
  • Interleukin-15 gene modified natural killing cell strain and its preparation method

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Embodiment

[0025] 1. Construction of pcDNA3 / IL-15 recombinant plasmid

[0026] Total RNA extracted from peripheral blood mononuclear cells after stimulation was used as a template for reverse transcription polymerase chain reaction (RT-PCR) to amplify the cDNA fragment of human IL-15, which is expected to be 486bp in length ( figure 1 ), wherein the RT parameters are: 37°C for 1 hour, 95°C for 10 minutes; the PCR parameters are: 94°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, cycled 30 times, and the last extension is 7 minutes. The construction process of pcDNA3 / IL-15 recombinant plasmid ( figure 2 ): Firstly, the RT-PCR product was connected with T-vector, and the product was transformed into competent Escherichia coli DH5α, positive clones were screened, and Shanghai Boya Bioengineering Company was entrusted to conduct DNA sequence determination. The results showed that the cDNA sequence of IL-15 was completely consistent with the report. Then, T-IL-15 was digested with Eco...

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Abstract

An interleukin-15 (IL-15) gene modified natural killing cell strain for immunotherapy of tumor is prepared through inserting the cDNA coding region of IL-15 in pcDNA3 eucaryotic expression carrier, configuring secretion-type recombinant eucaryotic expression carrier pc DNA3 / IL-15, using liposom to transfecte cell NK-92, adding Geneticin (G418) to screening culture medium, screening, limited dilution to cloned cells, detecting activity, choosing positive cell clones, and amplification.

Description

1. Technical field [0001] The invention relates to the field of cell adoptive immunotherapy for tumors, and more specifically, the invention relates to a genetically modified natural killer cell line and a preparation method thereof. 2. Technical background [0002] Natural killer (NK) cells play an important role in the body's anti-tumor and anti-virus processes. In the past, NK cells were isolated from human peripheral blood for clinical treatment. However, there are technical difficulties in the isolation, purification and in vitro culture of NK cells. Therefore, there is a need in the art for an NK cell strain that is convenient to culture in vitro, grows rapidly, has high killing activity against tumor cells, has high use efficiency, and has low toxic and side effects. Among them, NK-92 cells are a kind of NK cell line with high-efficiency and broad-spectrum anti-tumor effect established from non-Hodgkin's lymphoma patients, and have good clinical application prospects...

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Application Information

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IPC IPC(8): C12N5/10C12N15/79
Inventor 田志刚张建魏海明张建华孙汭
Owner UNIV OF SCI & TECH OF CHINA
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