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Natural killer cell line modified by human interferon-alpha gene and establishing method thereof

A technology of natural killer cells and gene modification, applied to cells modified by the introduction of foreign genetic material, methods based on microorganisms, genetic engineering, etc., can solve the problems of no NKL cell lines, etc., to facilitate screening and promotion, and improve the use of The effect of high efficiency and transfection efficiency

Inactive Publication Date: 2011-01-19
SHANDONG UNIV
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Problems solved by technology

So far, there is no relevant report on the establishment of NKL cell lines modified by human interferon-alpha gene

Method used

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  • Natural killer cell line modified by human interferon-alpha gene and establishing method thereof
  • Natural killer cell line modified by human interferon-alpha gene and establishing method thereof
  • Natural killer cell line modified by human interferon-alpha gene and establishing method thereof

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Embodiment

[0034] Example Construction of human interferon-alpha gene-modified natural killer cell line

[0035] (1) The construction of human interferon-alpha secretory eukaryotic expression vector, according to the construction process ( figure 1 ), the specific steps are as follows:

[0036] (1) Isolate human peripheral blood lymphocytes, extract the total RNA of the cells, amplify the cDNA fragment of human IFN-α, the length is 569bp, and the primer sequence used for the amplification is:

[0037] IFN-α-F: 5′-TTAGGATCCATGGCCTCGCCCTTT-3′;

[0038] IFN-α-R: 5'-CGCGAATTCGTTATTCCTTCCTCC-3';

[0039] The PCR reaction conditions are: denaturation at 95°C for 5 minutes, 1 minute at 94°C, 1 minute at 58°C, 1 minute at 72°C, a total of 30 cycles, and extension at 72°C for 10 minutes;

[0040] (2) The RT-PCR product was connected with T-vector, the product was transformed into competent Escherichia coli DH5α, positive clones were screened, and Shanghai Boya Bioengineering Company was entrus...

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Abstract

The invention discloses a natural killer cell line (NKL) modified by a human interferon (IFN)-alpha gene, which can stably excrete and express the human IFN-alpha and is established by transfecting the human IFN-alpha gene into an NKL cell. The cell line can be expressed in cells according to the RT-PCR and ELISA detection and identification, and the synthesized IFN-alpha is excreted in extracellular culture supernate. Further research indicates that the NKL cell line modified by the human IFN-alpha gene can effectively kill liver cancer cells, and such anti-tumor effect is related to the expression level improvement of the NKL-IFN-alpha cell, IFN-gamma, Granzyme, TNF-alpha and Perforin genes and proteins. Moreover, the modified NKL cell line is more sensitive to external stimulus. Therefore, the biological characteristics of the cell line are more favorable for the application of clinical adoptive immunotherapy.

Description

technical field [0001] The invention relates to a human interferon-alpha gene-modified natural killer cell line and a construction method thereof, belonging to the field of adoptive immunotherapy of tumors. Background technique [0002] As we all know, NK cells, as an important part of the body's innate immunity, are the body's first line of defense against tumors and infections. Therefore, the role of NK cells in adoptive immunotherapy of tumors has attracted much attention. Cells have become a new strategy for clinical transplantation adjuvant therapy and anti-tumor biological therapy. However, the expansion efficiency of NK cells in vitro is low, so the establishment of NK cell lines has become an important means of tumor biotherapy. [0003] The NKL cell line is derived from the peripheral blood of CD3-CD16+CD56+ large granular lymphocytic leukemia patients. It has broad-spectrum cytotoxic activity and has a good clinical application prospect. [0004] Interferon is a ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/21C12N15/63C12R1/91
Inventor 张建蒋雯田志刚
Owner SHANDONG UNIV
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