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Fusion protein for NKT (natural killer T) cell culture, encoding gene and application

A technology of NKT cells and fusion proteins, applied in cell culture active agents, animal cells, fusion polypeptides, etc., can solve the problem of low proportion of CD3/CD56 double-positive cells, and achieve enhanced tumoricidal activity and improved tumoricidal activity. Effect

Inactive Publication Date: 2017-06-09
深圳市艾克希尔生物科技有限公司
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Problems solved by technology

[0007] Aiming at the problems in the prior art that the in vitro expansion efficiency of peripheral blood mononuclear cells (PBMC) and the proportion of CD3 / CD56 double-positive cells in NKT cells are both low, the present invention provides a fusion protein for NKT cell culture, The coding gene and its application are used to improve the in vitro expansion efficiency of human peripheral blood mononuclear cells and the proportion of CD3 / CD56 double positive cells in NKT cells

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  • Fusion protein for NKT (natural killer T) cell culture, encoding gene and application
  • Fusion protein for NKT (natural killer T) cell culture, encoding gene and application
  • Fusion protein for NKT (natural killer T) cell culture, encoding gene and application

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Embodiment 1

[0053] 1. Construction of ICAM1-FN plasmid

[0054] According to the human ICAM1-1 and FN gene sequences, two pairs of primers were designed: forward primer P1 and reverse primer P2 of human ICAM1-1; forward primer P3 and reverse primer P4 of human FN. And the target gene sequence was obtained by two-step PCR. The primer sequences are as follows:

[0055] ICAM1-F: 5′-CATCATCATCATCATCAT CAGACATCTGTGTCCCC-3′

[0056] ICAM1-R:5′– GTAGGGGGCCGAGGTGTT-CT-3′

[0057] FN-F:5′- CTTAGGGGTCCGGGGCCCGG-GCT-3′

[0058] FN-R:5′-AGACTGCAGGTCGAC GTATCCAAGGTTTCTGGGTGGGATACTC-3′

[0059]The 5' end of the ICAM1-F primer was introduced with an NdeI restriction site (underlined and italicized part), and the 5' end of the ICAM1-R primer was introduced with a cDNA encoding a flexible peptide (the underlined bold part), and the plasmid containing the human ICAM1-1 cDNA sequence ( purchased from Origene) as a template. The 5' end of the FN-F primer introduced the cDNA sequence of the flex...

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Abstract

The invention relates to the field of biotechnology and medical science, in particular to a fusion protein for NKT (natural killer T) cell culture, an encoding gene and an application. The fusion protein is used for preparing NKT cells which are high in proliferating activity and killing activity. The fusion protein ICAM1-FN (intercellular adhesion molecule-1-fibronectin) can effectively stimulate proliferation of lymphocytes in the culture process of the NKT cells, the proportion of CD3 / CD56 double-positive cells is improved, so that the in-vitro amplification efficiency of peripheral blood mononuclear cells is twice that of a traditional method, the proportion of CD3 / CD56 double-positive cells is 2-10 times that of the traditional method, tumor killing activity of the NKT cells is enhanced, and the tumor killing activity of the prepared method of the NKT cells is improved by 15%-35% as compared with the traditional method.

Description

technical field [0001] The present invention relates to the fields of biotechnology and medicine. Specifically, the present invention relates to a fusion protein, coding gene and application for NKT cell culture, which are used to prepare NKT cells with high proliferative activity and high killing activity. Background technique [0002] The incidence of cancer has been on the rise since it was recorded, especially in the past two to three decades, with an annual growth rate of 3%-5%. Nearly half of new cancer cases worldwide are in Asia, most of them in China. About 6 people are diagnosed with cancer every minute in the whole country. [0003] In recent years, with the rapid development of molecular biology and immunology and other disciplines, tumor cell immunotherapy has become another new tumor treatment mode after surgery, radiotherapy and chemotherapy. Cellular immunotherapy, as a new technology to treat tumors, is to isolate, activate, and culture the patient's own a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N5/0783C12R1/19
CPCC07K14/70525C07K14/78C07K2319/00C12N5/0646C12N15/70C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/515C12N2501/727C12N2800/101
Inventor 肖洪宝
Owner 深圳市艾克希尔生物科技有限公司
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