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Preparation method of antigen-specific cytotoxicity T lymphocytes

A lymphocyte and cytotoxic technology, applied in the field of preparation of multi-antigen broad-spectrum CTL, can solve problems such as limited use

Inactive Publication Date: 2014-08-27
SHENZHEN HORNETCORN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adenoviral vectors have high transgenic efficiency and are not limited by whether the target cells are dividing cells. It is easy to produce high-titer viral vectors, and the evaluation of biological activity is also relatively simple. However, due to its high immunogenicity, its use is limited

Method used

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  • Preparation method of antigen-specific cytotoxicity T lymphocytes
  • Preparation method of antigen-specific cytotoxicity T lymphocytes
  • Preparation method of antigen-specific cytotoxicity T lymphocytes

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Experimental program
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Effect test

Embodiment 1

[0027] Taking targeting carcinoembryonic antigen (CEA) as an example, a CEA-specific gene fragment was obtained through in vitro synthesis and molecular cloning methods. The nucleotide sequence of the carcinoembryonic antigen gene fragment is shown in SEQ ID NO:1; the nucleotide sequence of the melanoma-associated antigen A3 gene fragment is shown in SEQ ID NO:2; the mucin 1 antigen gene fragment is shown in SEQ ID NO : As shown in 3.

Embodiment 2

[0029] The gene fragment of described CEA is introduced into lentiviral vector Lv, such as figure 1 The lentiviral vector Lv-CEA carrying the CEA antigen gene was obtained as shown. The same method was used to construct lentiviral vectors with MAGE-A3 and MUC1 antigen gene fragments. Such as figure 1 As shown, among them, the Lv-CEA lentiviral vector is the expression vector carrying the CEA antigen fragment; the Lv-MAGE-A3 lentiviral vector is the expression vector carrying the MAGE-A3 antigen fragment; the Lv-MUC1 lentiviral vector is the expression vector carrying the MUC1 antigen fragment expression vector.

[0030] The lentiviral vectors carrying different chimeric antigen receptor gene fragments were identified by single / double digestion, and the results were as follows: figure 2 and shown in Table 1.

[0031] Table 1 Identification results of lentiviral vectors carrying different tumor antigen gene fragments by enzyme digestion

[0032] carrier name ...

Embodiment 3

[0034] The lentiviral vectors constructed above carrying different tumor antigen genes were transduced into Hela cells. After 48 hours, the cells were lysed, and the expression of each antigen tag protein was detected by Western blot method. The results are as follows: image 3 shown. The results showed that Hela cells themselves did not express CEA, MAGE-A3 and MUC1. After transduction with recombinant vectors, recombinant tumor antigen proteins of the expected size could be detected, indicating that tumor antigen proteins could be expressed in Hela cells after transduction with recombinant vectors. Express correctly.

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Abstract

The invention discloses a preparation method of antigen-specific cytotoxicity T lymphocytes. The method comprises the following steps: 1, constructing a recombinant vector carrying tumor antigen gene; 2, separating and culturing DC cells; 3, transducing immature DC cells in step 2 by using the recombinant vector in step 1; 4, inducing the immature DC cells in step 3 through cytokines to form mature DC cells; and 5, coculturing the mature DC cells in step 4 and T lymphocytes to obtain the antigen-specific cytotoxicity T lymphocytes. The method preferably selects a lentivirus mixed vector realizing transduction to be used in the expression of three wide spectrum tumor antigens comprising CEA, MAGE-A3 and MUC1 to obtain activated and proliferated tumor antigen-specific T cells, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a novel multi-antigen broad-spectrum CTL. Background technique [0002] At present, the anti-tumor immunotherapy strategies for cancer mainly include: whole-tumor cell vaccines (Whole-tumor cell vaccines), dendritic cell vaccines (DC vaccines), recombinant proteins vaccines (recombinant proteins vaccines), peptide vaccines ( Peptide vaccines), DNA vaccines and virus vaccines, etc. Studies have shown that DC vaccine combined with adoptive T-cell immunotherapy (adoptive T-cell therapy) has the best effect. DC is the most powerful professional antigen-presenting cell found so far. The surface of mature DC highly expresses MHC-Ⅰ / Ⅱ molecules, co-stimulatory molecules (CD40, CD80, CD86, etc.) and adhesion molecules (LFA-3, ICAM-1, etc.) etc.), not only can effectively present tumor antigens to activated or memory T cells, but also presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10C12N15/867
Inventor 张鸿声张艳磊罗晓玲王宇环
Owner SHENZHEN HORNETCORN BIOTECH
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