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Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof

A technology that fuses cells and genes, applied in botany equipment and methods, biochemical equipment and methods, gene therapy, etc., can solve problems such as unsatisfactory therapeutic effects of fusion vaccines

Pending Publication Date: 2016-08-03
彭霞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the therapeutic effect of fusion vaccines is still unsatisfactory, and new methods are needed to enhance the effectiveness and targeting of fusion cells to induce CTL

Method used

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  • Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof
  • Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof
  • Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0176] Example 1. Preparation of Fusion Cells

[0177] Preparation methods of fusion cells, including S1) and S2);

[0178] S1) Introduce the coding gene of transforming growth factor-β receptor related protein and α1,3-galactosyltransferase related gene into tumor cells to obtain recombinant cells;

[0179] S2) Fusion of the recombinant cell and dendritic cell to obtain the fused cell.

[0180] The specific method is as follows:

[0181] 1. Preparation of recombinant cells

[0182] 1. Preparation of recombinant cells

[0183] Replace the fragment between the BamHI and XbaI recognition sites on the pLVX-Puro vector with the DNA molecule shown in SEQ ID No. 5 in the sequence list, and keep the other sequences unchanged, to obtain the recombinant vector pLVX-Puro / GT-Eng, the recombinant vector pLVX -Puro / GT-Eng expresses the Endoglin protein shown in SEQIDNo.1 and the α1,3 galactosyltransferase (alpha(1,3)Galactosyltransferase, alpha(1,3)GT) shown in SEQIDNo.3. The recombinant vector pLVX...

Embodiment 2

[0218] Example 2: DC / HepG2 (GT-Eng+) induces the production of IFN-γ-secreting T lymphocytes in vitro

[0219] The experiment was repeated three times, and the specific steps for each repeated experiment were as follows:

[0220] T lymphocytes secreting IFN-γ induced by DC / HepG2 (GT+) were obtained according to the following method, and the IFN-γ secreted by T lymphocytes was detected by enzyme-linked immunospot assay (ELISPOT). The 1×Washing buffer, biological The labeled antibody and enzyme-labeled avidin are all reagents in HumanIFN-gammaprecoatedELISPOTkit (HumanIFN-gammaprecoatedELISPOTkit is a product of Daktronics Biotechnology Co., Ltd., the catalog number is DKW22-1000-048). The specific steps are as follows:

[0221] 1) Take out the well plate in the kit, add 200 μL of incomplete RIPM1640 medium to each well, let it stand at room temperature for 5-10 minutes, and pour out the liquid.

[0222] 2) Add DC / HepG2(GT+)3×10 of Example 1 suspended in incomplete RIPM1640 medium to ea...

Embodiment 3

[0235] Example 3. Treatment of tumors by T lymphocytes secreting IFN-γ induced by the fusion cell DC / HepG2 (GT-Eng+)

[0236] The experiment was repeated three times, and the specific steps for each repeated experiment were as follows:

[0237] T lymphocytes secreting IFN-γ induced by DC / HepG2 (GT+) in Example 2, T lymphocytes secreting IFN-γ induced by DC / HepG2 (GT-Eng+), and IFN-γ secreted by DC / HepG2 T lymphocytes, T lymphocytes secreting IFN-γ induced by DC / HepG2 (Eng+), T lymphocytes secreting IFN-γ induced by DC / HepG2 (pLVX-Puro), T lymphocytes secreting IFN-γ induced by DC Lymphocytes, T lymphocytes induced by HepG2, T lymphocytes induced by HepG2 (Eng+), T lymphocytes induced by HepG2 (GT+) and T lymphocytes induced by HepG2 (pLVX-Puro) were suspended in PBS, and the cells were obtained separately The content is 10 5 A / μL DC / HepG2(GT+)-induced IFN-γ-secreting T lymphocyte suspension, DC / HepG2(GT-Eng+)-induced IFN-γ-secreting T lymphocyte suspension, DC / HepG2-induced secret...

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Abstract

The invention discloses a heterologous fusion gene modified cancer cell / dendritic cell fusion tumor vaccine and its preparation method. According to the heterologous fusion gene modified cancer cell / dendritic cell fusion tumor vaccine and its preparation method, a method for cell fusion comprises a step of fusing cancer cell which contains Endoglin and alpha1,3 galactosyl transferase related gene and is used for expression of Endoglin and alpha1,3 galactosyl transferase and dendritic cell to obtain fusion cell. The alpha1,3 galactosyl transferase gene encodes protein with its amino acid sequence being SEQ ID No.3. The Endoglin is protein with its amino acid sequence being SEQ ID No.1. The alpha1,3 galactosyl transferase related gene is formed by connecting the alpha1,3 galactosyl transferase gene and GTTI. Nucleotide sequence of GTTI is from the 1st nucleotide to the 548th nucleotide in the SEQ ID No.5 of the sequence table.

Description

Technical field [0001] The invention relates to a cancer cell / dendritic cell fusion tumor vaccine modified by a heterologous fusion gene in the field of biomedicine and a preparation method thereof. Background technique [0002] Immunotherapy for malignant tumors has become a new treatment method besides surgery, radiotherapy and chemotherapy. Among them, adoptive therapy of tumor-specific cytotoxic T lymphocytes (cytotoxic Tlymphocyte, CTL) has always been the focus of tumor immunotherapy. Fusion of tumor cells and dendritic cells (DC) has been widely accepted as an effective method to induce CTL. However, the therapeutic effect of fusion vaccines is still not ideal, and new methods are needed to enhance the effectiveness and targeting of CTL induced by fusion cells. [0003] Endoglin (also known as CD105, differentiation group 105) is a homodimeric transmembrane glycoprotein tumor vascular marker molecule with a molecular weight of 180kDa and a part of transforming growth facto...

Claims

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Application Information

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IPC IPC(8): C12N15/07C12N5/22A61K35/17A61K48/00A61P35/00
Inventor 彭霞卢小玲陈玥周源
Owner 彭霞
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