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DNA vaccine for anti-liver cancer

A DNA vaccine and anti-liver cancer technology, applied in recombinant DNA technology, DNA/RNA fragments, anti-tumor drugs, etc., can solve the problems of non-expression, inability to effectively activate the T cell system, and inability to recognize and kill effector T cells.

Active Publication Date: 2008-06-18
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the AFP antigenic determinant hAFP542-550 has good immunogenicity, it is not expressed on the membrane surface of liver cancer cells, and the liver cancer cells themselves cannot become antigenic particles, so the T cell system cannot be effectively activated and cannot be recognized and killed by effector T cells

Method used

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  • DNA vaccine for anti-liver cancer
  • DNA vaccine for anti-liver cancer
  • DNA vaccine for anti-liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, preparation of the active ingredient of the DNA vaccine of anti-liver cancer

[0048] The active ingredient of the DNA vaccine against liver cancer is

[0049] pcDNA3.1(+) / KOZAK-GPC3-N-hAFP 542-550 -EGFP-GPC3-C.

[0050] pcDNA3.1(+) / KOZAK-GPC3-N-hAFP 542-550 -The construction process of EGFP-GPC3-C is shown in Figure 1, and the method is as follows:

[0051] 1. Extract the total RNA from isolated human fetal liver, fetal kidney and placenta tissue with Trizol, reverse transcribe into cDNA for future use according to the instructions of the ReverseTranscription System kit (Promega Company).

[0052] 2. Use the sequence of Genbank Accession number: NM_004484 as a template to design primers (upstream primer TF: 5'-ctgccactctcccgcgctctcc-3' and downstream primer TR: 5'-gtggttccctttatcgaggaagac-3') to amplify from the cDNA of placental tissue with pyrobest enzyme The gene fragment L-GPC3 (1.851 kb) containing the open reading frame of the GPC3 gene. The ab...

Embodiment 2

[0060] Example 2, the detection of the expression and localization of the DNA vaccine against liver cancer in vivo

[0061] 1. Detection of fusion gene expression from RNA level and protein level respectively

[0062] 1. Detection of fusion gene expression in transfected cells at the RNA level

[0063] The liver cancer cell line HepG2 [HLA-A 2.1 (+)] (purchased from China Culture Depository Animal Cell Bank of Wuhan University (CCTCC) number GDC024), when the confluence of the cells is greater than 95%, digest and passage with 0.25% trypsin and EDTA, and inoculate into 6 wells respectively plate (two repetitions) and 25cm 2 In culture flasks (two replicates), when the confluence of the cells reached 80-90%, the pcDNA3.1(+) / KOZAK-GPC3-N-hAFP 542-550 - The EGFP-GPC3-C plasmid was transfected according to the method described in the instructions of lipofectamine 2000 (Invitrogen Company), and the cells were harvested 24 hours and 48 hours after transfection, respectively. Liv...

Embodiment 3

[0072] Example 3. Preliminary detection of the immune effect of lymphocyte activation by DNA vaccine against liver cancer

[0073] 1. Extract 20ml of whole blood from a healthy person and add it to a heparin anticoagulant tube, dilute it with an equal volume of normal saline (0.9% Nacl), and add 3ml of lymphocyte separation solution Lymphoprep (Norwegian AXIS-SHIELD company) according to the ratio of 6ml of diluted blood. Mix the two, centrifuge at 800g at room temperature for 20min, absorb the white lymphocyte layer in the middle, and add it to the 25cm 2 Culture in a culture flask with DMEM+10% FBS (fetal bovine serum), and set aside.

[0074] 2. Inoculate liver cancer cells HepG2 into 6-well culture plates and culture until the confluence is 80-90%, transfect plasmid pcDNA3.1(+) and plasmid pcDNA3.1(+) / KOZAK-GPC3-N-hAFP 542-550 -EGFP-GPC3-C, 4.0μg DNA was transfected with 5ul lipofectamine 2000 per well; 48 hours after transfection, separately cultured lymphocytes were a...

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Abstract

The invention discloses an anti-hepatocarcinoma DNA vaccine. Based on the membrane-anchoring property of primary hepatocarcinoma high-expression carcinoembryonic antigen GPC3, the invention uses one antigenic determinant Hafp542-550 in neoplastic alpha fetoprotein (AFP) of the organism itself as immunogen to construct the DNA vaccine and expression of fusion protein, so as to stimulate the T-cell immune response of the organism and exert the immune effect and damaging effect to hepatoma cells.

Description

technical field [0001] The invention relates to a DNA vaccine against liver cancer. Background technique [0002] Primary liver cancer (Primary Hepatocellular Carcinoma, PHC) is a common malignant tumor. At present, surgical resection is still used in clinical practice, supplemented by anhydrous ethanol tumor injection, percutaneous hepatic arterial chemotherapy and embolization. method, but the curative effect is not good, only 20% of tumors can be removed, and the 5-year survival rate of patients with unresectable tumors is less than 5%. [0003] Gene therapy is expected to be an effective new method for the treatment of liver cancer. Because tumor cells express low levels of histocompatibility antigen molecules (MHC), they lack signaling molecules that can effectively activate lymphocytes. At the same time, tumor cells themselves can produce immunosuppressive factors or certain cytokines (such as TGF-β) to inhibit the growth of lymphocytes. Activated to avoid the immune...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/00C12N15/62C12N15/79A61P35/00A61P1/16
Inventor 羊东晔黄来强蓝方
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV