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Mannan-modified hTRT (human Telomerase Reverse Transcriptase) gene-carrying and adeno-associated virus (AAV)-inducing targeting dendritic cell (DC) vaccine and preparation method thereof

A technology of dendritic cells and mannan, applied in the field of medical bioengineering, can solve the problems of short half-life, weak ability to activate immune effector cells, and poor targeting, and achieve good anti-tumor effect

Inactive Publication Date: 2012-07-11
郑骏年
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The problem to be solved by the present invention is to overcome the shortcomings of DC vaccines prepared by existing methods, such as short half-life of in vitro antigen peptide transfection DC, low transduction efficiency, poor targeting, and weak ability to activate immune effector cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of recombinant adeno-associated virus AAV / hTRT modified with mannan

[0016] 1. Obtain a human TRT gene (hTRT, 3.5kb in length) by RT-PCR technology;

[0017] 2. Clone the hTRT gene into the adeno-associated virus vector AAV (d16-95), construct the recombinant plasmid AA (d16-95) (abbreviated as AAV / hTRT), and prepare the AAV / hTRT virus with the pSH3 packaging system;

[0018] 3. AAV / hTRT amplification and purification AAV / hTRT virus repeatedly infected and lysed 293 cells to amplify the virus. Ultracentrifugation was used to collect and obtain purified virus bands. The PBS solution was dialyzed and desalted and stored at -80°C for later use. The virus titer was determined by half the tissue culture infectious dose (TCID 50) method;

[0019] 4. Mannan modification of AAV / hTRT Dissolve mannan in 0.1mol / L PBS solution (pH6.0) to a final concentration of 14mg / mL, add 0.01mol / L sodium periodate and oxidize at 4°C for 60min to form Oxidized mannan....

Embodiment 2

[0020] Example 2: Preparation of immature DC cells

[0021] The anticoagulant blood of the patient was collected by the blood cell separator under sterile conditions, centrifuged at 1500rpm / min for 10 minutes to absorb the upper plasma, inactivated at 56°C for 30 minutes and then centrifuged for later use, the blood cell pellet was double-diluted with normal saline, and the human lymphocytes with a specific gravity of 1.077 were separated The solution and the diluted blood were added to the centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 12 minutes, carefully extracted the white blood cell layer, washed twice with normal saline, and obtained PBMC after low-speed centrifugation. After culturing for 3 hours, the suspended T cells were washed away, and the adherent cells were placed in the culture medium containing fresh GM-CSF (800,000 U / L), IL-4 (1,000,000 U / L) and AIM V, at 37°C for 5 %CO 2 After incubation for 3 h in an incubator, immature DC cells can be...

Embodiment 3

[0022] Example 3: Mannan-modified AAV / hTRT infected DC cells and induced CTL

[0023] Adherent cells were infected with mannan-modified AAV / hTRT, and replaced with AIM V medium (containing GM-CSF and IL-4) after 5 hours to induce DC cells to obtain tumor-associated antigen DC vaccine; on the 6th day, the original Frozen T cells were mixed with cultured DC cells at the ratio of DC:T=1:20, AIMV was used as the medium, and GM-CSF (800,000 U / L), IL-2 (10,000 U / L) were added at the same time. ) and TNF-α (20 μg / L), co-cultured for 7 days to induce cytotoxic T lymphocytes (CTL).

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Abstract

The invention belongs to the technical field of medical biological engineering, and relates to a novel dendritic cell (DC) vaccine preparation technology. In the technology, an hTRT (human Telomerase Reverse Transcriptase) gene is inserted into an adeno-associated virus (AAV), and mannan surface modification is performed, so that an infected DC cell can be targeted in vitro. Along with stable expression of the hTRT gene in the DC, the DC can be continuously stimulated by a large quantity of hTRT antigen peptides. An hTRT antigen is a tumor-related antigen, so that a lymphocyte stimulated by the DC vaccine almost acts strong killing effects on all tumor cells. The vaccine prepared with a method disclosed by the invention has the characteristics of strong inducting function, high safety, low price, easiness for large-scale production, wide clinical application and the like. The problems of short half-life period, low transduction efficiency and poor activation-immune effect cell ability existing in the conventional DC transfected with an in-vitro antigen peptide are solved.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering, and relates to a novel dendritic cell (DC) vaccine preparation technology. In this method, the hTRT gene is inserted into the adeno-associated virus (AAV), and its surface is modified with mannan, so that it can target and infect DC cells in vitro. With the stable expression of hTRT gene in DC cells, DC cells can be continuously stimulated by a large amount of hTRT antigen peptides. Since the hTRT antigen is a broad-spectrum tumor-associated antigen, it is expressed in almost all tumor cells, while normal cells hardly express hTRT, so the lymphocytes stimulated by the DC vaccine have a strong killing effect on almost all tumor cells, avoiding the need for treatment. toxic side effects. Background technique [0002] In recent years, with the understanding of the mechanism of tumor immune escape and the discovery and identification of tumor-associated antigens, active immunotherapy...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K47/36A61P35/00C12N15/12C12N7/01C12N5/0783C12N5/0784
Inventor 郑骏年李连涛李慧忠张宝福刘俊杰张青杨洁章龙珍裴冬生
Owner 郑骏年
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