Dendritic cell targeted peptide, coding gene and application

A technology of dendritic cells and targeting peptides, applied in the direction of peptides, hybrid peptides, specific peptides, etc., can solve the problems of unsatisfactory curative effect, large side effects of radiotherapy and chemotherapy, etc., and achieve improved antigen presentation ability, high safety, The effect of broad application value

Inactive Publication Date: 2015-03-04
SHENZHEN TONGKANG BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its treatment is mainly based on surgery, chemotherapy and radiotherapy, but the side effects of radiotherapy and chemotherapy are large, and the curative effect is not very satisfactory

Method used

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  • Dendritic cell targeted peptide, coding gene and application
  • Dendritic cell targeted peptide, coding gene and application
  • Dendritic cell targeted peptide, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The dendritic cell-targeting peptide of the present invention is obtained by screening dendritic cells as target cells. The present invention utilizes a commercialized phage display heptapeptide library (New England BioLabs) to screen and obtain the target dendritic cell-targeting peptide. To the peptide:

[0035] 1. Screening with dendritic cells as target cells:

[0036] (1) Obtain dendritic cells:

[0037] Mononuclear cells (peripheral blood mononuclear cells, PBMCs) were isolated from the peripheral blood of healthy people, and mature dendritic cells were obtained by in vitro induction and culture. The method:

[0038] ① Take fresh peripheral blood from healthy people, add lymphocyte separation medium, and separate by density gradient centrifugation to obtain mononuclear cells.

[0039] ②Cultivate in RPMI-1640 medium containing 10% fetal bovine serum. Place at 37°C, 5% CO 2 After standing in the incubator for 2 hours, the suspended cells were removed.

[0040] ③...

Embodiment 2

[0051] Example 2: Obtaining fusion peptides containing dendritic cell targeting peptides

[0052] 1. Synthesis of a fusion peptide (NP-MUC1 for short) containing a dendritic cell targeting peptide:

[0053] The dendritic cell targeting peptide screened in Example 1 is connected to the MUC1 tumor antigen polypeptide through a flexible linker (Cys), and a fusion peptide comprising the dendritic cell targeting peptide is synthesized, such as SEQ ID NO .3 shown. At the same time, prepare the fusion peptide labeled with fluorescein FITC for future use.

[0054] 2. Identification of the binding ability of the fusion peptide to dendritic cells:

[0055] Dendritic cells were used as the target cells to identify the binding ability of the prepared fusion peptide NP-MUC1, and Hela cells were used as the control. The specific steps are as follows:

[0056] Dendritic cells were divided into 1 × 10 5 Seed on a 96-well plate at a density of 37°C, 5% CO 2 In the incubator; the cells wer...

Embodiment 3

[0058] Example 3: Evaluation of Fusion Peptide Effects

[0059] 1. Absorption of fusion peptide NP-MUC1 by antigen-presenting cells (dendritic cells)

[0060] The efficiency of fusion peptide into dendritic cells was observed by confocal laser microscopy. Specific steps are as follows:

[0061] Dendritic cells were seeded in six-well plates containing slides at 37°C, 5% CO 2 Culture in an incubator; then add fluorescein (FITC)-labeled MUC1 polypeptide or FITC-labeled fusion peptide NP-MUC1. After incubation for 6 hours, wash away the medium; 10 minutes after 4% paraformaldehyde fixation, 0.1% Triton-x100 for 5 minutes; then add DAPI staining, use laser confocal microscope to observe antigen-presenting cells for MUC1 polypeptide and Uptake efficiency of targeting fusion peptide NP-MUC1. Experimental results such as Figure 4 As shown, the results indicated that the uptake efficiency of the fusion peptide NP-MUC1 by antigen-presenting cells was significantly higher than tha...

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Abstract

The invention discloses a dendritic cell targeted peptide, a coding gene and an application. The dendritic cell targeted peptide is named NP. The amino acid sequence of the dendritic cell targeted peptide is shown in SEQ ID NO.1 in a sequence table. One efficiently targeted dendritic cell targeted peptide is screened out of numerous short peptides by using a dendritic cell as the target cell and reducing and screening phage random peptide libraries in a subtractive manner. The antigen presenting capacity of the dendritic cell and the capacity of the dendritic cell in killing most tumors, such as cervical cancers and breast cancers are obviously improved through fusion of the dendritic cell targeted peptide and a tumor-associated antigen MUC1. The dendritic cell targeted peptide has wide application value in diagnosis and treatment of most cancers.

Description

technical field [0001] The invention relates to a dendritic cell targeting peptide, a coding gene, a fusion peptide containing the dendritic cell targeting peptide, a recombinant expression vector, a transgenic cell line and applications. Background technique [0002] Cancer is one of the main causes of death in the world. In 2007, the number of people who died of cancer in the world reached 7.9 million, accounting for about 13% of all deaths, and the incidence rate is increasing year by year. Its treatment is mainly based on surgery, chemotherapy and radiotherapy, but the side effects of radiotherapy and chemotherapy are large, and the curative effect is not very satisfactory. Currently, immune cell therapy is becoming a new approach to cancer treatment. [0003] With the rapid development of basic disciplines such as molecular biology and immunology, the research on tumors has become increasingly in-depth. Especially in the past decade, a variety of tumor antigens closely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K19/00C12N15/11A61K47/42A61P35/00
Inventor 曹捷杨姗姗陈红波
Owner SHENZHEN TONGKANG BIOLOGICAL PHARMA
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