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Polypeptide with immune cell targeted recognition function and application thereof

A technology of immune cells and macrophages, applied in peptides and related application fields, can solve the problems of uncontrollable antibody quality, no uniform quality standard, cumbersome preparation and production steps, etc.

Active Publication Date: 2020-03-17
KEYANGLE LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since antibodies are macromolecular proteins, their preparation and production steps are cumbersome
During this process, there are many factors that affect the activity of the antibody, making the quality of the antibody uncontrollable, and the quality fluctuates greatly between batches.
There is no uniform standard for the quality of the same antibody sold by different manufacturers
Moreover, the fluorescent labeling of macrophages by the principle of antigen-antibody combination still has problems such as short fluorescence maintenance time during in vivo staining.

Method used

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  • Polypeptide with immune cell targeted recognition function and application thereof
  • Polypeptide with immune cell targeted recognition function and application thereof
  • Polypeptide with immune cell targeted recognition function and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The preparation of embodiment 1.EK24 fluorescent probe

[0066] The polypeptide used in the present invention is obtained by conventional solid-phase polypeptide chemical synthesis using a CEM automatic microwave polypeptide synthesizer according to the instructions provided by the instrument supplier. The polypeptide probe used in the present invention is from the C-terminus of human or non-human Triokinase / FMN cyclase.

[0067] In this example, a polypeptide having the following amino acid sequence was synthesized: EQPDPGAVAAAAILRAILEVLQSK.

[0068] Combine the above synthetic peptides with HOOK TM Dye rhodamine labeling kit (Cat. #786-142, Biosciences) was mixed with reagents, adjusted the pH value according to the manufacturer's instructions, and reacted for 1-2 hours. The product was purified by HPLC to obtain the target EK24 fluorescent probe. The chemical structure of the obtained fluorescent probe is as follows:

[0069] EQPDPGAVAAAAILRAILEVLQS-Y(EK24),

[...

Embodiment 2

[0071] The preparation of embodiment 2.EK24 fluorescent probe solution

[0072] Add 87 μL of DMSO (dimethyl sulfoxide) to dissolve 1 mg of EK24 fluorescent probe, then add 6873 μL of serum-free DMEM (Hyclone) incomplete culture solution, and pipette thoroughly to obtain a 50 μM EK24 fluorescent probe solution. When needed, the 50 μM EK24 fluorescent probe solution can be diluted by adding serum-free DMEM incomplete culture solution.

Embodiment 3

[0073] Example 3. Fluorescence-labeled staining of mouse macrophage cell lines cultured in vitro

[0074] Cultivate the mouse macrophage cell line RAW264.7 in a 96-well plate, wash the well once with 1×PBS buffer or DMEM incomplete culture solution without serum, remove the washing solution, add 100 μL DMEM without serum The EK24 solution with a concentration of 10 μM diluted in the complete culture solution was left in a cell culture incubator for 1 hour. Afterwards, the wells were washed twice with 1×PBS buffer or incomplete DMEM medium without serum. Then, add DMEM complete medium containing 10% FBS and 1% streptomycin-penicillin, Observe under FL Auto fluorescent microscope (Life Technologies, light source 10%, exposure 200ms, gain 5), the result is as follows figure 2 shown.

[0075] from figure 2 It can be seen that the macrophage cell line RAW264.7 can be labeled by the EK24 fluorescent probe (shown as red fluorescence in the original fluorescent color map).

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Abstract

The invention discloses a polypeptide for recognizing immune cells. The polypeptide comprises the following amino acid sequences: (a) an amino acid sequence of a C-terminal fragment sequence EQPDPGAVAAAAILRAILE containing human Triokinase / FMN cyclase; or (b) an amino acid sequence which is basically the same as the amino acid sequence in (a), and the basically the same means that the amino acid sequence in (a) has more than 80% of identity with that in (b). The invention also discloses a nucleic acid sequence for encoding the polypeptide; the polypeptide for targeted recognition of immune cells comprising the polypeptide and a polypeptide probe with a reporter group; a kit comprising the probe; and an application of the polypeptide or the probe in preparation of the kit for imaging livingcells of mammals.

Description

technical field [0001] The invention belongs to the field of molecular biology and cell detection and imaging. Specifically, the present invention relates to a polypeptide capable of targeting and recognizing immune cells and its related applications. Background technique [0002] Monocytes / macrophages originate in the bone marrow and circulate throughout the mammalian body through blood and lymph. After leaving blood vessels, monocytes / macrophages can enter almost all tissues of mammals, with strong plasticity and rich functional diversity. These cells play a variety of roles in normal tissue development, homeostasis, tissue repair, and immune responses to pathogens. Mature monocytes / macrophages have strong mobility in the body. In related tissues, after monocytes / macrophages phagocytize bacteria, pathogens and dead cells, they can enter lymph nodes or lymph organs such as the spleen through nearby lymphatic vessels Present the antigen of the phagocytosis to other lympho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54A61K49/00
CPCC12N9/1205A61K49/0056C12N9/88C12Y207/01029C12Y406/01015A61K49/0002G01N21/6428G01N21/6458G01N33/56966G01N2021/6439G01N2333/91215C12Y207/01028A61K49/0041G01N33/56972G01N21/76
Inventor 魏远安刘学术
Owner KEYANGLE LIFE TECH CO LTD
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