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Polypeptide having immune cell target recognition function and use thereof

An immune cell, targeted recognition technology, applied in general/multifunctional contrast agents, preparations for in vivo experiments, and analysis by chemical reaction of materials, etc., can solve the problem of no uniform quality standard, uncontrollable antibody quality, Cumbersome preparation and production steps

Active Publication Date: 2020-04-21
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, when the fluorescent staining and labeling of living monocytes / macrophages is carried out through the principle of antigen-antibody combination, there are still problems such as short fluorescence maintenance time.

Method used

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  • Polypeptide having immune cell target recognition function and use thereof
  • Polypeptide having immune cell target recognition function and use thereof
  • Polypeptide having immune cell target recognition function and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] The preparation of embodiment 1.EK24 fluorescent probe

[0078] 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 used in the present invention is derived from the C-terminus of human or non-human Triokinase / FMN cyclase.

[0079] In this example, a polypeptide having the following amino acid sequence was synthesized: EQPDPGAVAAAAILRAILEVLQSK.

[0080] 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:

[0081] EQPDPGAVAAAAILRAILEVLQS-Y(EK24),

...

Embodiment 2

[0083] The preparation of embodiment 2.EK24 fluorescent probe solution

[0084] 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

[0085] Example 3. Fluorescence-labeled staining of mouse macrophage cell lines cultured in vitro

[0086] 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 culture medium containing 10% FBS and 1% streptomycin-penicillin, add nuclear dye Nucblue (Invitrogen) for counterstaining, in Observe under FL Auto fluorescent microscope (LifeTechnologies, light source 10%, exposure 200ms, gain 5), the result is as follows figure 2 shown.

[0087] figure 2 Shown are images of fluorescently labeled EK24 fluorescent probes (shown as red fluorescence in the origina...

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Abstract

Disclosed is a polypeptide that recognizes immune cells, the polypeptide comprising the following amino acid sequence: (a) an amino acid sequence containing C terminal fragment sequence EQPDPGAVAAAAILRAILE of human Triokinase / FMN cyclase and the homologous sequence thereof; or (b) an amino acid sequence in which the amino acid sequence is substantially the same as described in (a), the substantially the same referred to amino acid sequence having at least 80% sequence identity to the amino acid sequence as described above in (a). Also disclosed in the present invention are a nucleotide sequence encoding the polypeptide; a polypeptide probe that targets recognized immune cells, comprises the polypeptide as described above and has a reporter group; a kit comprising the above-mentioned probe;and the use of the polypeptide or probe as described above in the preparation of the kit for mammalian living cell imaging.

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, a probe comprising the polypeptide, a kit, and an immunolabeling method thereof. Background technique [0002] Immune cells are cells that mediate the body's immune response and include many different types of cells. These cells circulate through the blood and lymphatic systems and are recruited to sites of tissue injury and infection. Different types of immune cells are distinguished by their function and physical characteristics, but immune cells all arise from the same origin in hematopoietic stem cells and develop in different ways in response to internal and external signals. The classic immunological view holds that the mononuclear phagocytic system is a myeloid cell population derived from bone marrow, in which monocytes circulate in the blood, bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/88A61K49/00G01N21/64G01N33/569
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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