A peptide targeting and recognizing immune cells and its application

An immune cell, targeted recognition technology, applied in applications, medical preparations with non-active ingredients, preparations for in vivo testing, etc. problem, to achieve the effect of good target specificity, low immunogenicity and excellent selectivity

Active Publication Date: 2022-04-15
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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  • A peptide targeting and recognizing immune cells and its application
  • A peptide targeting and recognizing immune cells and its application
  • A peptide targeting and recognizing immune cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] The preparation of embodiment 1.AK9 fluorescent probe

[0094] 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.

[0095] In this example, a polypeptide having the following amino acid sequence was synthesized: AILEVLQSK.

[0096] 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 AK9 fluorescent probe. The chemical structure of the obtained fluorescent probe is as follows:

[0097] AILEVLQS-Y (AK9),

[0098] Wherein Y is lysine+fluo...

Embodiment 2

[0099] The preparation of embodiment 2.AK9 fluorescent probe solution

[0100] Add 177 μL DMSO (dimethyl sulfoxide) to dissolve 1 mg AK9 fluorescent probe, then add 13981 μL serum-free DMEM (Hyclone) incomplete culture solution, and pipette thoroughly to obtain a 50 μM AK9 fluorescent probe solution. When needed, the 50 μM AK9 fluorescent probe solution can be diluted by adding serum-free DMEM incomplete culture solution.

Embodiment 3

[0101] Example 3. In vivo fluorescent labeling of macrophages in the peritoneal cavity of mice using AK9 fluorescent probe

[0102] 300 μL of 50 μM AK9 fluorescent probe solution was diluted to 1 mL with serum-free DMEM incomplete culture medium, and then injected into the peritoneal cavity of Kunming mice aged 5-8 weeks. After 4 hours, 5 mL of 1×PBS was injected and rubbed. After 10 minutes, the mice were sacrificed by cervical dislocation. Cut the skin of the abdominal cavity of the mouse, pierce the abdominal wall muscle with a syringe, and extract the fluid in the abdominal cavity of the mouse. The extracted peritoneal fluid was centrifuged at 1000rpm for 5min and washed twice with 1×PBS. Apply the appropriate amount of cells to Alexa After counterstaining with 488 anti-mouse F4 / 80 antibody (Biolegend), add it to a 96-well plate, add nuclear dye Nucblue (Invitrogen) for counterstaining, and wait for the suspended cells to settle at the bottom of the plate, Observed un...

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Abstract

The present invention discloses a polypeptide for recognizing immune cells. The polypeptide includes the following amino acid sequence: (a) the amino acid sequence containing the C-terminal fragment sequence AILEVLQS of human Triokinase / FMN cyclase and its homologous sequence; or, (b) An amino acid sequence substantially identical to the amino acid sequence described in (a), said substantially identical means having a sequence identity of more than 70% with the amino acid sequence described in (a). The present invention also discloses a nucleic acid sequence encoding the polypeptide; a targeted recognition immune cell, including the polypeptide as described above and a polypeptide probe with a reporter group; a kit including the above probe; and, as above Related applications of the polypeptide or probe.

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 mostly 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,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/11A61K49/00A61K47/64
CPCC12N9/1205A61K49/0056A61K47/64C12Y207/01028
Inventor 魏远安刘学术
Owner KEYANGLE LIFE TECH CO LTD
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