Polypeptide for target identification of immunocyte and application thereof

An immune cell, targeted recognition technology, applied in applications, medical preparations with non-active ingredients, preparations for in vivo tests, etc. question

Active Publication Date: 2020-02-07
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 for target identification of immunocyte and application thereof
  • Polypeptide for target identification of immunocyte and application thereof
  • Polypeptide for target identification of immunocyte and application thereof

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 synthesized above polypeptide 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...

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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PUM

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Abstract

The invention discloses polypeptide for target identification of immunocyte. The polypeptide comprises following amino acid sequences: (a) an amino acid sequence of a C terminal fragment sequence AILEVLQS containing human Triokinase/FMN cyclase of and a homologous sequence of the amino acid sequence; or (b) an amino acid sequence which is basically the same as the amino acid sequence as shown in (a), wherein the basic same meaning lies in that the amino acid sequence in (a) has 70% or above of sequence identity. The invention further discloses a nucleic acid sequence for coding the polypeptide, a polypeptide probe for target identification of immunocyte, containing the polypeptide and with a reported group, and a reagent kit comprising the probe, and applications of the polypeptide or theprobe.

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 Applications(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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