Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Enzymes and methods for labeling cell membrane surfaces and studying cell-cell interactions

A cell and labeling technology, applied in the field of bioengineering, can solve the problem of low reactivity

Active Publication Date: 2021-07-09
PEKING UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, sortase A can also catalyze the reaction between LPXTG and α-Gly, however, compared to its catalyzed α-Gly n reaction, the reactivity is lower

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Enzymes and methods for labeling cell membrane surfaces and studying cell-cell interactions
  • Enzymes and methods for labeling cell membrane surfaces and studying cell-cell interactions
  • Enzymes and methods for labeling cell membrane surfaces and studying cell-cell interactions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1, Cell Membrane Labeling

[0038] The eGFP-LPETG and the Staphylococcus aureus sortase A mutant involved in the present invention were simultaneously expressed on the pet28a vector by a cloning method, and the protein was purified using a 6×His tag. Cell membrane labeling reaction was performed in PBS buffer or Tris buffer (30mM Tris, 150mM NaCl, 5mM CaCl 2 , pH 7.4), the reaction conditions are as follows: 20 μM mgSrtA, 20 μM eGFP-LPETG, room temperature for 1 hour, cell density 2×10 7 cells / mL. After the reaction was completed, the buffer was used to wash three times, and then the flow cytometer was used for characterization. The result is as figure 2 As shown, it can be seen that the fluorescence intensity of eGFP in cells is stronger after adding mgSrtA (+mgSrtA) compared with adding only eGFP-LPETG (-mgSrtA), which proves that eGFP-LPETG is labeled on the cell surface. The experimental results of the six mutants shown in SEQ ID No: 1 to 6 in the seque...

Embodiment 2

[0039] Example 2. Recording and detection of model HER2 positive cell-cell interaction

[0040] First, ZHER and / or mgSrtA were displayed on the surface of HEK293T cells by genetic modification, and eFluor670 was labeled as a signal for later sorting. MDA-MB-231 cells were stably transfected with HER2 receptor (human epidermal growth factor receptor-2) by lentiviral transfection as Her2 positive cells (Her2+ cells). The above two kinds of cells (cell density 2×10 7 cells / mL) were co-incubated in PBS buffer at room temperature for 30 minutes, and 100 μM biotin-LPETG was added for labeling, and MDA-MB-231 cells themselves were Her2-negative cells (Her2-cells) as a control. After labeling, wash with PBS three times, then incubate with Streptavidin PE on ice for 15 min, and finally use flow cytometry to characterize.

[0041] In this experiment, Her2-positive cells interacted with cells expressing the Her2 ligand ZHER, allowing mgSrtA to label biotin on the surface of Her2-positi...

Embodiment 3

[0042] Example 3. Detection of model Raji cell-cell interaction

[0043] First, CD40L (or CD40L* or CTLA4) and mgSrtA are displayed on the surface of HEK293T cells by genetic modification, and cells expressing CD40L or CTLA4 can induce the interaction between HEK293T and Raji cells by pairing with CD40 or B7 on the surface of Raji cells , CD40L* is a mutant of CD40L that does not interact with CD40L and serves as a negative control. The above-mentioned HEK293T cells expressing different ligands were co-incubated with Raji cells (Raji cells were pre-labeled with eFluor670) in PBS buffer at room temperature for 30 minutes, and at the same time, 100 μM biotin-LPETG was added for labeling. After labeling, wash with PBS three times, then incubate with Streptavidin PE on ice for 15 min, and finally use flow cytometry to characterize.

[0044] In this experiment, the interaction between cells expressing CD40L, CD40L* or CTLA4 and Raji cells expressing CD40 and B7 allowed mgSrtA to l...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an enzyme and a method for marking the surface of cell membrane and studying cell-cell interaction, and relates to a mutant of Staphylococcus aureus sortase A evolved through genetic engineering, which can realize labeling mediated by proximity effect Based on this reaction, the labeling of non-genetically modified cell membrane surfaces can be achieved, and cell-cell interactions can be accurately captured and labeled in vivo and in vitro.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an enzymatic labeling technique, in particular to an enzymatic cell membrane labeling method, and a method for researching and discovering cell-cell interactions using enzymatic reactions. Background technique [0002] Cell membrane labeling is an important means to study cell function. Usually, cell membrane labeling needs to be genetically modified, which greatly limits the application of cell membrane labeling technology. In addition, cell-cell interactions are crucial to life processes, mediating many important life processes. However, it is relatively difficult to discover cell-cell interactions, so the development of technologies that can study / detect cell-cell interactions is of great significance for revealing some signal transduction in life processes. [0003] Staphylococcus aureus sortase A (Sa-SrtA) is a class of cysteine ​​transpeptidase ubiquitous in Gram-positive bac...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48C12Q1/02C12N9/10
Inventor 陈鹏陈龙葛韵刘士博
Owner PEKING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com