A Rapid Drug Screening Method Based on CD47/Sirpα Blocking Function and Its Biological Effects

A screening method and biological effect technology, applied in biochemical equipment and methods, botany equipment and methods, microbial measurement/inspection, etc., can solve problems such as fast, efficient and accurate screening systems for new drugs that have not yet been seen, and achieve low cost effect

Active Publication Date: 2022-04-29
杭州科兴生物科技有限公司
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no rapid, efficient and accurate screening system for new drugs based on CD47 / SIRPα blocking function and its biological effects

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
  • A Rapid Drug Screening Method Based on CD47/Sirpα Blocking Function and Its Biological Effects
  • A Rapid Drug Screening Method Based on CD47/Sirpα Blocking Function and Its Biological Effects
  • A Rapid Drug Screening Method Based on CD47/Sirpα Blocking Function and Its Biological Effects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of recombinant CD47-tagRFP fusion protein

[0052]The red fluorescent protein tagRFP gene and the human full-length CD47 gene were constructed in the same reading frame, so that the red fluorescent protein tagRFP was connected to the C-terminal of the CD47 protein to form a CD47-tagRFP fusion protein, and at the same time, 6 histidines were connected to the C-terminal of the tagRFP His tag. Since the binding site of CD47 and SIRPα is at the N-terminal of the extracellular domain, tagRFP is connected to the C-terminal of the intracellular domain of CD47, which does not affect the binding of CD47 and SIRPα. The restriction endonucleases EcoRI (GAATTC) and SexAI (ACCTGGT) at the front and rear ends were used for gene synthesis and then connected to the downstream of the CMV promoter of the lentiviral expression vector pLV-Puro to obtain the plasmid pCMV-CD47tagRFP( figure 1 ), the PGK promoter and Puro resistance gene in the original plasmid are remo...

Embodiment 2

[0065] Example 2 Construction of plasmid pCMV-SIRPαCyPet

[0066] The cyan fluorescent protein CyPet gene was constructed downstream of the SIRPα protein, and after gene synthesis, EcoRI (GAATTC) and SexAI (ACCTGGT) were used to connect to the downstream of the CMV promoter of the lentiviral expression vector pLV-Puro to obtain the plasmid pCMV-SIRPαCyPet (such as figure 2 shown). The DNA gene sequence of the SIRPαCyPet fusion protein is shown in SEQ ID NO:3, and the amino acid sequence of the expressed SIRPαCyPet fusion protein is shown in SEQ ID NO:4.

Embodiment 3

[0067] Example 3 pCMV-SH2 PTPN11 Construction of Ypet plasmid

[0068] The yellow fluorescent protein gene Ypet was passed through a connecting peptide (G 4 S) 3 Construct the downstream of the two SH2 domain genes at the N-terminal of the PTPN11 protein, and clone (EcoR I / SexA I) into the downstream of the CMV promoter of the lentiviral expression vector pLV-Puro to obtain the plasmid pCMV-SH2 PTPN11 Ypet (such as image 3 shown). The DNA gene sequence of the fusion protein is shown in SEQ ID NO:5, after expression SH2 PTPN11 The sequence of the Ypet fusion protein is shown in SEQ ID NO:6.

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

PropertyMeasurementUnit
fluorescenceaaaaaaaaaa
fluorescenceaaaaaaaaaa
fluorescenceaaaaaaaaaa
Login to View More

Abstract

The present invention establishes a rapid drug screening method based on CD47 / SIRPα blocking function and its biological effects: red fluorescent protein RFP is coupled to the carboxyl terminal (C-terminal) of CD47 protein; cyan fluorescent protein gene CyPet is constructed on SIRPα protein downstream of PTPN11 gene, and the yellow fluorescent protein gene Ypet was constructed downstream of the PTPN11 gene through a connecting peptide (G4S)3, and eukaryotic expression vectors were constructed respectively, and a new stable transfection cell line SIRPαCyPet.SH2 was obtained PTPN11 Ypet / THP‑1, which uses the affinity of CD47‑RFP to cell lines for fluorescence detection to screen blockers. This method can accurately reflect the effects of anti-SIRPα and anti-CD47 drugs, can simultaneously obtain blocking function and biological effect data of CD47 / SIRPα signaling pathway, and construct an experimental model for rapid screening of anti-CD47 / SIRPα drugs and evaluation of their biological effects.

Description

technical field [0001] The invention relates to a rapid drug screening method based on the CD47 / SIRPα target, belonging to the field of biotechnology. Background technique [0002] CD47 (Cluster of Differentiαtion 47, Cluster of Differentiation 47), also known as integrin-associated protein (IAP), is a transmembrane protein. CD47 belongs to the immunoglobulin superfamily and can bind the ligands thrombospondin-1 (TSP-1) and signal regulatory protein α (SIRPα, Signal Regulatory Protein α, signal regulatory protein α). SIRPα is a regulatory membrane glycoprotein mainly expressed in myeloid cells, stem cells or neurons. The intracellular domain of SIRPα contains 4 ITIMs (Immunoreceptor tyrosine-based inhibitory motifs, immunoreceptor tyrosine-based inhibitory domain), after the ectodomain of SIRPα binds to CD47, it transmits a signal to the cell to phosphorylate ITIMs, and then SHP phosphorylates Enzymes, such as PTPN11 (PTPN11, SHP2: Tyrosine-protein phosphatase non-receptor...

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 & AuthorityPatents(China)
IPC IPC(8): C12Q1/02C12N15/62C12N15/867C12N5/10C07K19/00G01N21/64
CPCG01N21/6428G01N33/5047C12N9/16C12N15/86C07K14/70503C07K14/70596C12N2510/00C12N2740/15043C07K2319/60C07K2319/21
Inventor范春雷吴王亲武虎匡红刘美星莫一平
Owner杭州科兴生物科技有限公司