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Coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competitive chromatography

A coronavirus and protein ligand technology, applied in the direction of retroRNA virus, virus, biological testing, etc., can solve the problems of long research and development cycle and cannot use epidemic detection in time, so as to ensure biological safety, improve detection sensitivity, and emit peak narrow effect

Active Publication Date: 2020-06-12
浙江诺迦生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for quicker and more sensitive testing than current methods like ELISA due to its ability to detect different types of virions without requiring expensive equipment. Additionally, this method provides multiple benefits including increased efficiency when analyzed at low concentrations compared to traditional techniques (ELISA), enhanced accuracy during diagnoses based on genetic material from infected individuals, reduced risk of false negative results caused by non-virus particles, and better control over sample size. Overall, these technical improvements make the test faster and easier to perform while maintaining high levels of precision.

Problems solved by technology

Technics: There were technical problem addressed by this patented study relating to detecting coronavirus like COVID 19. Current methods involve testing samples from individuals or animals suspected of being sick, which takes too much effort and requires specialist instruments. Colloid gold assay tests provide high sensitivity but may result in delayed diagnosis due to poor reproduction rates caused by previous exposure.

Method used

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  • Coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competitive chromatography
  • Coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competitive chromatography
  • Coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competitive chromatography

Examples

Experimental program
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Embodiment 1

[0033] Example 1 Construction of pCDH-ACE2mFc.copGFP plasmid

[0034] The C-terminal sequence of the human ACE2 ectodomain gene is connected with a protein-directed biotinylation sequence, a specific protease cleavage site tag and a purification tag to form an artificially designed sequence, wherein the human ACE2 ectodomain gene is the cell membrane of human ACE2 The outer part, that is, the coding gene sequence of the 1-739aa part of GenBank: AB046569.1; the protein-directed biotinylation sequence is biotin protein ligase BirA, and its recognition site sequence is GSGGSGGSAGGGLNDIFEAQKIEW; the specific protease is EK The enzyme, the cleavage site tag is a Flag tag; the purification tag is a mouse IgG Fc segment (mFc). Finally, the nucleic acid sequence of the genetically designed ACE2-Flag-mFc fusion protein is obtained. After host (human) codon optimization, the sequence is as shown in SEQ ID NO: 1. EcoR I and NotI restriction endonucleases are designed at both ends, and th...

Embodiment 2

[0038] Example 2 Establishment of ACE2mFc.copGFP / 293 Stably Transduced Cell Line

[0039] The pCDH-ACE2mFc.copGFP plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into the lentiviral packaging line cell 293V to prepare ACE2mFc.copGFP lentivirus, and transfected into HEK293 cells. The clones were picked under a fluorescent microscope to establish ACE2mFc.copGFP / 293 stable cells. transfected cell lines. Specific steps are as follows:

[0040] 1) The day before the experiment, five 15cm dishes were plated to ensure that the cells reached 70%-80% confluence before transfection.

[0041] 2) 1-2 hours before transfection, replace the medium in the dish with serum-free and antibiotic-free DMEM medium.

[0042] 3) Prepare a 15mL centrifuge tube, add 5mL 1×HBS, then add 100μg pCDH-ACE2mFc.copGFP plasmid, 100μg PH1 / PH2 mixed plasmid (PH1:PH2=3:1, mass ratio), and mix gently .

[0043] 4) Add 4mL PEI working solution (10μM), mix gently, and incubate at 37°C for 20min.

[00...

Embodiment 3A

[0054] Embodiment 3 ACE2-Biotin protein preparation

[0055] Use protein-free 293 cell culture medium to amplify and culture ACE2mFc.copGFP / 293 stably transfected cells, collect supernatant culture fluid, and use proteinA / G column to purify ACE2mFc fusion protein, use EK enzyme to cut ACE2 from the column, pass through biotin protein The ligase couples biotin to the (GSGGSGGSAGGGLNDIFEAQKIEW) site of ACE2 to obtain ACE2-Biotin protein. Specific steps are as follows:

[0056] 1) Expand the ACE2omFc.copGFP / 293 stably transfected cells into a five-layer cell factory, and culture them with HektorHEK293 cell culture medium without proteins and peptides;

[0057] 2) Collect the culture medium, centrifuge at 1200g, 4°C for 20min, take the supernatant, and use saturated ammonium sulfate method to precipitate protein;

[0058] 3) Precipitated protein was reconstituted with PBS (pH7.4) of 1 / 10-1 / 100 volume of the original solution, desalted, concentrated, and replaced with binding / was...

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Abstract

The invention discloses a coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competitive chromatography. The coronavirus rapid detection kit comprises quantum dot labeled ACE2protein, quantum dot labeled rabbit IgG, recombinant coronavirus spinous process protein S1, goat anti-rabbit IgG polyclonal antibody, an immunochromatographic test strip and other materials. The detection sensitivity is improved through quantum dot fluorescence labeling and multistage coupling amplification signals, the detection specificity is improved and the antibody research and developmentcycle is avoided by utilizing the ligand and receptor binding principle, the kit capable of rapidly detecting coronavirus is provided, and the biosafety in the detection process is guaranteed by establishing a virus inactivation system. The kit disclosed by the invention is suitable for detecting various biological samples and environmental samples such as oral mucosa liquid, respiratory tracts, whole blood, plasma, serum, excrement and the like, and can be applied to rapid detection of coronaviruses taking ACE2 as a receptor, such as SARS-CoV-19, SARS-CoV, HCoV-NL63 and the like.

Description

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Claims

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Application Information

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Owner 浙江诺迦生物科技有限公司
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