A kit for rapid detection of coronavirus based on s protein ligand and ace2 receptor competition chromatography

A coronavirus and protein ligand technology, applied in the direction of reverse transcription RNA virus, virus, biological testing, etc., can solve the problem of long research and development cycle and cannot use epidemic detection in time, and achieve the effect of improving detection specificity and detection sensitivity.

Active Publication Date: 2021-06-15
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

Problems solved by technology

The advantage of this type of reagent is that it is fast, simple, does not require equipment and professional operation, and the cost of reagents is low; but the disadvantage is that for new viruses, protein antigens and specific antibodies of new viruses must first be prepared, resulting in a long development cycle and cannot be used in an epidemic situation in time detection, and the detection accuracy of the kit is closely related to the quality of the protein antigen and specific antibody

Method used

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  • A kit for rapid detection of coronavirus based on s protein ligand and ace2 receptor competition chromatography
  • A kit for rapid detection of coronavirus based on s protein ligand and ace2 receptor competition chromatography
  • A kit for rapid detection of coronavirus based on s protein ligand and ace2 receptor competition chromatography

Examples

Experimental program
Comparison scheme
Effect test

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 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 / washing buf...

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Abstract

The invention discloses a coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competition chromatography, including quantum dot-labeled ACE2 protein, quantum dot-labeled rabbit IgG, recombinant coronavirus spike protein S1, Goat anti-rabbit IgG polyclonal antibodies, and immunochromatographic test strips and other materials. The present invention improves detection sensitivity through quantum dot fluorescent labeling and multi-stage coupling amplification signals, uses the principle of ligand-receptor binding to improve detection specificity and avoids the antibody development cycle, and provides a kit that can quickly detect coronaviruses. Establish a virus inactivation system to ensure biological safety during the detection process. The kit of the present invention is suitable for the detection of various biological samples and environmental samples such as oral mucosal fluid, respiratory tract, whole blood, plasma, serum, excrement, etc., and can be applied to SARS‑CoV‑19, SARS‑CoV, and HCoV‑NL63, etc. Rapid detection of coronaviruses using ACE2 as a receptor.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography. Background technique [0002] Coronaviruses belong to the genus Coronavirus of the family Coronaviridae in systematic classification. Viruses of the genus Coronavirus are positive-sense single-stranded RNA viruses with an envelope (envelope), with a diameter of about 80-120nm. Pangolins, dogs, wolves, chickens, cattle, snakes, birds and other vertebrates. The 2019 novel coronavirus (SARS-CoV-19, formerly known as 2019-nCoV) is the seventh known coronavirus that can infect humans, and the remaining six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Studies have shown that SARS-CoV-19, SARS-CoV and HCoV-NL63 infect humans through the viral envelope spike glycoprotein (S-protein) mediated interaction between the virus and the ACE2 recept...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/569G01N33/558G01N33/533C12N15/867C12N15/62
CPCC07K14/705C07K2319/00C07K2319/43C07K2319/50C12N15/86C12N2740/15043C12N2800/107C12N2800/22G01N33/533G01N33/558G01N33/56983G01N33/582G01N33/588G01N2333/165
Inventor 范春雷朱晓进程向荣
Owner 浙江诺迦生物科技有限公司
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