Nanoparticle-rhACE-2 compound for blocking coronavirus from infecting target cells, preparation method and application of nanoparticle-rhACE-2 compound

A -rhace-2, nanoparticle technology, applied in the field of medicine, to achieve strong anti-degradation ability, high safety, and the effect of inhibiting virus infection

Inactive Publication Date: 2021-11-05
南京纳科生物材料有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in a study of soluble ACE-2 protein (hrsACE-2), it was found that it can inhibit the infection of SARS-CoV-2 on Vero 6 cells, but this study still needs further

Method used

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  • Nanoparticle-rhACE-2 compound for blocking coronavirus from infecting target cells, preparation method and application of nanoparticle-rhACE-2 compound
  • Nanoparticle-rhACE-2 compound for blocking coronavirus from infecting target cells, preparation method and application of nanoparticle-rhACE-2 compound
  • Nanoparticle-rhACE-2 compound for blocking coronavirus from infecting target cells, preparation method and application of nanoparticle-rhACE-2 compound

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0037] Embodiment 1 plasmid construction

[0038] Artificially synthesized human angiotensin-converting enzyme 2 (GenBank: AB046569.1) corresponding to the nucleic acid sequence from 18 to 740 amino acids, expressed His tag protein at the C-terminus for purification, and expressed Avi tag behind the His tag for Biotin labeling. The specific steps are as follows: 100 ng of PCR product; 100 ng of Topo carrier; 1 ml of BP clonase; TEbuffer to 5 μl; Then, the above ligation product was transformed into Escherichia coli competent cells: the specific steps were: the volume of competent cells was 100 μl, adding 2 μl of the above reaction product, incubating on ice for 30 min, and heat shocking at 42°C for 90 sec. Incubate on ice for 2 min. Add 250 μl of SOC medium. Shake the bacteria at 225 rpm for 1 h at 37°C, and spread 100 μl of the product onto an LB plate. Incubate overnight at 37°C. Pick cloned colonies for PCR identification, and shake positive clones for plasmid extracti...

Embodiment 2

[0040] Example 2 Packaging produces pseudoviruses of the new coronavirus and protein purification

[0041] The day before will be about 4*10 7Cells were plated in a 10cm cell culture dish and prepared for toxin production. Use lipofectamine-2000 to transfect the plasmid packaging virus. According to the instructions, the vector expression plasmid (pLV-CMV-Luciferase), integration plasmid (pSPAX2) and envelope plasmid (PRP-CMV-human-beta-globin-intron-S) were added to 4ug :2ug:1ug was added to 1ml Opti-MEM, and 12ul lipofectamine-2000 was added to another tube of 1ml Opti-MEM at the same time, the two were mixed and left at room temperature for 15 minutes, then added dropwise to the prepared 293T cells After 8 hours, change the fresh medium, collect the supernatant after 48 hours, centrifuge at 3000g for 30 minutes, discard the precipitate, the supernatant is the virus solution, and store at -80°C. stand-by. ACE-2 purification: the expression vector pRP-EGFP-hACE-2[18-740aa]...

Embodiment 3

[0042] Example 3 Detection of the toxicity of different concentrations of nanoparticles and soluble ACE-2 to cells

[0043] The streptavidin-coated nanoparticles were purchased from sigma company, invitrogen company, and Xi'an Ruixi Biomaterials Co., Ltd., and the streptavidin-modified soluble nanomicelles were prepared by our laboratory. The specific steps were as follows: firstly, Commercially purchased streptavidin-labeled distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000-streptavidin) was placed at room temperature for 30 minutes, then 5 mg was weighed and dissolved in 1 ml DMSO until fully dissolved Finally, add it dropwise to 2ml of ultrapure water, stir while adding, stir evenly, put it in a 500WM dialysis bag for dialysis for 6 hours, then take out the dialysis bag, and then use the probe to sonicate for 1 minute and 1 minute, the product is Streptavidin-modified nanomicelles, stored at 4°C until use. Spread 1X104 wells of 293T cells into a 96-...

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Abstract

The invention discloses a nanoparticle-rhACE-2 compound for blocking coronavirus from infecting target cells, a preparation method and application of the nanoparticle-rhACE-2 compound. The preparation method comprises the following steps: S1, preparing and purifying rhACE-2 protein; S2, labelling the rhACE-2 protein with biotin; and S3, preparing the nanoparticle-rhACE-2 compound: washing nanoparticles with PBS for three times, specifically, washing the nanoparticles with 1ml of PBS for the first time, washing the nanoparticles with PBS with the same amount as a sample for the later two times, adding the soluble rhACE-2 protein in the S2 according to different molar ratios, performing incubating for 30 minutes at 37 DEG C to enable the rhACE-2 to be effectively combined to the surfaces of the nanoparticles, and then performing washing with PBS for three times, wherein the product at the moment is the nanoparticle-rhACE-2 compound. According to the invention, a coronavirus receptor angiotensin converting enzyme 2 is used as an antagonist; and the nanoparticles are used as a carrier to block and neutralize virus particles, so that the infection of viruses to target cells is inhibited.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a nanoparticle-rhACE-2 complex for blocking coronavirus infection of target cells, a preparation method and application thereof. Background technique [0002] Coronaviruses can be divided into 4 genera of α, β, γ, and δ. In addition to the recent outbreak of SARS-CoV-2, there are currently 7 types of coronaviruses that are infectious to humans, and the other 6 are HCoV- 229E and HCoV-NL63; beta HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV, the latter two of which can cause more severe clinical symptoms. SARS-CoV-2 is a β-coronavirus, which is an unsegmented single positive-strand RNA virus. According to the genome structure, the complete sequence homology of SARS-CoV-2 and SARS genome is about 79.5% and both Both use ACE-2 as the main receptor. The novel coronavirus (SARS-CoV-2) caused by the novel coronavirus (SARS-CoV-2) is still spreading globally, and the novel ...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/65A61K47/54A61K38/48C07K19/00C12N15/85A61P31/14B82Y5/00B82Y40/00
CPCA61K47/6923A61K47/65A61K47/545A61K38/4813A61P31/14C12N9/485C12N15/85C12Y304/17023B82Y5/00B82Y40/00C07K2319/21
Inventor 褚玲玲
Owner 南京纳科生物材料有限公司
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