Preparation method and application of exosome for expressing ACE2 protein

An exosome and protein technology, applied in the medical field, can solve the problems of inability to use on a large scale and limited plasma sources.

Active Publication Date: 2021-03-02
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to small molecule drugs, plasma therapy is good, but it has certain risks, because plasma is a complex mixture that varies from person to person, and the source of plasma is limited, so it cannot be used on a large scale

Method used

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  • Preparation method and application of exosome for expressing ACE2 protein
  • Preparation method and application of exosome for expressing ACE2 protein
  • Preparation method and application of exosome for expressing ACE2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Extraction and identification of hMSC-derived exosomes from human bone marrow mesenchymal stem cells stably expressing ACE2 protein.

[0039] 1. Construction of hMSC human bone marrow mesenchymal stem cell line stably expressing ACE2

[0040] (1) Transfer pVSV, pGAG, pREV and the constructed pLV-puro plasmid containing the human ACE2 gene into HEK293T cells with a density of 70% in a 10cm cell culture dish by polyethyleneimine PEI transfection system, 8 After ~12 hours, replace 10 mL of fresh DMEM medium and culture in a 37°C constant temperature CO2 incubator for more than 48 hours. During this period, the virus packaging system will assemble in the cells to form lentiviral particles containing the ACE2 genome and release them into the cell culture medium.

[0041] (2) Transfer the virus suspension, that is, the cell culture medium, to a 15mL centrifuge tube, centrifuge at 1500rpm at room temperature for 3-5min, carefully absorb the supernatant and transfer to another ...

Embodiment 2

[0052] The neutralization effect of the exosomes enriched in human ACE2 extracted in Example 1 on the new coronavirus was detected.

[0053] In this embodiment, pseudoviruses (pseudoviruses), also known as "pseudoviruses", are a type of chimeric virus particles that express recombinant sugars of another virus on the surface of a replication-defective virus (viral vector). protein chimeric virus particles. Pseudoviruses are recommended as positive quality controls for viral nucleic acid detection kits.

[0054] 1. Construction of HEK293T human embryonic kidney cell line stably expressing ACE2

[0055] (1) Transfer pVSV, pGAG, pREV and the constructed pLV-puro plasmid containing the human ACE2 gene into HEK293T cells with a density of 70% in a 10cm cell culture dish by polyethyleneimine PEI transfection system, 8 After ~12h, change 10mL of fresh DMEM medium, and keep the temperature at 37°C in CO 2 Cultivate in the incubator for more than 48 hours, during which the virus pack...

Embodiment 3

[0066] The neutralization effect of the exosomes enriched in human ACE2 extracted in Example 1 on the new coronavirus was detected.

[0067] (1) Spread VERO E6 Vero E6 kidney cells in a 96-well plate at a rate of 10,000 per well.

[0068] (2) After the cells adhere to the wall the next day, mix 40 μl of exosomes of different concentrations with 40 μl of the new coronavirus containing 100 times the 50% tissue cell infection amount (TCID50), and place it at 37°C for 2 hours, and then mix the Solution was added to VERO E6 cells, and each group was repeated for 3 wells.

[0069] (3) Continue to culture for about 5 days, and analyze and evaluate the neutralization ability of exosomes enriched in human ACE2 to the new coronavirus by observing the survival of VERO E6 African green monkey kidney cells. See Figure 6, Figure 6 ELISA results of exosomes expressing ACE2 protein in the new crown neutralizing antibody detection kit. like Figure 6 As shown, the exosomes in the control ...

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Abstract

The invention discloses a preparation method of an exosome for expressing ACE2 protein. The preparation method comprises the following steps: constructing an hMSC human mesenchymal stem cell line forstably expressing human ACE2 protein; and extracting exosome rich in ACE2 protein from a culture supernatant of an hMSC human bone marrow mesenchymal stem cell line through an ultracentrifugation method. The exosome can neutralize new coronavirus, and has treatment and prevention potential on the novel coronavirus. Therefore, the exosome for expressing the ACE2 protein, which is prepared by the method, can be applied to the preparation of anti-novel coronavirus SARS-CoV2 medicines.

Description

technical field [0001] The invention belongs to the field of medical technology, and relates to a preparation method and application of exosomes expressing ACE2 protein. Background technique [0002] The novel coronavirus belongs to the genus Coronavirus, and its basic structure consists of M protein, S protein, N protein, E protein, genomic single-stranded RNA and envelope. Studies have shown that the mechanism of SARS-CoV-2 infecting the human body is similar to that of the S protein, which combines with the ACE2 protein of human cells to mediate the virus infecting the host. [0003] Angiotensin-converting enzyme 2 (ACE2) is a homologue of carboxypeptidase ACE. Under physiological conditions, ACE converts angiotensin I (Ang I) to angiotensin II (Ang II), thereby activating renin-vascular Tensin system (RAS), and ACE2, as a negative regulator of RAS, negatively regulates the activation of RAS by cutting and inactivating Ang II, thereby regulating the homeostasis of the bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/57C12N15/867A61K35/28A61P31/14
CPCC12N5/0663C12N9/485C12N15/86A61K35/28A61P31/14C12Y304/17023C12N2510/00C12N2740/15043Y02A50/30
Inventor 谢枫周芳芳张龙
Owner SUZHOU UNIV
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