ACE2 mutant as well as mutation method and application thereof

A technology of mutants and uses, applied in the field of ACE2 mutants and their mutations and applications, can solve the problems of blood pressure and blood sugar disorders in patients, and achieve the effect of avoiding myocardial fibrosis, avoiding RAAS disorders, and high affinity

Active Publication Date: 2022-06-28
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Injecting a large amount of ACE2 into the blood is likely to cause disturbance of the patient's blood pressure and blood sugar
More importantly, overexpression of ACE2 in the mouse heart leads to severe myocardial fibrosis (22) , with the risk of heart rupture (23,24)
These serious side effects are a serious obstacle to the clinical application of ACE2, and a problem that the industry is concerned about and does not want its positive and negative effects to coexist. So far, there is no solution

Method used

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  • ACE2 mutant as well as mutation method and application thereof
  • ACE2 mutant as well as mutation method and application thereof
  • ACE2 mutant as well as mutation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: SARS-Cov-2 spike protein RBD entry into cells is dependent on ACE2 protein on the cell membrane

[0077] The density of cultured cells (HEK293T, human kidney embryo cell line, purchased from Concordia Cell Bank) was 60-80%, and the serum component accounted for 10% of the total volume;

[0078] Transfect an empty plasmid (control) or a plasmid overexpressing ACE2 (for the construction method of this plasmid, please refer to the "Plasmid Construction" section);

[0079] After transfection, 1ug / ml recombinant SARS-Cov-2 spike protein RBD (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., 40592-V05H) was added, and the cells were incubated for 6 hours;

[0080] Using immunoblotting to observe the entry of the SARS-Cov-2 spike protein into cells;

[0081] The results showed that the SARS-Cov-2 spike protein RBD could significantly enter ACE2-overexpressing cells, but not HEK293T cells without ACE2 expression (such as figure 1 shown). The above resul...

Embodiment 2

[0082] Example 2: Inhibitory effect of incubation of ACE2 extracellular segment on the entry of SARS-Cov2 pseudovirus into human lung epithelial cells

[0083] Culture cells (293T and BEAS-2B human lung epithelial cells, purchased from Concord Cell Bank) density to 60-80%;

[0084] Add SARS-Cov-2 pseudovirus (1*10 -6 pfu / L) (Beijing Yiqiao Shenzhou Technology Co., Ltd.), the serum component accounts for 10% of the total volume;

[0085] While adding the SARS-Cov-2 pseudovirus, add 0, 0.25, and 1 μg / ml of ACE2 extracellular segment protein (the construction of this protein is described in the method), and incubate the cells with the pseudovirus;

[0086] After incubating the cells for 6 hours, use the luciferase detection kit (Beijing Biolab Technology Co., Ltd., SY0058) to observe that under the incubation of different concentrations of extracellular segment ACE2, the stronger the intracellular fluorescence, it means that SARS- The more the Cov-2 pseudovirus enters the cell;...

Embodiment 3

[0088] Example 3: ACE2 mutants eliminate the degradation of angiotensin II by ACE2;

[0089] Culture cells (293T cells, purchased from Concordia Cell Bank) at a density of 60-80%;

[0090] Transfect or not transfect a plasmid overexpressing wild-type ACE2 protein or mutant ACE2 into the culture medium (for the construction method of the plasmid, refer to the "plasmid construction" section), and the serum component accounts for 10% of the total volume;

[0091] 48 hours after transfection of the plasmid, 1 ng / ml of recombinant angiotensin II (promocell) was added, and the cells were incubated for 12 hours;

[0092] After incubating the cells for 12 hours, the cell supernatant was obtained, and the concentration of angiotensin II in the culture supernatant was measured using an angiotensin II ELISA kit (purchased from Raybiotech, ELA-ANGII-1);

[0093] The results show that cells overexpressing ACE2 can significantly reduce the level of angiotensin II in the culture medium, whi...

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Abstract

The invention relates to a mutant of an angiotensin converting enzyme 2 (ACE2) protein or an extracellular domain thereof, which is a mutant obtained by mutating one or more histidines (namely histidines at any one or more positions of the 345th site, the 374th site, the 378th site and the 505th site of the N end) in zinc ion dependent active sites of the N end of the ACE2 extracellular domain into non-polar amino acids. As bait protein of the coronavirus, the mutant can play a role in trapping the coronavirus outside cells and prevent the virus from entering the cells; and meanwhile, the level of angiotensin II cannot be degraded due to mutation of a specific site, so that the simultaneous accompanying side effects such as RAAS disorder and myocardial fibrosis caused by rapid increase of a large amount of ACE2 or extracellular fragments thereof are avoided.

Description

technical field [0001] The present invention relates to a mutant of ACE2 protein or its extracellular segment, and the application of the mutant in preventing or treating pneumonia caused by coronavirus, pulmonary edema, respiratory distress, sepsis and even multiple organ failure, In particular, while fighting the coronavirus, the mutant avoided the side effects of RAAS disorder and myocardial fibrosis caused by the rapid increase of ACE2 or its extracellular segment. Background technique [0002] The new coronavirus is a positive-stranded RNA single-stranded virus. The spike protein (SP) on its surface is a glycoprotein containing 180 amino acids, and its domain is divided into N-terminal S1 and C-terminal S2. Under the action of furin and transmembrane protease serines 2 (TMPRSS2), the spike protein contains RBD (receptor binding domain, receptor recognition domain, which can recognize and bind to human cell membrane surface ACE2) The S1 segment is cleaved, and the S2 se...

Claims

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Application Information

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IPC IPC(8): C12N9/48A61K38/48A61P31/14A61P11/00A41D13/11
CPCC12N9/485C12Y304/17023A61P31/14A61P11/00A41D13/1192A61K38/00
Inventor 徐涛宋婀莉冯寒杨临溥
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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