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High-affinity human angiotensin converting enzyme 2 (ACE2) mutant and application thereof

A mutant, T27W technology, applied in the field of bioengineering, can solve the problems that affect the therapeutic effect, cannot effectively block the combination of the virus and the airway epithelial cell membrane receptor ACE2, and low affinity, and achieve the effect of wide application prospects

Pending Publication Date: 2022-03-01
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ACE2-Fc fusion proteins disclosed in the prior art have low affinity and cannot effectively block the binding of the virus to the airway epithelial cell membrane receptor ACE2 in the treatment of viral infections, thereby affecting the therapeutic effect.

Method used

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  • High-affinity human angiotensin converting enzyme 2 (ACE2) mutant and application thereof
  • High-affinity human angiotensin converting enzyme 2 (ACE2) mutant and application thereof
  • High-affinity human angiotensin converting enzyme 2 (ACE2) mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Angiotensin-converting enzyme 2 (ACE2) mutant design

[0065] Two interacting proteins are bound by hydrogen bonds, ionic bonds, van der Waals forces and hydrophobic interactions between their amino acid side chains. By observing and analyzing the structures of ACE2 and SARS-CoV-2 (PDB ID: 6M18), the present invention has designed a series of ACE2 mutants (the amino acid sequences thereof are respectively as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ shown in ID NO:32 and SEQ ID NO:34). The mutated ACE2 variant is expressed as a dimer (i.e. ACE2-Fc Protein, its sequence is as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO :25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, shown in SEQ ID NO:33 and SEQ ID NO:35).

[0066] Table 1 ACE2 mutants and ACE2-Fc protein

[0067]

Embodiment 2

[0068] Example 2 The plasmid construction of the S protein of the new coronavirus SARS-CoV-2

[0069] The plasmid p3XFLAG-CMV14-2019nCoV-S expressing the S protein of the new coronavirus SARS-CoV-2:

[0070] The DNA sequence (SEQ ID NO: 11) of the S protein ORF of SARS-CoV-2 synthesized by genes was digested with restriction DNA endonucleases HindIII and XbaI, and the plasmid vector p3XFLAG was digested with the same restriction enzymes at the same time -5CMV14 (Sigma, Cat. No. E4901), the S protein ORF with cohesive ends obtained after enzyme digestion and the plasmid vector fragment were ligated with T4 ligase, and transformed into E. coli competent cells to obtain the plasmid p3XFLAG-CMV14-2019nCoV-S (DOI : https: / / doi.org / 10.1371 / journal.pone.0076469).

Embodiment 3

[0071] Example 3 Production and purification of wild-type and mutant ACE2-Fc dimers

[0072] 3.1 Cell culture and transient transfection

[0073] CHO-3E7 cells were grown in serum-free FreeStyle™ CHO expression medium (Life Technologies, Carlsbad, California, USA). Cells were incubated on an orbital shaker (VWR Scientific, Chester, PA) at 37°C, 5% CO 2held in an Erlenmeyer flask (Corning Inc., Acton, MA). On the day of transfection, DNAs encoding ACE2-Fc proteins of SEQ ID NO: 5-8, SEQ ID NO: 13, 21, 23, 25, 27, 29, 31, 33 and 35 (sequences such as SEQ ID NO: 16- 19) and PEI (Polysciences, Eppelheim, Germany) were mixed in a certain ratio, and then added to the flask together with the cells to be transfected. On day 5 of transfection, about 1 ml of supernatant was collected for detection of expression level. The supernatant collected on day 6 was used for further purification.

[0074] 3.2 Purification and analysis

[0075] Centrifuge the cell culture fluid, then filter....

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Abstract

The invention belongs to the technical field of bioengineering, and discloses a high-affinity human angiotensin converting enzyme 2 (ACE2) mutant and application thereof. The high affinity ACE2 mutant or ACE2-Fc protein comprises, relative to the wild type ACE2 protein, one or more mutations selected from the group consisting of sites 25, 27, 31, 34, 79, 82, 324, 326, 330, and 387. The mutant can be effectively combined with SARS-CoV-2 virus S protein, has high affinity, can be effectively used for treatment and prevention of viruses, especially novel coronavirus, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to high-affinity ACE2 mutants and applications thereof. Background technique [0002] Angiotensin-converting enzyme 2 (ACE2) is a membrane receptor expressed on the surface of airway epithelial cells. In the early 21st century, scientists began to conduct in-depth research on ACE2. ACE2 has been confirmed to be the host cell receptor of human coronaviruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63), and can interact with the spike protein (spike, S) of coronaviruses This interaction is mediated by the receptor-binding domain (RBD) of the spike protein S and is considered to be a key step in membrane fusion between the virus and the cell. Recently, ACE2 has once again attracted people's attention, and studies have shown that ACE2 is the host cell receptor of a newly discovered deadly novel coronavirus (NCP), the nove...

Claims

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Application Information

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IPC IPC(8): C12N9/48C07K19/00A61K38/48A61K47/68A61P31/12A61P31/14
CPCC12N9/485A61K47/6815A61P31/12A61P31/14C12Y304/17023C07K2319/30A61K38/00
Inventor 郑茜史敏龙杨芸芸
Owner NANJING GENSCRIPT BIOTECH CO LTD
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