Coronavirus neutralizing effect protein and application thereof

A coronavirus and protein technology, applied in antiviral agents, virus/bacteriophage, peptide/protein components, etc., to achieve stable molecular structure and overcome poor specificity

Active Publication Date: 2022-06-10
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]However, both classes of coronavirus inhibitors have their own disadvantages: small molecule inhibitors are less specific, and thus have negative effects on other similar coronavirus targets when inhibiting coronavirus targets. Physiological activity also produces inhibition
Excessive use of large-scale monoclonal antibodies can lead to viruses prone to more immune evasion and may be ineffective against new mutant strains

Method used

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  • Coronavirus neutralizing effect protein and application thereof
  • Coronavirus neutralizing effect protein and application thereof
  • Coronavirus neutralizing effect protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the preparation of ACE2 mutant protein C4-2

[0061] 1. Construction of recombinant expression vector pCDNA3.1 / C4-2

[0062] In the present invention, the 30th, 78th, 321st and 474th positions of the amino acid sequence of angiotensin-converting enzyme 2 (ACE2) are subjected to point mutations, and then ACE2 mutant protein C4-2 is obtained after fusion with the Fc segment of the mouse IgG antibody. The amino acid sequence is shown in SEQID No.1.

[0063] Design and artificially synthesize the eukaryotic expression DNA sequence (SEQ ID No.2) according to the amino acid sequence of the C4-2 protein, and add NheI (GCTAGC) and XbaI (TCTAGA) double restriction sites and homology arms were connected to the pCDNA3.1 vector with double restriction enzymes (NheI and XbaI) through Gibson Assembly, and then transferred into DH5α cells to amplify and extract the plasmid, and after sequencing, it was verified that it was correct The recombinant expression vector pCDN...

Embodiment 2

[0070] Example 2, ACE2 mutant protein C4-2 in vitro activity assay

[0071]1. Construction of cell lines expressing five S protein mutants of coronaviruses by lentiviral transfection

[0072] The five S proteins of SARS-CoV-2 come from: wild-type SARS-CoV-2, alpha, beta, gamma and delta epidemic strains of SARS-CoV-2. The amino acid sequence of the S protein from wild-type SARS-CoV-2 (ie Wuhan-Hu-1 strain) is the same as NCBI Reference Sequence: YP_009724390.1, and the corresponding coding gene sequence is NCBI Reference Sequence: NC_045512.2 (21563..25384) , NCBIGeneID is 43740568; the amino acid sequence of the S protein from the alpha epidemic strain has the following changes compared with YP_009724390.1: amino acid residues 69-70 are missing, amino acid residue 144 is missing, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, the corresponding coding genes have the following changes compared with NC_045512.2 (21563..25384): 205-210 nucleotide deletion, 430-432 nucleotide ...

Embodiment 3

[0077] Example 3, Affinity Determination of ACE2 Mutant Protein C4-2 and SARS-CoV-2S Protein RBD

[0078] For the determination of typical affinity, the Biacore 2000 instrument was used in the experiment, and its detection principle is based on surface plasmon resonance (SPR), which can sensitively reflect the change of the refractive index on the chip surface. To study intermolecular interactions, one molecule is immobilized on the surface of the chip, and the other molecule flows across the surface in the form of a solution. The response value of SPR is proportional to the change of mass concentration near the chip.

[0079] In a typical test, the CM5 chip was chosen to measure the protein-protein interaction between soluble ACE2 protein and SARS-CoV-2 Spike-RBD. The CM5 chip is based on a covalent conjugation strategy with medium capacity and versatile properties. The running buffer was HBS-EP (Cytiva). RBD-his (Shenzhou: 40592-V08H105) (20ng / μl) was dissolved in sodium ...

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Abstract

The invention discloses a coronavirus neutralizing effect protein and application thereof. The invention provides a protein which is a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues on the 1st-740th sites of an amino acid sequence such as SEQ ID No.1, or having 80% or more of identity and having the same function, or connecting a protein tag on the basis of the protein. The C4-2 protein provided by the invention can be combined with the coronavirus S protein and inhibit the combination of the coronavirus S protein and an ACE2 receptor of a human cell, so that the coronavirus is effectively neutralized, and the C4-2 protein is proved at the cellular level. Meanwhile, the corresponding DNA sequence is beneficial to soluble expression in a human cell line, and a new direction is provided for treatment of the coronavirus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a coronavirus neutralizing effector protein and its application. Background technique [0002] Since the 21st century, human beings have experienced three coronavirus pandemics that caused severe respiratory symptoms and high mortality, namely SARS, Middle East Respiratory Syndrome (MERS), and Novel Coronavirus Pneumonia (COVID-19). Among them, the new type of coronavirus pneumonia that broke out at the end of 2019 was caused by a new type of coronavirus (SARS-CoV-2, severe acute respiratory syndrome-related coronavirus). Existing research evidence has shown that SARS-CoV-2 is one of the three coronaviruses. Easiest to spread. The World Health Organization (WHO) announced in March 2020 that COVID-19 has constituted a global pandemic. Today, more than 200 countries and regions have confirmed cases of COVID-19. Statistics released by the WHO show that there have been more than 200 mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C07K19/00C12N15/57C12N15/62C12N15/85C12N5/10G01N33/569A61K38/48A61K39/395A61K47/68A61P31/14
CPCC12N9/485C12N15/85C12N5/0686C12Y304/17023G01N33/56983A61K47/6801A61P31/14C07K2319/30C12N2510/00C12N2800/107G01N2333/948G01N2333/165G01N2469/10A61K38/00
Inventor 席建忠王博仑赵俊轩
Owner PEKING UNIV
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