Recombinant receptor binding protein and recombinant receptor protein for detecting new coronavirus neutralizing antibody

A receptor binding and receptor protein technology, applied in the field of biomedicine, can solve problems such as insufficient binding stability, false negatives, and restrictions on the continuous detection of neutralizing antibodies against the new coronavirus, and meet the needs of tracking and detection of neutralizing antibodies. The effect of simple operation and low detection cost

Active Publication Date: 2021-04-27
SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the binding affinity between RBD protein and ACE2 protein, the sVNT method has certain false negatives in detecting the neutralizing antibody of the new coronavirus; and the binding stability of the two proteins is insufficient, which limits the neutralizing antibody of the new coronavirus. Continuous testing

Method used

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  • Recombinant receptor binding protein and recombinant receptor protein for detecting new coronavirus neutralizing antibody
  • Recombinant receptor binding protein and recombinant receptor protein for detecting new coronavirus neutralizing antibody
  • Recombinant receptor binding protein and recombinant receptor protein for detecting new coronavirus neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0266] Example 1: Plasmid construction and protein expression and purification of recombinant ACE2 monomeric protein

[0267] 1. Experimental method and steps

[0268] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-His plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and C Terminal His tag. The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0269] 1.2 Protein expression and purification

[0270] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-His plasmid extraction

[0271] Use the endotoxin-free plasmid large-scale extraction kit to extract the plasmid according to the steps. After measuring the OD value, store at -20°C.

[0272] 1.2.2 Expi 293F cell culture an...

Embodiment 2

[0293] Example 2: Plasmid construction and protein expression and purification of recombinant ACE2-FFc protein

[0294] 1. Experimental method and steps

[0295] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-FFc plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and Fc tag, and the ACE2 protein extracellular region and the Fc tag are connected through a flexible linker (Flexible linker) (reference doi:10.1016 / j.addr.2012.09.039.). The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0296] 1.2 Protein expression and purification

[0297] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-FFc plasmid extraction

[0298] Use the endotoxin-free plasmid large-scale extracti...

Embodiment 3

[0323] Embodiment 3: Expression plasmid construction and protein expression purification of ACE2-RFc protein

[0324] 1. Experimental method and steps

[0325] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-RFc plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and Fc tag, and the extracellular region of ACE2 protein and the Fc tag are connected by a rigid linker (reference doi:10.1016 / j.addr.2012.09.039.). The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0326] 1.2 Protein expression of ACE2-RFc

[0327] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-RFc plasmid extraction

[0328] Use the endotoxin-free plasmid large-scale extraction kit to extract the plasmid ...

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Abstract

The invention belongs to the field of biological medicines, and relates to a recombinant receptor binding protein and a recombinant receptor protein for detecting a new coronavirus neutralizing antibody, in particular to the recombinant receptor binding protein, the recombinant receptor protein, a nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a reagent composition, a kit and application. The recombinant receptor binding protein is an RBD trimer protein, the RBD trimer protein has an optimized spatial structure and higher immunogenicity, the recombinant receptor protein is an ACE2 dimer protein, the ACE2 dimer protein has a natural protein conformation capable of simulating ACE2, after the recombinant receptor binding protein and the recombinant receptor protein are combined, the new coronavirus neutralizing antibody can be detected through the principle of enzyme linked immunosorbent assay, the sensitivity, specificity and stability are remarkably improved, and the requirement for tracking detection of the new coronavirus neutralizing antibody on the market can be greatly met.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a recombinant receptor-binding protein and a recombinant receptor protein for detecting neutralizing antibodies against the new coronavirus; specifically, the disclosure relates to a recombinant receptor-binding protein, a recombinant Receptor protein, nucleic acid molecule, recombinant expression vector, recombinant host cell, reagent composition, kit and application. Background technique [0002] Novel coronavirus disease 2019 (COVID-19), referred to as "new coronary pneumonia", is a pneumonia caused by severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). SARS-CoV-2 is a single-stranded RNA (ssRNA) virus, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus, MERS- CoV), the third outbreak of coronavirus among peo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/569G01N33/68G01N33/58A61K39/215A61P31/14
CPCC07K14/005G01N33/56983G01N33/6854G01N33/581A61K39/12A61P31/14C12N2770/20022C07K2319/735C07K2319/02C07K2319/30C07K2319/61C07K2319/21G01N2333/165G01N2469/20C12N2770/20034
Inventor 孙振华左炽健高树峰李娜
Owner SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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