Bifunctional fusion protein combined with coronavirus as well as preparation method and application of bifunctional fusion protein

A coronavirus and fusion protein technology, applied in the biological field, can solve problems affecting the effect and interaction of antibodies

Pending Publication Date: 2021-09-24
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the drug described in this patent is a combination, the antibodies that make up the combination are all antibodies against amino acids 318-510 of the S protein (Spike protein) that bind SARS-CoV, even if each The specific binding sites of different antibodies are different amino acids in positions 318-510, and there will be disadvantages of mutual influence on each other
[0018] Since antibodies belong to macromolecular proteins, and due to the characteristics of their spatial structure, when antibodies and antigens combine with each other, there will be interaction between antibodies. The compositions provided by this patent are all bound to the S protein of SARS-CoV (Spike protein) amino acids 318-510, when combined with the virus, will affect the effect of the antibody on neutralizing the SARS-CoV coronavirus

Method used

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  • Bifunctional fusion protein combined with coronavirus as well as preparation method and application of bifunctional fusion protein
  • Bifunctional fusion protein combined with coronavirus as well as preparation method and application of bifunctional fusion protein
  • Bifunctional fusion protein combined with coronavirus as well as preparation method and application of bifunctional fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, the gene synthesis of the bifunctional fusion protein that specifically binds coronavirus

[0065] (1) Construction of expression vector for antibody binding to coronavirus S protein (Spike protein)

[0066] CoV022 is an antibody that binds to the coronavirus S protein (Spike protein).

[0067] CoV022 light chain full-length expression vector construction:

[0068] Whole gene synthesis CoV022 light chain full-length cloning vector was double-digested with HindIII and EcoR1, and the pL101 expression vector was double-digested at the same time, the target fragment of about 750bp and the expression vector fragment of about 9kb were recovered by gel respectively, and the two were ligated with T4 DNA ligase and merged After transformation, a small amount of plasmid was extracted and identified by double digestion with HindIII and EcoR1.

[0069] The full-length CoV022 light chain has a nucleotide sequence shown in SEQ ID NO:1 and an amino acid sequence shown ...

Embodiment 2

[0117] Example 2, Preparation of a bifunctional fusion protein specifically binding to coronavirus

[0118] (1) Preparation of ACE2-609-Fc / CoV022 bifunctional fusion protein specifically binding to coronavirus:

[0119] The expression vectors of CoV022, ACE2-609-Fc, and ACE2-609-Fc / CoV022 bifunctional proteins were column-purified, and the obtained plasmids were transiently transfected into host cells Expi CHO-S cells, and fed-in medium was added for continuous culture for 8 For ~12 days, use serum-free and animal-derived component-free medium for host cell culture and target protein expression; collect the host cell culture supernatant, and use Protein A affinity chromatography column to separate and purify.

[0120] SDS-PAGE confirmed the molecular weight of purified CoV022, ACE2-609-Fc, ACE2-609-Fc / CoV022 bifunctional protein.

[0121] (2) Preparation of ACE2-615-Fc / CoV022 bifunctional fusion protein specifically binding to coronavirus:

[0122] The expression vectors of ...

Embodiment 3

[0125] Example 3, detection of the binding ability of the bifunctional fusion protein specifically binding to coronavirus and coronavirus

[0126] ELISA assay detects the binding ability of the bifunctional fusion protein specifically binding to coronavirus and coronavirus protein:

[0127] Use the coating solution to add SARS-CoV-2 S protein (Spike protein) to the microtiter plate, and coat at 37°C for 1 hour; discard the coating solution, add blocking solution, 350 μl / well, and block for 1.5 hours at 37°C; CoV022, ACE2-609-Fc, ACE2-609-Fc / CoV022 bifunctional protein, and ACE2-615-Fc, ACE2-615-Fc / CoV022 bifunctional protein were diluted step by step with liquid, added to the microtiter plate, and placed in a 37°C water bath 1 hour; wash 5-7 times with plate washer; dilute rabbit anti-human-HRP with diluent and add to ELISA plate, 37°C water bath for 1 hour; discard supernatant, wash 5-7 times with plate washer; add TMB chromogenic solution Protect from light for color develo...

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Abstract

The invention discloses a bifunctional fusion protein combined with coronavirus. Binding regions at two ends of the bifunctional fusion protein are respectively combined with different epitopes of a Spike protein, and the bifunctional fusion protein simultaneously has the functions of resisting SARS-CoV and SARS-CoV-2 and the function of combining ACE2. The bifunctional fusion protein combined with coronavirus has the capability of treating virus infected cells, and also has the effects of resisting viruses, reducing lung inflammation, reducing cytokine storm and treating lung injury.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention discloses a bifunctional fusion protein binding to coronavirus, its preparation method and application. Background technique [0002] 2019-novel coronavirus (2019 novel coronavirus, 2019-nCoV, SARS-CoV-2) is an enveloped, positively charged single-stranded RNA virus belonging to the genus Betacoronavirus and subgenus Sarbecovirus. After humans are infected with SARS-CoV-2 (2019-nCoV, SARS-CoV-2), most patients will develop symptoms such as fever, cough, vomiting, diarrhea, and mild pneumonia, and some patients will develop severe pneumonia and become seriously ill. Patients may suffer from acute respiratory failure, shock, or even death. [0003] In the whole process of SARS-CoV-2 (2019-nCoV, SARS-CoV-2) infection and pathogenesis, an important metalloproteinase-angiotensin converting enzyme 2 (ACE2) in the human body plays a role Important role (Lu R, Zhao X, Li j, et ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K38/48A61K47/68A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2319/30C07K2317/51C07K2317/515A61K38/00A61K38/48A61K47/68C07K16/00C12N5/10C07K14/705A61K38/43A61K39/395C12N15/62C07K19/00C07K16/1003
Inventor 王皓钱卫珠张大鹏李晶陶静李伟李继阳张浔敏李云峰袁秀珍焦树阁
Owner TAIZHOU MABTECH PHARM CO LTD
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