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Nano antibody for resisting novel coronavirus and variant thereof as well as preparation method and application of nano antibody

A technology of nano-antibody and coronavirus, applied in the field of biomedicine, to achieve the effects of reducing production costs, simple transformation of multi-combination forms, and wide application range

Active Publication Date: 2022-07-22
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of this application is to provide a nanobody against novel coronavirus and its variants and its preparation method and application, aiming at solving the lack of a nanobody that can effectively combine with novel coronavirus and its variants in the prior art. antibody problem

Method used

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  • Nano antibody for resisting novel coronavirus and variant thereof as well as preparation method and application of nano antibody
  • Nano antibody for resisting novel coronavirus and variant thereof as well as preparation method and application of nano antibody
  • Nano antibody for resisting novel coronavirus and variant thereof as well as preparation method and application of nano antibody

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preparation example Construction

[0045] A second aspect of the embodiments of the present application provides a method for preparing a nanobody against a novel coronavirus and a variant thereof, comprising the following steps:

[0046] S01. Provide the target protein antigen, coat the target protein antigen on the immune tube and combine with the phage library to obtain the target protein antigen-phage library;

[0047] S02. The target protein antigen-phage library is eluted to obtain an eluate, and the eluate is mixed with the helper phage for enrichment screening, and monoclonal antibody screening to obtain specific nanobody sequences;

[0048] S03. Clone the specific nanobody sequence into an expression vector to obtain a recombinant plasmid, transfer the recombinant plasmid into a host cell to induce expression and purify to obtain a nanobody targeting the new coronavirus.

[0049] The second aspect of the examples of the present application provides a method for preparing a nanobody against a novel coro...

Embodiment 1

[0111] Nanobody Nb-H6 against novel coronavirus and its variant strain and preparation method thereof

[0112] preparation methods such as figure 1 shown, as follows:

[0113] 1. Amplification and titration of natural library:

[0114] 1) pick E. coli The TG1 monoclonal strain was cultured in 100 mL of 2×YT medium containing 10 mg / L Tet at 37°C and shaken at 200 rpm to the logarithmic growth phase (OD 600 nm = 0.5);

[0115] 2) Add 100 μL to 200 μL of the VHH phage natural library stored at -20°C to 1. E. coli In TG1 bacterial solution, incubate at 37°C for 1 h;

[0116] 3) Add 400 mL of 2×YT (containing 2% glucose, 100 mg / L Amp), shake at 37°C and cultivate at 200 rpm until OD600 nm = 0.5, then add the helper phage M13KO7 (the final concentration is 10 10 pfu / mL), incubate at 37°C for 1 h;

[0117] 4) Centrifuge the above-mentioned 500 mL bacterial solution at 6000 rpm for 15 min, discard the supernatant (remove glucose, free phage and free helper phage), and take ...

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Abstract

The invention relates to the technical field of biological medicines, in particular to a nano antibody for resisting a novel coronavirus and a variant thereof as well as a preparation method and application of the nano antibody, and provides a nano antibody Nb-H6 which can specifically aim at the novel coronavirus and a variant S1 thereof, can effectively inhibit the combination of S1 protein and ACE2, can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, and can be used for preparing a novel coronavirus-resistant nano antibody. Therefore, viruses are prevented from entering and infecting cells; and meanwhile, the Nb-H6 has very good affinity with Delta, Alpha and Omicron mutants, so that the Nb-H6 is beneficial to wide application.

Description

technical field [0001] The present application belongs to the technical field of biomedicine, and in particular relates to a nanobody against a novel coronavirus and its variant strains, as well as a preparation method and application thereof. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SARS-CoV-2 for short) is the causative agent of the coronavirus disease 2019 (COVID-19) that is currently ravaging the world. As of May 18, 2022, World Health Organization (WHO) statistics show that globally, COVID-19 has caused more than 520 million confirmed cases and 6.2 million deaths. The new coronavirus is a single-stranded RNA virus of the β-coronaviridae family. Its RNA genome encodes four major structural proteins: Spike protein (S), Nucleocapsid protein (N), and transmembrane protein. (Membraneprotein, M) and envelope protein (envelope protein, E). The study found that the S protein is responsible for attachment to the host cell surfa...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/70G01N33/569
CPCC07K16/10C12N15/70G01N33/56983C07K2317/569C07K2317/565C07K2317/567G01N2333/165Y02A50/30
Inventor 叶亮吴丹丹
Owner SHENZHEN UNIV
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