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Nano antibody for neutralizing toxicity of novel coronavirus as well as preparation method and application of nano antibody

A nanobody and virus technology, applied in the field of peptides, can solve the problems of limited sources and mismatch of blood types of donors and recipients, and achieve the effects of low production cost, strong neutralization ability, and small molecular weight

Active Publication Date: 2020-12-01
SYSVAX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the source of polyantibody plasma is not only limited, but its clinical application is also limited by conditions such as difficult quality control, donor-recipient blood type mismatch, and potential infectious factors. Human monoclonal antibodies can effectively overcome the above problems

Method used

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  • Nano antibody for neutralizing toxicity of novel coronavirus as well as preparation method and application of nano antibody
  • Nano antibody for neutralizing toxicity of novel coronavirus as well as preparation method and application of nano antibody
  • Nano antibody for neutralizing toxicity of novel coronavirus as well as preparation method and application of nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of New Coronavirus Neutralizing Toxicity Nanobodies

[0057] 1. Animal immunization program and results

[0058] Alpaca (llama) immunization program: an adult male healthy llama alpaca, number KZL007, is immunized by subcutaneous injection at multiple points. 0.9 mg of S1 protein was injected each time, once every 14 days, for a total of 3 injections. 14 days after the third immunization, 2 ml of blood was collected to measure the titer.

[0059] llama immune titer detection method: using enzyme-linked immunosorbent assay, the target antigen is the new coronavirus RBD protein, the primary antibody is anti-his-HRP, TMB color development, 450nm detection O.D. value). When the serum is diluted 128K times (ie, 128000 times, the meaning of the expression is similar in the following text), the ELISA result can be above 1.0. During detection, the serum was diluted with pH 7.4 PBS buffer, wherein the serum was diluted by 1K times, 2K times, 4K times, 8K...

Embodiment 2

[0122] Example 2 Affinity detection test of novel coronavirus neutralizing toxic nanobodies

[0123] 1. Instruments and reagents:

[0124] In this example, the instrument Gator probe life is used to detect the intermolecular interaction by thin film interference method, and the sample to be tested with a concentration of not less than 80% and the antigen RBD-FC protein (new coronavirus RBD protein) of a concentration of not less than 90% are prepared .

[0125] Probe species: Kinetic index of binding of anti-human Fc antibody and human Fc.

[0126] Buffer: Q buffer (PBS+0.02%Tween-20+0.2%BSA).

[0127] 2. Experimental method:

[0128] 1) NBS1-2 sample detection method: immobilized antigen RBD-Fc (100nM) to detect nanobody; probe type: anti-human Fc antibody; NBS1-2 was diluted to 60000nM, 30000nM, 15000nM, 7500nM, 3750nM with Q buffer , 1875nM, 937.5nM, OnM.

[0129] 2) NBS1-3 sample detection method: immobilized antigen RBD-Fc (100nM) to detect nanobody; probe type: anti...

Embodiment 3

[0137] Example 3 Virus Neutralization Test of Novel Coronavirus Neutralizing Toxicity Nanobodies

[0138] The virus neutralization test is a technology for detecting specific antibodies, that is, the interference ability of the biological activity of the antibody to be tested is determined by using a known virus. In the embodiment of the present invention, this technology is used to detect the activity of the screened nanobody.

[0139] Four nanobody samples with a purity of more than 80% were prepared: NBS1-2, NBS1-3, NBS1-10 and NBS1-57; the nanobody used was prepared in Example 1.

[0140] 1. Experimental method:

[0141] 1) First, take the original concentration of each protein sample as the initial concentration, use the sample diluent (20mM PB, pH7.2) to dilute each protein sample in a gradient, set 1mg / ml, 100μg / ml, 10μg / ml, 1μg in turn / ml, 0.1μg / ml, 0.01μg / ml and other dilution concentrations;

[0142] 2) The diluted sample was incubated with an equal volume of SARS...

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Abstract

The invention discloses a nano antibody for neutralizing toxicity of a novel coronavirus as well as a preparation method and application of the nano antibody. The nano antibody comprises a framework region FR and a complementarity determining region CDR, wherein the complementary determining region CDR comprises a CDR1, a CDR2 and a CDR3; the amino acid sequence of the CDR1 is selected from at least one of amino acid sequences shown as SEQ ID NO.1 and SEQ ID NO.2; the amino acid sequence of the CDR2 is selected from at least one of amino acid sequences shown as SEQ ID NO.3, SEQ ID NO.4 and SEQID NO.5; and the amino acid sequence of the CDR3 is selected from at least one of amino acid sequences shown in any one of SEQ ID NO.6 to SEQ ID NO.9. The nano antibody for neutralizing toxicity of novel coronavirus has the advantages of small molecular weight, high affinity with SARS-CoV-2 virus, low production cost and the like.

Description

technical field [0001] The invention relates to the technical field of polypeptides, in particular to a nanobody for neutralizing toxicity of the new coronavirus and its preparation method and application. Background technique [0002] Novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is caused by a new betacoronavirus-novel coronavirus (SARS-CoV-2 virus), with angiotensin converting enzyme 2 (Angiotensinconverting enzyme2, ACE- 2) Invasion of cells as a receptor leads to lung injury, and the aggravation of the condition is closely related to the secondary systemic inflammatory response. Severely ill patients will develop acute respiratory distress syndrome (Acuterespiratory distress syndrome, ARDS) and septic shock, and eventually multiple organ dysfunction Exhausted. At present, most therapeutic drugs such as baricitinib, remdesivir, and chloroquine are still in clinical trials, and there is no specific drug treatment. COVID-19 is highly contagious and ca...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14A61P11/00
CPCC07K16/10G01N33/56983A61P31/14A61P11/00C07K2317/569C07K2317/565C07K2317/567C07K2317/76A61K2039/505G01N2333/165C07K2317/22C07K2317/92C07K16/1003
Inventor 庄文超李福胜李倩卉凌志
Owner SYSVAX INC
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