阅读说明：本技术 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用 (Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof ) 是由 高威 高芳 刘晓宇 苟黎明 于 2020-04-27 设计创作，主要内容包括：本发明涉及抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用。该抗体至少具有重链CDR1、重链CDR2、重链CDR3之一。该抗体可用于制备针对COVID-19的诊断试剂或诊断试剂盒、抗体药物或药物组合物。本发明通过噬菌体展示技术获得了抗新型冠状病毒SARS-Cov-2的中和性单域抗体,该抗体能够阻断SARS-Cov-2-RBD与ACE2阳性细胞的结合,对SARS-Cov-2伪病毒具有显著的病毒中和作用,为COVID-19的预防和治疗提供了有效的备选抗体药物。(The invention relates to a neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof. The antibody has at least one of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3. The antibody can be used for preparing a diagnostic reagent or a diagnostic kit, an antibody medicament or a pharmaceutical composition aiming at COVID-19. The invention obtains the neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 by phage display technology, the antibody can block the combination of SARS-Cov-2-RBD and ACE2 positive cells, has obvious virus neutralizing effect on SARS-Cov-2 pseudovirus, and provides effective alternative antibody medicine for preventing and treating COVID-19.)

1. A neutralizing single domain antibody against the novel coronavirus SARS-Cov-2, said single domain antibody consisting of a heavy chain, characterized in that said antibody has at least one of the following technical characteristics:

i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: X1-F-X2-F-X3-X4-Y, wherein X1 is D or S, X2 is A, D or Y, X3 is S or A, and X4 is S or D;

ii. The heavy chain includes a heavy chain CDR2 having the amino acid sequence: I-G-X5-I-X6-H-S-G-S-T-Y-Y-N-P-S-L-K-S-X7-V, wherein X5 is E or S, X6 is H or Y, and X7 is L or R;

iii, the heavy chain comprises heavy chain CDR3 with the amino acid sequence: VKDFGHLGQMAS, VKDLGFADH, VKDFVVGETAEFSY, or AREWHSGYDY.

2. The neutralizing single domain antibody of claim 1 wherein the amino acid sequence of said heavy chain CDR1 is: DFAFSSY, SFDFSSY, SFDFSDY, or DFYFADY;

the amino acid sequence of the heavy chain CDR2 is: IGEIHHSGSTYYNPSLKSLV, IGEIHHSGSTYYNPSLKSRV, IGEIHHSGSTYYNPSLKSRV, or IGSIYHSGSTYYNPSLKSRV.

3. The neutralizing single domain antibody of claim 1, wherein said heavy chain comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR 3;

when the heavy chain CDR1 is DFAFSSY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSLV and the heavy chain CDR3 is VKDFGHLGQMAS;

when the heavy chain CDR1 is SFDFSSY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV and the heavy chain CDR3 is VKDLGFADH;

when the heavy chain CDR1 is SFDFSDY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV and the heavy chain CDR3 is VKDFVVGETAEFSY;

when the heavy chain CDR1 is DFYFADY, the heavy chain CDR2 is IGSIYHSGSTYYNPSLKSRV and the heavy chain CDR3 is AREWHSGYDY.

4. The neutralizing single domain antibody according to claim 1, wherein the amino acid sequence of said single domain antibody is represented by one of SEQ ID NO 5 to SEQ ID NO 8.

5. The neutralizing single domain antibody of claim 1, wherein said heavy chain has a label comprising a fluorescent label, an enzymatic label, and a radioactive label.

6. Nucleic acid encoding a neutralizing single domain antibody against the novel coronavirus SARS-Cov-2 according to any one of claims 1 to 5.

7. The nucleic acid of claim 6, wherein the sequence of the nucleic acid is as shown in any one of SEQ ID NO 1 to SEQ ID NO 4.

8. Use of the neutralizing single domain antibody against the novel coronavirus SARS-Cov-2 according to any one of claims 1 to 5 for the preparation of a diagnostic agent or diagnostic kit, a medicament or a pharmaceutical composition.

9. Use of the nucleic acid according to claim 6 or 7 for the preparation of a neutralizing single domain antibody, a medicament or a pharmaceutical composition against the novel coronavirus SARS-Cov-2.

10. Use according to claim 8 or 9, characterized in that the medicament or pharmaceutical composition has a neutralizing antiviral effect against the novel coronavirus SARS-Cov-2.

Technical Field

The invention relates to a neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof, belonging to the technical field of biological medicines.

Background

In 2019, the world infection cases of the novel coronavirus infectious diseases (COVID-19) exceed 200 ten thousand, and the fatality rate reaches 6% (the)https://covid19.who.int/) COVID-19 is induced by infection with a novel Coronavirus (Severe Acute Respirancy Syndrome Coronavir 2, SARS-CoV-2. SARS-CoV-2 is a positive-stranded RNA virus having a diameter of about 80-120nm and is a member of the β genus Coronavirus (Betaconovir)^[1]The homology with SARS-CoV is 79.5% in the Sarbecovirus subgenus^[2]. At present, the source and natural host of SARS-CoV-2 virus are not clear, and effective measures cannot be taken to prevent and stop the spread of the virus.

The present study shows that, like other coronaviruses, the host receptor of SARS-Cov-2 is angiotensin converting enzyme 2(ACE2)^[3]Abundant expression in lung tissue and small intestine tissue^[4]. When SARS-CoV-2 infects human body, it can induce the change of S2 subunit structure by combining Receptor Binding Domain (RBD) of Spike protein S1 subunit on virus outer shell with ACE2, and further promote the fusion of virus and host cell membrane, mediate virus invading host cell^[5]。

Spike proteins are expressed in trimeric form on the viral coat, and each monomeric structure consists of one S1 subunit and one S2 subunit. It has been found that the RBD of the S1 protein on the Spike protein trimer exists in different states of "closed" and "open". When in the "open" state, one RBD in the trimer is in the extended state^[6]This fine change in conformation mediates recognition and binding of the Spike protein to ACE 2.

Single domain antibodies (sdabs), heavy chain single domain antibodies VHHs, comprise only the variable region of the antibody heavy chain. The single domain antibody has small molecular weight, high protein expression level, weak immunogenicity and easy production and preparation^[7]And the like. More importantly, the single-domain antibody can recognize the fine structure hidden on the surface of the antigen, can accurately aim and capture a target spot, and can be specifically combined with a target molecule^[8]。

At present, no specific vaccine and neutralizing antibody of SARS-CoV-2 virus is applied clinically. Therefore, the human monoclonal antibody with neutralization function obtained by rapid and efficient screening of SARS-Cov-2 neutralizing antibody is an urgent need for preventing and treating COVID-19.

Disclosure of Invention

The main purposes of the invention are: overcomes the problems in the prior art, provides a neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2, and has high-efficiency antiviral capacity for the novel coronavirus SARS-Cov-2. Also, applications of the antibody are provided.

The technical scheme for solving the technical problems of the invention is as follows:

a neutralizing single domain antibody against the novel coronavirus SARS-Cov-2, said single domain antibody consisting of a heavy chain, characterized in that said antibody has at least one of the following technical characteristics:

i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: X1-F-X2-F-X3-X4-Y, wherein X1 is D or S, X2 is A, D or Y, X3 is S or A, and X4 is S or D;

ii. The heavy chain includes a heavy chain CDR2 having the amino acid sequence: I-G-X5-I-X6-H-S-G-S-T-Y-Y-N-P-S-L-K-S-X7-V, wherein X5 is E or S, X6 is H or Y, and X7 is L or R;

iii, the heavy chain comprises heavy chain CDR3 with the amino acid sequence: VKDFGHLGQMAS, VKDLGFADH, VKDFVVGETAEFSY, or AREWHSGYDY.

Preferably, the amino acid sequence of the heavy chain CDR1 is: DFAFSSY, SFDFSSY, SFDFSDY, or DFYFADY;

the amino acid sequence of the heavy chain CDR2 is: IGEIHHSGSTYYNPSLKSLV, IGEIHHSGSTYYNPSLKSRV, IGEIHHSGSTYYNPSLKSRV, or IGSIYHSGSTYYNPSLKSRV.

Preferably, the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3;

when the heavy chain CDR1 is DFAFSSY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSLV and the heavy chain CDR3 is VKDFGHLGQMAS;

when the heavy chain CDR1 is SFDFSSY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV and the heavy chain CDR3 is VKDLGFADH;

when the heavy chain CDR1 is SFDFSDY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV and the heavy chain CDR3 is VKDFVVGETAEFSY;

when the heavy chain CDR1 is DFYFADY, the heavy chain CDR2 is IGSIYHSGSTYYNPSLKSRV and the heavy chain CDR3 is AREWHSGYDY.

Preferably, the amino acid sequence of the single domain antibody is shown in one of SEQ ID NO 5 to SEQ ID NO 8.

Preferably, the heavy chain has a label, including fluorescent, enzymatic, and radioactive labels.

The present invention also provides:

nucleic acid encoding a neutralizing single domain antibody against the novel coronavirus SARS-Cov-2 as described hereinbefore.

Preferably, the sequence of the nucleic acid is as shown in one of SEQ ID NO 1 to SEQ ID NO 4.

The present invention also provides:

use of the neutralizing single domain antibody against the novel coronavirus SARS-Cov-2 as described hereinbefore for the preparation of a diagnostic agent or diagnostic kit, a medicament or a pharmaceutical composition.

Use of the nucleic acid as hereinbefore described for the preparation of a neutralizing single domain antibody, medicament or pharmaceutical composition against the novel coronavirus SARS-Cov-2.

Wherein the medicine or the medicine composition has a neutralizing antiviral effect against a novel coronavirus SARS-Cov-2.

The invention obtains the neutralizing single-domain antibody for resisting the novel coronavirus SARS-Cov-2 by the phage display technology, the antibody can block the combination of SARS-Cov-2-RBD and ACE2 positive cells, has obvious virus neutralizing effect on SARS-Cov-2 pseudo virus, provides an effective alternative antibody medicament for the prevention and treatment of COVID-19, and has potential clinical application prospect.

Drawings

FIG. 1 is a graph showing the binding of enriched phages to the antigen protein by ELISA in example 1 of the present invention.

FIG. 2 is a diagram of specific binding assay (ELISA) of the phage to SARS-Cov-2-RBD-hFc protein in example 2 of the present invention.

FIG. 3 is a schematic diagram of an expression vector of example 3 of the present invention.

FIG. 4 is a diagram showing the results of SDS-PAGE in example 3 of the present invention.

FIG. 5 is a graph showing the results of affinity analysis in example 4 of the present invention.

FIG. 6 is a graph showing the analysis of the blocking effect of the single domain antibody in example 5 of the present invention.

FIG. 7 is a graph showing the evaluation of neutralizing effect of the single domain antibody against pseudovirus according to example 6 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.