CN118440186A - A broad-spectrum antibody against SARS-like coronavirus and/or novel coronavirus mutant strains and its application - Google Patents

Abstract

The present invention relates to an antibody with a broad spectrum against SARS-like coronavirus and/or novel coronavirus mutant strains and its application, wherein the antibody comprises a heavy chain and a light chain, and the antibody has at least one of the following technical features: (1) the heavy chain comprises a heavy chain CDR1, and its amino acid sequence is: GGSISSSSYF; (2) the heavy chain comprises a heavy chain CDR2, and its amino acid sequence is: FHYSGST; (3) the heavy chain comprises a heavy chain CDR3, and its amino acid sequence is: ARGGYSGYFDY; (4) the light chain comprises a light chain CDR1, and its amino acid sequence is: QSISVW; (5) the light chain comprises a light chain CDR2, and its amino acid sequence is: KAS; (6) the light chain comprises a light chain CDR3, and its amino acid sequence is: QQSNSDLYT. The antibody with a broad spectrum against SARS-like coronavirus and/or novel coronavirus mutant strains of the present invention can effectively block the binding of the novel coronavirus RBD to the host receptor protein ACE2, and has a strong neutralizing ability against a variety of SARS-like coronaviruses and novel coronavirus mutant strains.

Description

A broad-spectrum antibody against SARS-like coronavirus and/or novel coronavirus mutant strains and its application

Technical Field

The present invention relates to a broad-spectrum antibody against SARS-like coronavirus and/or novel coronavirus mutant strains and application thereof, belonging to the field of biomedicine.

Background technique

Animal coronaviruses are ubiquitous in wild animals. Given their large reservoirs, frequent recombination, and high genomic plasticity in wild animals, more animal coronaviruses may be transmitted to humans in the future, especially SARS-like coronaviruses that have caused two global pandemics. Both SARS-CoV-2 and SARS-CoV belong to SARS-like coronaviruses, which share common origins with many SARS-like coronaviruses found in bats, civets, and pangolins, including bat coronaviruses RaTG13, RsSHC014, and WIV1, civet coronavirus SZ3, and pangolin coronaviruses GD18 and GX-P5L. These animal coronaviruses are very similar to SARS-CoV or SARS-CoV-2, and are even able to use the same receptors to enter cells and replicate efficiently in primary human airway cells. In addition, the receptor binding motif (RBM) in the pangolin coronavirus GD18 spike protein that mediates viral entry into cells has only one amino acid difference from SARS-CoV-2, which further highlights their potential to spill over into the human population and cause new epidemics.

The mortality and economic losses associated with SARS-like coronavirus infections highlight the importance of developing broadly effective countermeasures, which may be key to preventing and mitigating current and future zoonotic events. Cross-neutralizing antibodies against SARS-like coronaviruses are an attractive countermeasure to prevent or mitigate current and future spillover events. In fact, several anti-SARS-CoV-2 monoclonal antibodies isolated from COVID19 survivors and vaccines have broad cross-neutralizing activity against SARS-like coronaviruses. In general, broadly cross-neutralizing antibodies target conserved epitopes retained by SARS-like coronaviruses. Therefore, the development of multiple cross-neutralizing antibodies targeting different conserved epitopes will provide more options for the use of such antibodies for prevention and treatment.

In addition, SARS-CoV-2 is a positive-stranded single-stranded RNA virus. RNA viruses are characterized by a high mutation rate and the accumulation of mutations over time. This accumulation of mutations is the main driving force for viral evolution and genomic variation, thereby escaping host immunity and developing drug resistance (Duffy S, PLoS Biol, 2018 08; 16(8)). Currently, five new coronavirus mutants have been listed as mutants of concern (VOC) by the World Health Organization. In addition to the popular Omicron mutant, there are also Alpha, Beta, Gamma and Delta mutants. The biggest feature of the new coronavirus mutant is that it can escape vaccine immunity and monoclonal antibodies. At present, the new coronavirus Omicron mutant has developed resistance to multiple new coronavirus monoclonal antibodies that are on the market and in clinical trials. Therefore, it is necessary to discover potent and broad-spectrum neutralizing antibodies that can resist the escape of new coronavirus mutants to deal with the currently popular Omicron mutant.

Summary of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide a broad-spectrum antibody against SARS-like coronavirus and/or new coronavirus mutant strains and its application.

In order to solve the above technical problems, the technical solution of the present invention is as follows:

An antibody (VSCM16-12) with a broad spectrum against SARS-like coronavirus and/or novel coronavirus mutant strains, the antibody comprising a heavy chain and a light chain, and the antibody having at least one of the following technical features:

(1) The heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GGSISSSSYF;

(2) the heavy chain includes a heavy chain CDR2, whose amino acid sequence is: FHYSGST;

(3) the heavy chain includes a heavy chain CDR3, whose amino acid sequence is: ARGGYSGYFDY;

(4) the light chain includes a light chain CDR1, whose amino acid sequence is: QSISVW;

(5) The light chain includes a light chain CDR2, whose amino acid sequence is: KAS;

(6) The light chain includes a light chain CDR3, whose amino acid sequence is: QQSNSDLYT.

Furthermore, the antibody has at least one of the following technical features:

(a) The heavy chain comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is: GGSISSSSYF, the amino acid sequence of heavy chain CDR2 is:

The amino acid sequence of the heavy chain is: FHYSGST, and the amino acid sequence of the heavy chain CDR3 is: ARGGYSGYFDY;

(b) The light chain includes light chain CDR1-3, the amino acid sequence of light chain CDR1 is: QSISVW, the amino acid sequence of light chain CDR2 is: KAS, and the amino acid sequence of light chain CDR3 is: QQSNSDLYT.

Furthermore, the antibody has the following technical characteristics: the heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GGSISSSSYF; the heavy chain includes a heavy chain CDR2, whose amino acid sequence is: FHYSGST; the heavy chain includes a heavy chain CDR3, whose amino acid sequence is: ARGGYSGYFDY; the light chain includes a light chain CDR1, whose amino acid sequence is: QSISVW; the light chain includes a light chain CDR2, whose amino acid sequence is: KAS; the light chain includes a light chain CDR3, whose amino acid sequence is: QQSNSDLYT.

Furthermore, the heavy chain also includes heavy chain FR1-4, and the amino acid sequence of heavy chain FR1 is

QVQLQESGPGLVKPSETLSLTCTVS, the amino acid sequence of the heavy chain FR2 is WGWIRQPPGKGLEWIGS, the amino acid sequence of the heavy chain FR3 is FYNPSLKSRITISVDTSKNQFSLSLISVTAADTAVYYC, and the amino acid sequence of the heavy chain FR4 is WGQGTLVTVSS.

Furthermore, the light chain also includes light chain FR1-4, and the amino acid sequence of light chain FR1 is

DIQMTQSPSTLSASVGDRVTITCRAS, the amino acid sequence of the light chain FR2 is LAWYQQKPGKAPKLLIY, the amino acid sequence of the light chain FR3 is SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC, and the amino acid sequence of the light chain FR4 is FGQGTKLEIK.

Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2.

Furthermore, the gene sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 4.

Nucleic acid molecules encoding broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants as described above.

Furthermore, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:3 and/or SEQ ID NO:4.

The use of the broad-spectrum antibodies against SARS-like coronavirus and/or new coronavirus mutant strains as described above in the preparation of diagnostic reagents or diagnostic kits or drugs.

Furthermore, the drug has a neutralizing effect on SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the new coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, new coronavirus Alpha mutant strain, new coronavirus Beta mutant strain, new coronavirus Gamma mutant strain, new coronavirus Delta mutant strain, and new coronavirus Omicron mutant strain.

The use of the nucleic acid molecules as described above in the preparation of broad-spectrum antibodies or drugs against SARS-like coronaviruses and/or new coronavirus mutant strains.

Furthermore, the drug has a neutralizing effect on SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the new coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, new coronavirus Alpha mutant (B.1.1.7), new coronavirus Beta mutant (B.1.351), new coronavirus Gamma mutant (P1), new coronavirus Delta mutant (B.1.617.2), and new coronavirus Omicron mutant (B.1.1.529).

An expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the nucleic acid molecule as described above.

Optionally, the method for preparing broad-spectrum antibodies against SARS-like coronavirus and/or novel coronavirus mutant strains of the present invention comprises the following steps:

S1. SARS-CoV-2-specific Memory B cells were isolated from the peripheral blood of patients who recovered from SARS-CoV-2 infection;

S2, amplify the Ig variable sequence of SARS-CoV-2-specific memory B cells to obtain specific amplification products;

S3. Construct expression plasmids, perform in vitro transfection, expression, and purification to obtain broad-spectrum antibodies against SARS-like coronaviruses and/or novel coronavirus mutant strains.

The present invention uses flow cytometry single-cell analysis and sorting technology and single B cell PCR amplification antibody preparation technology to sort single memory B cells specific to the spike protein from PBMCs of recovered COVID-19 patients, directly clone the antibody heavy chain and light chain variable region sequences, construct an expression plasmid, and express and purify the antibody (VSCM16-12) against SARS-like coronavirus.

The broad-spectrum anti-SARS-like coronavirus and/or novel coronavirus mutant antibody of the present invention is an antibody that specifically targets RBD, can specifically bind to a variety of SARS-like coronaviruses and novel coronavirus mutants, and has strong binding ability. This specific antibody can effectively block the binding of a variety of novel coronavirus mutants to receptor proteins and is an effective broad-spectrum neutralizing active monoclonal antibody.

Compared with the prior art, the present invention has the following beneficial effects:

1) The preparation process of the broad-spectrum antibodies against SARS-like coronavirus and/or new coronavirus mutant strains of the present invention is simple and rapid, and the obtained antibodies are fully human antibodies and have no immunogenicity.

2) The broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants of the present invention can effectively block the binding of the new coronavirus RBD to the host receptor protein ACE2, and have a strong binding ability to a variety of SARS-like coronaviruses.

3) The broad-spectrum anti-SARS-like coronavirus and/or novel coronavirus mutant antibodies of the present invention have a strong and broad-spectrum neutralizing effect on a variety of SARS-like coronaviruses and popular novel coronavirus mutants (Alpha, Beta, Gamma, Delta and Omicron).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the flow cytometry method for sorting SARS-CoV-2 S protein-specific memory B cells in the present invention.

Figure 2 is a sequence diagram of the heavy chain variable region of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibody of the present invention.

Figure 3 is a sequence diagram of the light chain variable region of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibody of the present invention.

Figure 4 is a diagram showing the binding of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibodies of the present invention to different subunits of the SARS-CoV-2S protein.

Figure 5 is a diagram of the binding of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant strain antibody of the present invention to the SARS-like coronavirus, wherein the abscissa is the antibody dilution concentration after log conversion, and the ordinate is the absorbance at a wavelength of 450nm.

Figure 6 is a graph showing the neutralization of SARS-like coronavirus by antibodies with different concentrations of broad-spectrum anti-SARS-like coronaviruses and/or new coronavirus mutant strains of the present invention, wherein the horizontal axis is the antibody concentration and the vertical axis is the antibody inhibition rate.

Figure 7 is a graph showing the neutralization of the novel coronavirus original strain, Alpha, Beta, Gamma, and Delta mutant strains by the broad-spectrum antibodies against SARS-like coronaviruses and/or novel coronavirus mutants of the present invention, wherein the horizontal axis represents the antibody concentration and the vertical axis represents the antibody inhibition rate.

Detailed ways

The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

Example 1

Referring to Figure 1, the specific method for obtaining broad-spectrum antibodies (VSCM16-12) against SARS-like coronavirus and/or novel coronavirus mutant strains is as follows:

(1) Flow cytometry single-cell sorting of SARS-CoV-2-specific memory B cells

a. Collect peripheral blood from several patients who have recovered from SARS-CoV-2 infection, separate PBMCs, and freeze them in liquid nitrogen for future use;

b. Resuscitate the collected peripheral blood PBMCs from patients who have recovered from the novel coronavirus SARS-CoV-2 infection from liquid nitrogen;

c. Add complete medium and incubate at 37°C overnight, wherein the complete medium is 1640 (Gibco, Catalog No.: C11875500CP) medium containing 10% fetal bovine serum (Gibco, Catalog No.: 10270-106);

d. Then, cells were stained with Live/dead (ThermoFisher, Catalog No.: L34962), CD3 (BD Biosciences, Catalog No.: 612752), CD19 (Biolegend, Catalog No.: 302230), IgD (Biolegend, Catalog No.: 348240), CD27 (Biolegend, Catalog No.: 356412), IgG (BD Biosciences, Catalog No.: 555787), and SARS-CoV-2 S probe, which was SARS-CoV-2 S ECD protein (Sino Biological, Catalog No.: 40589-V08B1) labeled with Alexa Fluor ^™ 488 Protein Labeling Kit (ThermoFisher, Catalog No.: A20181);

e. Flow cytometry sorting of Live/dead ^- CD3 ^- CD19 ⁺ IgD ^- CD27 ⁺ IgG ⁺ and SARS-CoV-2 S probe-positive B cells to obtain SARS-CoV-2 S protein-specific Memory B cells.

(2) Amplification of SARS-CoV-2-specific memory B cell Ig variable region sequences (reference: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.)

a. Add the prepared cell lysate into a 96-well PCR plate. The system is as follows;

b. Add the sorted SARS-CoV-2 S protein-specific Memory B cells to the 96-well PCR plate containing the above-mentioned cell lysate;

c. Place the 96-well PCR plate on dry ice and quickly freeze the lysed cells;

d. Incubate at 68°C for 1 minute and place on ice;

e. Configure the cDNA synthesis system as shown in the following table:

f. PCR amplification to obtain antibody variable region cDNA, wherein the PCR reaction conditions are: 42°C for 5 min, 25°C for 10 min, 50°C for 60 min, and 94°C for 5 min;

g. The first round of PCR amplification of Ig variable region, the reaction system is as follows:

The above 5’First PCR primer mix and 3’First PCR primer mix refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

The first round of PCR amplification obtained the first round of PCR amplification products, wherein the PCR reaction conditions were: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 55 s amplification for 50 cycles; 72°C for 10 min;

h. The second round of amplification of Ig variable region, the reaction system is as follows:

Among them, the above-mentioned 5'Second PCR primer mix and 3'Second PCR primer mix are both referenced from the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

The second round of PCR amplification obtained the second round of PCR amplification products, wherein the PCR reaction conditions were: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 45 s amplification for 50 cycles; 72°C for 10 min;

i. Recover the second round of PCR amplification products using an agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03);

j. Specific amplification of Ig variable region to obtain specific PCR products. The reaction system is as follows:

Among them, the above-mentioned 5’Specific PCR primer mix and 3’Specific PCR primer mix refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

PCR reaction conditions for specific amplification: pre-denaturation at 94°C for 15 min; 50 cycles of amplification at 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), and 72°C for 45 s; 72°C for 10 min;

k. Detection of specific PCR products by agarose gel electrophoresis, the results are shown in Figure 2;

l. Recover specific PCR products using an agarose gel DNA recovery kit (Tian Gen, catalog number: DP209-03).

(3) Construction of expression plasmid, in vitro transfection, expression and purification of antibodies

3.1 Construction of expression plasmid

a. Take 30.4 μL of each of the above specific PCR products (IgH, Igκ and Igλ), add 3.4 μL of CutSmart buffer (NEB, catalog number: B7204S) and mix well to obtain the corresponding mixture;

b. Configure the enzyme digestion system as follows:

Among them, AgeI-HF, SalI-HF, BsiWI-HF and XhoI are NEB restriction enzymes, and their catalog numbers are R3552L

(AgeI-HF), R3138L (SalI-HF), R3553L (BsiWI-HF) and R0146L (XhoI).

c. Add the corresponding configured mixture of step b to each mixture in step a;

d. Enzyme digestion at 37°C for 2h;

e. Use agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03) to recover the digested product;

f. Connect the specific PCR product after enzyme digestion with the corresponding vector. The connection system is as follows:

Among them, the above-mentioned IgH vector is AbVec2.0-IGHG1 (AddGene, catalog number: 80795), the Igκ vector is AbVec1.1-IGKC (AddGene, catalog number: 80796), and the Igλ vector is AbVec1.1-IGLC2-XhoI (AddGene, catalog number: 99575).

g. Connect overnight at 16°C;

h. Add the overnight ligation product to a centrifuge tube containing 100 μL DH5α competent cells (Tian Gen, Catalog No.: CB101-02) and place on ice for 30 minutes;

i. Place the mixture of the ligation product and competent cells in step h in a 42°C water bath and heat shock for 90 seconds;

j. Take out and place on ice for 3-5 minutes, then add 900 μL TB medium (ThermoFisher, catalog number: 22711022);

k. After shaking culture for 45-60 minutes, centrifuge at 6000 rpm for 1 minute;

l. Aspirate 800 μL of supernatant and use the remaining liquid to mix the bacteria (derived from the competent cells described above);

m. The bacterial suspension in step l is evenly applied on an agar plate with ampicillin;

n. Place the agar plate upside down in a 37°C bacterial incubator and culture for 16 hours;

o. Pick a single full colony on the agar plate and send it to the company for sequencing together with the second round of PCR products;

p. Select the colonies that match 100% of the sequence of the second-round PCR product, and use the endotoxin-free plasmid mini-extraction kit (Tian Gen, catalog number: DP118-02) to extract the plasmids to obtain the heavy chain plasmid and the light chain plasmid respectively.

3.2 In vitro transfection and expression

a. One day before transfection, the density of 293 F cells was adjusted to 1×10 ⁶ cells/mL using FreeStyle ^™ 293 Expression Medium (Gibco, Cat. No.: 12338018);

b. Count the cells on the day of transfection and adjust the 293 F cell density to 2×10 ⁶ cells/mL using fresh FreeStyle ^™ 293 Expression Medium (Gibco, Cat. No.: 12338018);

c. Prepare PEI mixed solution: Add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) to OptiPRO ^TM SMF medium (ThermoFisher, Catalog No.: 12309019) to a concentration of 4 μg/mL during transfection;

d. Prepare plasmid mixture: add the extracted paired heavy chain plasmid and light chain plasmid into OptiPRO ^TM SFM medium (ThermoFisher, catalog number: 12309019) at a mass ratio of 1:2 and mix well to make the total plasmid concentration at the time of transfection to be 1 μg/mL;

e. Finally, add the PEI mixture to the plasmid mixture and mix gently to form a transfection mixture system, and let it stand at room temperature for 20 minutes;

f. Add the transfection mixture in step e to the 293 F cells in step b and shake gently to mix;

g. Place the 293 F cells from step f in a 37°C suspension incubator containing 8% CO ₂ and culture at 125 rpm for 7 days.

3.3 Antibody Purification

a. The transfected suspension culture medium was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;

b. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);

c. Open Protein purification instrument, use PBS to balance the Protein A column, flow rate 3mL/min;

d. The filtered expression supernatant was then loaded onto the Protein A column at a flow rate of 3 mL/min;

e. Use PBS to wash away non-specific binding proteins on the Protein A column at a flow rate of 3 mL/min;

f. Elute the antibody bound to the Protein A column using glycine buffer at pH 3.0 at a flow rate of 1 mL/min;

g. Collect the eluate to obtain the purified antibody.

(4) ELISA screening of antibodies that bind to the S protein of the original strain of the new coronavirus SARS-CoV-2

a. Coat the ELISA plate with SARS-CoV-2Wuhan-Hu-1 S protein (Sino Biological, Cat. No. 40589-V08B1) at a concentration of 2 μg/mL (100 μL/well);

b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d. After washing away the blocking solution with 1×PBST, add the purified antibody to the ELISA plate at a concentration of 1 μg/mL and incubate at 37°C for 1 hour;

e. After washing away the unbound antibodies with 1×PBST, add RHP-labeled anti-human IgG antibody (Jacksonimmunoresearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h. The OD value was measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific), and the OD value was used to reflect the binding strength of the antibody.

Antibodies with a broad spectrum of activity against SARS-like coronaviruses and/or new coronavirus mutants (VSCM16-12) were selected based on the binding strength of the antibodies to the SARS-CoV-2 S protein.

After sequencing and identification, the amino acid sequence of the heavy chain variable region of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant strain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The gene sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 4. The amino acid sequence of the full-length heavy chain of the antibody is shown in SEQ ID NO: 5; the amino acid sequence of the full-length light chain of the antibody is shown in SEQ ID NO: 6.

SEQ ID NO: 1:

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYFWGWIRQPPGKGLEWIGSFHYSGSTFYNPSLKSRITISVDTSKNQFSLSLISVTAADTAVYYCARGGYSGYFDYWGQGTLVTVSS.

SEQ ID NO: 2:

DIQMTQSPSTLSASVGDRVTITCRASQSISVWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSNSDLYTFGQGTKLEIK.

SEQ ID NO: 3:

CAGGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTTCTGGGGCTGGATCCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTTTCCATTACAGTGGGAGCACCTTCTACAACCCGTCCCTCAAGAGTCGAATCACCATATCCGTAGACACGTCCAAGAACCAATTCTCCCTGAGTCTGATCTCAGTG ACCGCCGCAGACACGGCTGTATATTACTGTGCGAGAGGTGGATATAGTGGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC.

SEQ ID NO: 4:

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGTCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACT TATTACTGCCAACAGTCTAATAGTGATCTGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA.

SEQ ID NO: 5:

MGWSCIILFLVATATGVHSQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYFWGWIRQPPGKG

LEWIGSFHYSGSTFYNPSLKSRITISVDTSKNQFSLSLISVTAADTAVYYCARGGYSGYFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

SEQ ID NO: 6:

MGWSCIILFLVATAVHSDIQMTQSPSTLSASSVGDRVTITCRASQSISVWLAWYQQKPGKAPKLL

IYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSNSDLYTFGQGTKLEIKRTVAAPSVF

IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Example 2: Binding Experiment

(1) ELISA to determine the targets of broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants and SARS-CoV-2 S protein binding

a. Coat the ELISA plate with SARS-CoV-2 S1 protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV-2 S2 protein (Sino Biological, Catalog No.: 40590-V08B) at a concentration of 2 μg/mL (100 μL/well);

b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d. After washing away the blocking solution with 1×PBST, add the above purified antibodies and control antibodies to the ELISA plate at a concentration of 1 μg/mL and incubate at 37°C for 1 hour;

e. After washing away the unbound antibodies with 1×PBST, add RHP-labeled anti-human IgG antibody (Jacksonimmunoresearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h. The OD value was measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific), and the OD value was used to reflect the binding strength of the antibody.

The results are shown in Figure 4. VSCM16-12 exhibited strong binding ability to SARS-CoV-2 S1 protein, but had no binding to SARS-CoV-2S2, indicating that the antibody is an antibody targeting S1.

(2) Expression and purification of SARS-like S1 protein

a. One day before transfection, the density of 293 F cells was adjusted to 1×10 ⁶ cells/mL using FreeStyle ^™ 293 Expression Medium (Gibco, Cat. No.: 12338018);

b. Count the cells on the day of transfection and adjust the 293 F cell density to 2×10 ⁶ cells/mL using fresh FreeStyle ^™ 293 Expression Medium (Gibco, Cat. No.: 12338018);

c. The gene sequences encoding the SARS-like coronavirus RaTG13, WIV1, GX-P5L, GD18, SZ3, and Civet007 S1 proteins were synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector with a Strep-Tactin tag (SA-WSHPQFEK-(GGGS)2-GGSAWSHPQFEK). The gene numbers of the above SARS-like coronaviruses are: RaTG13 (Genebank: MN996532.2), WIV1 (Gendbank: KC881007.1), GX-P5L (Genebrank: MT040335.1), GD18 (Genebbank: MT799524.1), SZ3 (Geneban k: AY304486.1), and Civet007 (Genebang: AY572034.1).

d. Prepare transfection mixture: add the plasmid synthesized in step c to Opti-MEM ^TM (ThermoFisher, Catalog No.: 31985062) medium and mix well to make the transfection plasmid concentration 1 μg/mL, then add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) and mix well to make the PEI transfection concentration 4 μg/mL;

e. Add the transfection mixture in step d to the 293 F cells in step a and shake gently to mix;

f. The 293 F cells from step e were placed in a 37°C suspension incubator containing 8% CO ₂ and cultured at 125 rpm for 7 days;

g. The transfected suspension culture was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;

h. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);

i. Open Protein purification instrument, using Buffer W (IBA Lifesciences, catalog number: 2-1003-100) to separate Strep- The column was balanced with a high capacity cartridge (IBA Lifesciences, catalog number: 2-1237-001) at a flow rate of 1 mL/min.

j. Then the filtered expression supernatant was loaded with Strep- High capacity cartridge column, flow rate 1mL/min;

k. Use Buffer W to wash away non-specific binding proteins on the column at a flow rate of 1 mL/min;

l. Use Buffer BXT (IBA Lifesciences, Catalog No.: 2-1042-025) to elute the protein bound to the column at a flow rate of 1 mL/min;

m. Collect the eluate to obtain the purified SARS-like S1 protein.

(3) ELISA test of the half effective concentration (EC ₅₀ ) of the constructed antibody against the novel coronavirus SARS-CoV-2 and SARS-like S1 protein

a. Coat the ELISA plate with the above-mentioned purified SARS-like S1 protein or SARS-CoV-2 S1 protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV S1 protein (Sino Biological, Catalog No.: 40150-V08B1) at a concentration of 2 μg/mL (100 μL/well);

b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d. After washing away the blocking solution with 1×PBST, the purified antibody in Example 1 was diluted (using a blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933)) to obtain antibody solutions with concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, 0.0005, 0.0017 and 0 μg/mL, respectively. The antibody solutions were added to the ELISA plate and incubated at 37°C for 1 h.

e. Unbound antibodies were washed away with 1× PBST, and then RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Catalog No.: 109-035-003) was added and incubated at 37°C for 1 h;

f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h. OD values were measured using Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

i. The data were calculated using Prism 8.0 software (GraphPad) to obtain the half effective concentration (EC ₅₀ ) of the antibody against the novel coronavirus SARS-CoV-2 Wuhan-Hu-1 and Omicron mutants.

The results are shown in Figure 5. The VSCM16-12 antibody showed strong binding ability to the new coronavirus SARS-CoV-2 and SARS-CoV as well as the animal-derived SARS-like coronavirus S1, with EC ₅₀ ranging from 35.10-58.18 ng/mL.

Example 3: Virus Neutralization Experiment

(1) Pseudovirus Encapsulation

a. One day before infection, 293T cells were seeded into a 10 cm cell culture dish at a density of 5 × 10 ⁶ cells/well;

b. The gene sequence encoding the S protein of SARS-CoV-2 or SARS-CoV or animal-derived SARS-like virus RaTG13 or WIV1 or GX-P5L or GD18 or SZ3 or Civet007 or Alpha mutant strain or Beta mutant strain or Gamma mutant strain or Delta mutant strain was synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector. Among them, the gene numbers of the above-mentioned SARS-like coronaviruses and mutants are: SARS-CoV-2 (Genebank: YP_009724390.1), SARS-CoV (GenBank: Y463060.1), RaTG13 (Genebank: MN996532.2), WIV1 (Gendbank: KC881007.1), GX-P5L (Genebrank: MT040335.1), GD18 (Genebbank: MT799524.1), SZ3 (Geneban k: AY304486.1), Civet007 (Genebang: AY572034.1), SARS-CoV-2Alpha (Genebank: OV054768.1), SARS-CoV-2Beta (Genebank: MZ433432.1), SARS-CoV-2Gamm (Genebank: MZ427312.1), SARS-CoV-2Delta (Genebank: OK091 006.1);

c. Prepare a plasmid mixture: add the plasmid synthesized in step b and plasmid PNL4-3 (Genebank: AF324493.2) at a mass ratio of 1:3 to Opti-MEM ^TM (ThermoFisher, catalog number: 31985062) culture medium and mix well to make the total plasmid concentration 1 μg/mL;

d. Prepare PEI mixed solution: Add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) to Opti-MEM ^TM medium (ThermoFisher, Catalog No.: 31985062) to a concentration of 4 μg/mL during transfection;

e. Finally, add the PEI mixture to the plasmid mixture and mix gently to form a transfection mixture system, and let it stand at room temperature for 20 minutes;

f. Discard the 293T cell culture medium in step a, and add the transfection mixture in step d to the 293T cells;

g. The 293T cells from step f were placed in a 37°C cell culture incubator containing 5% CO2 for 6 h;

h. Aspirate the transfection mixture and replace with freshly prepared DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106);

i. After culturing in a 37°C cell culture incubator containing 5% CO ₂ for 72 hours, the supernatant was collected by centrifugation at 12000 rpm for 15 minutes. The supernatant was the packaged pseudovirus.

(2) Pseudovirus neutralization assay to detect the half inhibitory concentration (IC 50 ) of antibodies against novel coronavirus SARS-CoV _- 2, SARS-CoV, animal SARS-like coronavirus RaTG13, WIV1, GX-P5L, GD18, SZ3, Civet007, and novel coronavirus Alpha, Beta, Gamma, and Delta mutants

a. One day before infection, 293T/hACE2 cells were seeded into 96-well plates at a density of 0.8×10 ⁵ cells/mL at 100 μL per well (the culture medium used was

For: DMEM containing 10% FBS;

b. On the day of infection, the purified antibodies obtained in Example 1 were mixed with the packaged pseudoviruses described above, and diluted to obtain multiple mixed solutions;

Each group of mixed solutions includes mixed solutions with antibody concentrations of 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.137, 0.046, 0.015, 0.005, 0.017 and 0 μg/mL; diluted with DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106);

c. Incubate the mixture of the antibody and pseudovirus obtained in step b at 37°C for 1 h;

d. Discard the medium in the 96-well plate in step a, add the antibody and virus mixture in step c, and centrifuge at 800g for 30min;

e. After incubation at 37°C in a cell culture incubator for 6-8 hours, the antibody and virus mixture was discarded and freshly prepared DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106) was added;

f. After the cells were cultured for 48 h, 50 μL of cell lysis buffer (Promega, catalog number: E153A) was added to each well of the 96-well culture plate and lysed at 37°C for 2 min;

g. The 96-well culture plate was then placed in a -40°C freezer for 30 min;

h. After freezing, the 96-well culture plate was taken out and placed at 37°C for 3 min for lysis, and then centrifuged at 2000 rpm for 1 min to obtain cell lysate;

i. Pipette 40 μL of the above cell lysate and add it into the 96-well black flat floor;

j. Add 50 μL of luciferase detection reagent (Promega, catalog number: E1501) and measure the OD value using a Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

k. Calculate the neutralization inhibition rate: inhibition rate = [1 - (OD value of the well with the mixture of antibody and virus - OD value of the blank well) / (OD value of the well with only virus but no antibody added - OD value of the blank well] × 100%.

1. Based on the results of neutralization inhibition rate, IC ₅₀ of the antibody was calculated using Prism 8.0 software (GraphPad).

As shown in Figure 6, VSCM16-12 showed strong neutralization ability against the new coronavirus SARS-CoV-2 and SARS-CoV, as well as SARS-like coronaviruses from animals, with IC ₅₀ values for RaTG13, GX-P5L, Civet007, WIV1, and SZ3 ranging from 20.26 to 89.94 ng/mL. And as shown in Figure 7, VSCM16-12 also had neutralization ability against the Alpha, Beta, Gamma, and Delta mutants of the new coronavirus, with IC ₅₀ values for the Gamma and Delta mutants reaching 67.2 ng/mL and 69.53 ng/mL, respectively.

The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims (10)

1. An antibody with broad spectrum against SARS-like coronavirus and/or novel coronavirus mutant strains, comprising a heavy chain and a light chain, characterized in that the antibody has at least one of the following technical features: (1) The heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GGSISSSSYF; (2) the heavy chain includes a heavy chain CDR2, whose amino acid sequence is: FHYSGST; (3) the heavy chain includes a heavy chain CDR3, the amino acid sequence of which is: ARGGYSGYFDY; (4) the light chain includes a light chain CDR1, whose amino acid sequence is: QSISVW; (5) The light chain includes a light chain CDR2, whose amino acid sequence is: KAS; (6) The light chain includes a light chain CDR3, whose amino acid sequence is: QQSNSDLYT. 2. The antibody according to claim 1, characterized in that the antibody has at least one of the following technical features: (a) the heavy chain comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is: GGSISSSSYF, the amino acid sequence of heavy chain CDR2 is: FHYSGST, and the amino acid sequence of heavy chain CDR3 is: ARGGYSGYFDY; (b) The light chain includes light chain CDR1-3, the amino acid sequence of light chain CDR1 is: QSISVW, the amino acid sequence of light chain CDR2 is: KAS, and the amino acid sequence of light chain CDR3 is: QQSNSDLYT. 3. The antibody according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2. 4. A nucleic acid molecule encoding a broad-spectrum antibody against SARS-like coronavirus and/or new coronavirus mutant strains as described in any one of claims 1 to 3. 5 . The nucleic acid molecule according to claim 4 , characterized in that the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 3 and/or the nucleotide sequence SEQ ID NO: 4. 6. Use of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibody as described in any one of claims 1 to 3 in the preparation of a diagnostic reagent or diagnostic kit or drug. 7. The use according to claim 6, characterized in that the drug has a neutralizing antiviral effect against SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the novel coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, novel coronavirus Alpha mutant, novel coronavirus Beta mutant, novel coronavirus Gamma mutant, novel coronavirus Delta mutant, and novel coronavirus Omicron mutant. 8. Use of the nucleic acid molecule as described in claim 4 or 5 in the preparation of broad-spectrum antibodies or drugs against SARS-like coronavirus and/or new coronavirus mutant strains. 9. The use according to claim 8, characterized in that the drug has a neutralizing effect on SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the novel coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, novel coronavirus Alpha mutant, novel coronavirus Beta mutant, novel coronavirus Gamma mutant, novel coronavirus Delta mutant, and novel coronavirus Omicron mutant. 10. An expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule according to claim 4 or 5.