CN115141272B

CN115141272B - Novel coronavirus monoclonal antibody XY1 and application thereof

Info

Publication number: CN115141272B
Application number: CN202210704198.6A
Authority: CN
Inventors: 徐义; 罗均利; 蔡开妹
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2021-01-31
Filing date: 2021-01-31
Publication date: 2024-06-25
Anticipated expiration: 2041-01-31
Also published as: CN115109150A; CN113150130B; CN115109150B; CN113150130A; CN115141271A; CN115141271B; CN115109151A; CN115141272A; CN115109151B

Abstract

The invention discloses a novel coronavirus monoclonal antibody XY1 and application thereof. The fully humanized antibody XY1 of the anti-SARS-Cov-2S 1 or RBD protein is screened, and the antibody has important scientific significance and clinical application prospect for preventing, treating and diagnosing SARS-Cov-2.

Description

Novel coronavirus monoclonal antibody XY1 and application thereof

The application is provided with the application number 2021101325814, the application date 2021, 1 month and 31 days, and the application is named as follows: novel coronavirus monoclonal antibodies and uses thereof.

Technical field:

The invention belongs to the technical field of monoclonal antibody screening and preparation, and particularly relates to a novel coronavirus monoclonal antibody XY1 and application thereof.

The background technology is as follows:

SARS-Cov-2 is a linear single-stranded positive strand RNA virus whose structural proteins include spike protein (S), small envelope protein (E), envelope protein (membrance, M) and nucleoprotein (N). The S protein is composed of S1 and S2 subunits.

SARS-Cov-2 binds to the membrane protein of Angiotensin converting enzyme 2 (ACE 2, angiotensin-converting enzyme) on the surface of pulmonary epithelial cells via spike protein (S protein) on the surface of the viral particle, and ACE2 subsequently undergoes a change in shape and structure, resulting in the entry of the virus into the cell. Based on a new coronavirus pathogenic mechanism, the binding of S protein and cell surface ACE2 is blocked by a neutralizing antibody of S protein, so that the virus is blocked from entering cells.

When SARS-Cov-2 invades the body, it induces the body to produce corresponding effector B cells and memory B cells, and the effector B cells produce antibodies, which can be used for diagnosis and treatment of SARS-Cov-2. The plasma obtained from SARS-Cov-2 patient is infused into SARS-Cov-2 patient, and the symptoms can be improved. However, the number of plasma of SARS-Cov-2 healers is limited in the face of a huge number of patients, and monoclonal antibodies can be produced in vitro on a large scale by screening effective neutralizing antibodies against SARS-Cov-2 in the SARS-Cov-2 healers, and by genetic engineering and protein expression techniques.

The invention comprises the following steps:

The main object of the present invention is to provide a specific monoclonal antibody against SARS-Cov-2 and its use. The mononuclear cells are obtained from SARS-Cov-2 rehabilitee to construct phage library, and the antibody specific to SARS-Cov-2 is fast screened, and the antibody can be used in clinical detection, diagnosis, prevention and treatment of SARS-Cov-2.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

extracting peripheral blood from SARS-Cov-2 rehabilitee, obtaining mononuclear cells, extracting total RNA, synthesizing cDNA, amplifying heavy chain variable region and light chain variable region by using heavy chain variable region and light chain variable region combining primer, randomly combining obtained heavy chain variable region and light chain variable region, recombining with phage vector, converting XL1-Blue by recombination product, adding auxiliary phage VCSM13 to obtain SARS-Cov-2 phage antibody library with titer of about 10 ¹³. The antigen S1 or RBD is used for screening anti-Fab antibody, and the heavy chain variable region and the light chain variable region sequence which can be combined with the S1 or RBD are obtained through sequencing, the heavy chain variable region is combined with an Fc fragment of IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express the fully humanized anti-S1 or RBD antibody. Monoclonal antibodies which can be used for SARS-Cov-2 detection, diagnosis, prevention or treatment are screened through in vitro affinity assay and pseudovirus neutralization experiments.

The novel coronavirus antibodies screened by the invention comprise 11 antibodies in total of XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY 11.

The invention is screened:

the XY1 antibody heavy chain variable region comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY1 antibody heavy chain variable region amino acid sequence:

GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS;

the XY1 antibody light chain variable region comprises:

CDRL1 amino acid sequence: QGIRNS A

CDRL2 amino acid sequence: DAS (DAS)

CDRL3 amino acid sequence: QHYFGTPLT A

Preferably the XY1 antibody light chain variable region amino acid sequence:

GSTGDAEIVMTQSPSSLSASEGDRVIITCRASQGIRNSLAWYQQKPGKAPKLLLYDASKLESGVPSRFSGSGSGTHFTLTIDSLQPEDFATYYCQHYFGTPLTFGGGTKVEIK;

the XY2 antibody heavy chain variable region comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY2 antibody heavy chain variable region amino acid sequence:

GSTGDEVQLVESGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS;

the XY2 antibody light chain variable region comprises:

CDRL1 amino acid sequence: QTISKY A

CDRL2 amino acid sequence: EAS (electronic article surveillance)

CDRL3 amino acid sequence: QQSYSSRFT A

Preferably the XY2 antibody light chain variable region amino acid sequence:

GSTGDAAIRLTQSPSSLSASVGDTVTITCRASQTISKYLHWYQQKPGEAPKLLISEASTFQGGVSSRFSGSRSGTDFTLTIYSLQPEDSATYYCQQSYSSRFTFGPGTKVEIK;

the XY3 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY3 antibody heavy chain variable region amino acid sequence:

The XY3 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: QGISSW A

CDRL2 amino acid sequence: AAS (architecture for service)

CDRL3 amino acid sequence: QQANSFPLT A

Preferably the XY3 antibody light chain variable region amino acid sequence:

GSTGDAAIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGQGTKLEIK;

the XY4 antibody heavy chain variable region amino acids comprise:

CDRH1 amino acid sequence: GGTFSSIA A

CDRH2 amino acid sequence: IIPIFGTA A

CDRH3 amino acid sequence: ARDVIEATIYGMDV A

Preferably XY4 antibody heavy chain variable region amino acid sequence:

GSTGDQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSIAINWVRQAPGQGLAWMGKIIPIFGTANYAQKFQGRVTMTADESTNTAYMELSSLRSEDTAVYYCARDVIEATIYGMDVWGQGTTVTVSS;

The XY4 antibody light chain variable region amino acids comprise:

CDRL1 amino acid sequence: QSISTY A

CDRL2 amino acid sequence: GAS (GAS)

CDRL3 amino acid sequence: QQSYSAPYT A

Preferably the XY4 antibody light chain variable region amino acid sequence:

GSTGDADIVMTQSPSSLPASVGDRVTITCRTSQSISTYVNWYQQKSGNAPELLMYGASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFAQGTKLEIR;

the XY5 antibody heavy chain variable region amino acids comprise:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY5 antibody heavy chain variable region amino acid sequence:

the XY5 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: QGVSNY A

CDRL2 amino acid sequence: AAS (architecture for service)

CDRL3 amino acid sequence: QHYDSPPYT A

Preferably the XY5 antibody light chain variable region amino acid sequence:

GSTGDAVIWMTQSPSSLSASMGDRVTITCRASQGVSNYLAWYQHKPGKAPELLIYAASTLQSGVPSRFSASRSGTDFTLTISSLQPEDIATYYCQHYDSPPYTFGQGTKLEVK;

the XY6 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY6 antibody heavy chain variable region amino acid sequence:

GSTGDEVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS;

The XY6 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: ALAKHF A

CDRL2 amino acid sequence: KDT (KDT)

CDRL3 amino acid sequence: QSPDTTGRI A

Preferably the XY6 antibody light chain variable region amino acid sequence:

GSTGDASYELTQPPSVSVSPGQTARITCSGDALAKHFGHWYQQRPGQAPVLVIYKDTERPLGIPERFSGSSSGATVTLTISAVEAEDEADYYCQSPDTTGRIFGGGTKVTVLGQPKA;

the XY7 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY7 antibody heavy chain variable region amino acid sequence:

The XY7 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: QGISSW A

CDRL2 amino acid sequence: AAS (architecture for service)

CDRL3 amino acid sequence: QQSYSIPRT A

Preferably the XY7 antibody light chain variable region amino acid sequence:

GSTGDAAIRLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPRTFGQGTKLEIK;

the XY8 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably the XY8 antibody heavy chain variable region amino acid sequence:

QVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS;

the XY8 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: QDISDW A

CDRL2 amino acid sequence: RAV (RAV)

CDRL3 amino acid sequence: QQTNTFPIT A

Preferably the XY8 antibody light chain variable region amino acid sequence:

GSTGDAVIWMTQSPIQMTQSPSSVSAYVGDRVTITCRASQDISDWLAWYQQAPGKAPKLLIYRAVTLQDDVPSRFSGSGSGTDFSLTITGLQREDFATYYCQQTNTFPITFGHGTRLEIK;

the XY9 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY9 antibody heavy chain variable region amino acid sequence:

the XY9 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: SSDVGSYNL A

CDRL2 amino acid sequence: EVT (EVT)

CDRL3 amino acid sequence: ISYAGNNNLV A

Preferably the XY9 antibody light chain variable region amino acid sequence:

GSTGDAQSALTQPPSVSGAPGQTVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIIEVTKRPPGVPDRFSGSKSGNTASLTVTGLQAEDEADYHCISYAGNNNLVFGGGTQLTVLGQPKA;

the amino acid sequence of the heavy chain variable region of the XY10 antibody comprises

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY10 antibody heavy chain variable region amino acid sequence:

The XY10 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: SSDIGRSS A

CDRL2 amino acid sequence: RNN (RNN)

CDRL3 amino acid sequence: AAWDNTLRGYV A

Preferably XY10 antibody light chain variable region amino acid sequence:

GSTGDASYELTQLPSASGTPGQRVTISCSGSSSDIGRSSVNWYQQLPGTAPKLLIYRNNQRPSGVPDRLSGSKSGTSGSLAISGLQSEDEADYYCAAWDNTLRGYVFGTGTKVTVLGQPKA;

the XY11 antibody heavy chain variable region amino acid sequence comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV A

Preferably XY11 antibody heavy chain variable region amino acid sequence:

The XY11 antibody light chain variable region amino acid sequence comprises:

CDRL1 amino acid sequence: QSVLFSPNNKNY A

CDRL2 amino acid sequence: WAS (WAS)

CDRL3 amino acid sequence: QQYDSSPWT A

Preferably the XY11 antibody light chain variable region amino acid sequence:

GSTGDADIQLTQSPDSLAVSLGERATINCKSSQSVLFSPNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYDSSPWTFGQGTKVEIK.

the antibodies of the invention comprise conventional constant regions in addition to the heavy and light chain variable regions described above.

The novel coronavirus antibody comprises XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11, and 11 antibodies, wherein the 11 antibodies have better affinity reaching nM level and have neutralization effect on pseudoviruses through detection affinity and in-vitro neutralization experiments; it is further preferred to include a combination of two antibodies XY4, XY10, the additive neutralization effect of which is very pronounced.

The novel coronavirus antibody, preferably at least one of XY1, XY4, XY7 and XY10 antibodies, is an antibody for novel coronavirus treatment; IC50 values for these four antibodies have significant advantages; it is further preferred that the combination of XY4 and XY10 antibodies is an antibody for use in novel coronavirus therapy. The detection phase of XY4 suitable for antigen detection is shown according to the ELISA detection results of different antibody combinations; at least one of the XY1, XY2, XY3, XY5, XY6, XY7, XY8, XY9 antibodies is suitable as a coating antibody for detecting an antigen.

The invention further preferably provides the use of a combination of coated and detected antibodies in the preparation of a reagent for detecting novel coronaviruses:

the coating antibody XY1 is used to detect at least one of antibodies XY4, XY7, XY8, XY9, XY10, and XY11, preferably at least one of antibodies XY4 and XY10, and more preferably to detect antibody XY4.

Coating the antibody XY2 to detect at least one of the antibodies XY4, XY9 and XY10 in a combined way, and preferably detecting at least one of the antibodies XY4 and XY 10; further preferred is detection of antibody XY4.

The coating antibody XY3 is used to detect at least one of the antibodies XY4 and XY10, preferably the antibody XY4.

Coating the antibody XY4 to detect at least one of the antibodies XY2, XY8 and XY10 in combination, preferably detecting the antibody XY10;

coating the antibody XY5 and combining at least one of the detection antibodies XY4 and XY10, preferably detecting the antibody XY4;

Coating the antibody XY6 to detect at least one of the antibodies XY4 and XY10 in a combined way, and preferably detecting the antibody XY4;

coating the antibody XY7 and combining at least one of the detection antibodies XY4 and XY10, preferably detecting the antibody XY4;

coating the antibody XY8 to detect at least one of the antibodies XY4 and XY10 in combination, preferably detecting the antibody XY4;

Coating the antibody XY9 and combining at least one of the detection antibodies XY4 and XY10, preferably detecting the antibody XY4;

Coating an antibody XY10 and detecting an antibody XY4 in a combined manner;

coated antibody XY11 antibody XY4 was detected in combination.

The novel coronavirus neutralizing antibody of the present invention further comprises sequences having at least 80% identity between the amino acid sequences of the heavy chain variable region and the light chain variable region and the amino acid sequences of the 11 kinds of antibody heavy chain variable region and the amino acid sequences of the antibody light chain variable region.

Antibodies of the invention that also include conservative sequence variants of the amino acid sequence of the antibody are also included within the scope of the invention. Conservative amino acid sequence variants include modifications of the amino acid sequence that do not significantly alter the properties of the neutralizing antibodies of the invention, such as variants resulting from similar amino acid substitutions, deletions, additions of amino acids, as are well known in the art.

The neutralizing antibodies of the present invention also include both human and non-human antibodies, as well as all antibodies that have the same function or are engineered and optimized as the neutralizing antibodies described above. Further comprises: glycosylation or polyethylene glycol modification, and the like.

The novel coronavirus monoclonal antibody also comprises any one of Fab, fab '-SH, fv, scFv and (Fab') 2 fragments, and any one of the heavy chain variable region and the light chain variable region of the antibody.

Fab refers to a portion of an antibody molecule comprising a variable and constant region of a light chain and a variable and constant region of a heavy chain joined by disulfide bonds.

Fab' -SH refers to Fab fragments comprising a portion of the hinge region.

Fv refers to the smallest antibody fragment that contains the antibody heavy chain variable region, light chain variable region and has all antigen binding sites.

ScFv refers to an engineered antibody in which the light chain variable region is linked directly to the heavy chain variable region or through a peptide chain.

(Fab ') 2 refers to the dimer of Fab'.

The invention also provides nucleic acid sequences of the heavy and light chain variable regions of the novel coronavirus neutralizing antibodies.

Also included are nucleic acid sequences that have greater than 80% identity thereto or that contain a degenerate codon that expresses the same amino acid.

The invention also provides a preparation for detecting, preventing or treating the novel coronavirus, which comprises the novel coronavirus neutralizing antibody.

The invention also provides application of the novel coronavirus neutralizing antibody, which comprises any one or more of the following components;

(1) For preparing novel coronavirus detection formulations;

(2) For the preparation of a formulation for the prevention of novel coronavirus infections;

(3) Is used for preparing a preparation for treating novel coronavirus infection.

The invention obtains mononuclear cells from a new coronary rehabilitee, extracts RNA, then reversely transcribes cDNA, amplifies heavy chain and light chain of an antibody by doubling as a heavy chain variable region and a light chain variable region primer, and assembles the heavy chain variable region and the light chain variable region onto a phage vector to construct a SARS-Cov-2 Fab phage antibody library; antibodies against Fab were screened using antigen S1 or RBD (RBD is a protein of S1 that plays a key role in binding to ACE 2), and by sequencing, heavy and light chain variable region sequences were obtained that bind to S1 or RBD, the heavy chain variable region being combined with an Fc fragment of IgG1, and the antibody heavy and light chains being expressed simultaneously in mammalian cells, the fully humanized antibodies against RBD or S1 were expressed. Through in vitro affinity measurement and pseudo-virus neutralization experiments, monoclonal antibodies with stronger affinity and virus neutralization capacity can be screened and used for SARS-Cov-2 detection, diagnosis, prevention or treatment. Plays a positive role in preventing and treating the novel coronavirus.

Drawings

FIG. 1 is an antibody heavy chain variable region amplification electrophoresis strip;

FIG. 2 is an antibody light chain variable region kappa chain amplification electrophoresis band;

FIG. 3 is an antibody light chain variable region lambda chain amplification electrophoresis band;

FIG. 4 is an electrophoresis band after fusion of antibody heavy and light chains;

FIG. 5 shows the electrophoresis bands after SfiI cleavage of pC3C plasmid;

FIG. 6 shows the results of colony PCR identification of the fusion products of the heavy chain and the light chain of the pC3C plasmid-linked antibodies transformed by XL 1-Blue;

FIG. 7 is a run 5 enriched phage antibody library ELISA assay;

helper phage (VCMS 13) was added to control wells and the experimental group was added to enrich phage for round 5;

FIG. 8 shows the results of PCR identification of monoclonal bacterial solutions by adding RBD phage to XL 1-Blue;

FIG. 9 shows expression of monoclonal antibodies and Coomassie brilliant blue staining;

FIG. 10 shows the results of ELISA assays for monoclonal antibodies;

Control wells were not added with antibody, but with anti-human Fab-HRP secondary antibody;

FIG. 11 is a monoclonal antibody IC50 assay;

FIG. 12 shows the detection of the antibody superposition neutralization effect using the combination of antibodies XY1, XY4, XY7, XY 10.

The specific embodiment is as follows:

the invention is further described below in connection with specific examples without limiting the invention.

The RBD protein of SARS-CoV-2 used in the embodiment of the invention is purchased from Beijing Yiqiao China biotechnology Co., ltd, and S1 of SARS-Cov-2 is purchased from Beijing Bai Caesalpis biotechnology Co., ltd.

The phage antibody library construction method of the invention is referred to in ：Antony S.Dimitrov(ed.),Generation and Selection of Rabbit Antibody Libraries by Phage Display,2009,Therapeμtic Antibodies:Methods and Protocols,vol.525,101-128.

Example 1

1. Construction of phage antibody library

18 Parts of SARS-Cov-2 convalescence patient peripheral blood (blood sample from convalescence patient within 1 month of discharge) were obtained and mononuclear cells were isolated using human peripheral blood lymph isolate. Total RNA was extracted, 18 parts of RNA were mixed in equal amounts, reverse transcribed into cDNA, heavy and light chain variable region fragments were obtained by amplification using heavy and light chain variable region-combining primers, antibody heavy and light chain variable regions were randomly spliced and ligated with phage vectors, and transformed into XL1-Blue bacteria (available from the institute of biotechnology, ltd.) in the presence of helper phage VSCM (available from the NTCC national collection of typical cultures) to assemble phage antibody display libraries.

The method specifically comprises the following steps:

1. Monocyte isolation

10Ml of peripheral blood was collected from each of 18 SARS-Cov-2 patients, and mononuclear cells were isolated using a human peripheral blood lymph isolate (LTS 1077-1, tianjin-located ocean Biometrics, inc.; co., ltd.).

(1) Blood collection: 10mL of peripheral blood of a donor is aseptically extracted, an anticoagulant (30 u/mL heparin) is added, and the mixture is packaged in a 15mL centrifuge tube.

(2) A15 mL centrifuge tube containing anticoagulated whole blood was centrifuged at 3000rpm for 10min, acceleration 7, deceleration 6.

(3) The upper plasma was aspirated and the lower cells were diluted with equal volume of PBS.

(4) A50 mL centrifuge tube was taken and the same amount of separation solution as the blood sample was added.

(5) The blood sample was carefully aspirated with a pipette and added to the surface of the separation solution at 500-1100g (1800 rpm), centrifuged for 10min, acceleration 5, deceleration 4.

(6) After centrifugation, the centrifuge tube is divided into four layers from top to bottom; the second layer is a cyclic milky white lymphocyte layer.

(7) The second layer of annular milky white lymphocyte layer was carefully pipetted into a new 15mL centrifuge tube, 10mLPBS added to the resulting tube and the cells were mixed.

(8) 1800Rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(9) The supernatant was discarded, and 5ml of the erythrocyte lysate was added to each 15ml centrifuge tube, and the mixture was left standing at room temperature for 5 minutes after being slightly mixed.

(10) 1800Rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(11) The supernatant was discarded and the resulting cells resuspended in 10mL PBS.

(12) 1800Rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(13) The supernatant was discarded and precipitated as PBMC.

2. Total RNA extraction

The obtained monocytes were subjected to extraction of total RNA by adding 0.75ml of Trizol (Thermo filter) to 5X10 ⁶ cells, and then 18 parts of RNA were mixed in equal amounts by taking 1. Mu.g of each RNA.

(1) 0.75Ml Trizol was added to 5X10 ⁶ cells.

(2) Mixing the above and the below for several times, and incubating on ice for 5min to ensure the cell to be fully lysed.

(3) Centrifuge at 12000rpm for 10 minutes at 4℃and transfer the supernatant to a clean centrifuge tube.

(4) 1MlTrizol ml of chloroform was added thereto and mixed well.

(5) Incubating on ice for 2-3min.

(6) Centrifuge at 12000rpm at 4℃for 15 min.

(7) Transferring the supernatant phase to a clean centrifuge tube.

(8) Adding isopropanol with equal volume into the supernatant and uniformly mixing.

(9) Incubate for 10min at room temperature, centrifuge at 12000rpm at 4℃for 10-15 min.

(10) The supernatant was discarded and the pellet was retained.

(11) The precipitate was washed with 1ml of 75% ethanol.

(12) Centrifuge at 12000rpm at 4℃for 5 min.

(13) The supernatant was discarded as much as possible.

(15) Drying at room temperature for 5-10min, wherein no liquid is visible on the tube wall.

(16) Add 20-50. Mu.l of RNAse free water-soluble RNA.

(17) 1 Mu lRNA was used for electrophoresis detection and RNA concentration measurement.

(18) The RNA is packaged and stored in a refrigerator at the temperature of-80 ℃ to avoid repeated freezing and thawing.

3. CDNA Synthesis

Mu.g of RNA was taken and used with cDNA synthesis kit (K1612, thermo filter).

(1) CDNA was synthesized by taking 5. Mu.g of RNA.

(2) The 10. Mu.l RNA/primer mixt. Mu.re system is shown in Table 1 below and includes:

TABLE 1

System components	Volume of
		RNA	5μg
50μM Oligo(dT)₂₀	1μl
		Hex Random Primer	1μl
10mM dNTP	1μl
		DEPC-treated water	Total volume of 10. Mu.l

(3) Incubate at 65℃for 5min, place on ice for 1min.

(4) Additional reagents were added as shown in Table 2 below to prepare CDNA SYNTHESIS Mix for the next reaction.

TABLE 2

System components	1 Reaction
		10xRT buffer	2μl
25mM MgCl₂	4μl
		0.1M DTT	2μl
RNaseOΜT(40Μ/μl)	1μl
		Superscript III RT(200Μ/μl)	1μl

(5) 10 Μl CDNA SYNTHESIS Mix to RNA/primer Mix was added, gently mixed, centrifuged, and reacted under the following conditions of Table 3:

TABLE 3 Table 3

Step1	50℃	50min
			Step2	85℃	5min
		Cooling on ice, and briefly centrifuging
			Step3	RNaseH	1μl
Step4	37℃	20min

(6) The cDNA product was stored in aliquots at-20 ℃.

4. Antibody heavy chain variable region (VH) and light chain variable region (VL) amplification

The synthesized cDNA was diluted 10-fold and used as a template for amplifying the heavy and light chain variable regions.

(1) The system was formulated as follows in Table 4, taking VH as an example:

TABLE 4 Table 4

(2) The reaction conditions are shown in the following table

TABLE 5

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s
		Go to Step 2x34
			Step5	72℃	1m 30s
Step6	4℃	Forever

(3) Electrophoresis

Mu.l of the sample was subjected to 1% agarose gel electrophoresis to determine whether the target band had been amplified, and the size was about 400bp (see FIG. 1).

(4) All amplified VH were mixed in equal amounts, added with 0.1 volumes of 3M sodium acetate and 2.2 volumes of ethanol, mixed well and left at-20 ℃ overnight.

(5) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis with 1% gel.

(6) The 400bp size band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was determined.

(7) The amplification and recovery of VL was performed according to steps (1) to (6), and the results are shown in FIG. 2 and FIG. 3.

(8) The concentration of nucleic acid after purification of VH and VL was adjusted to 100 ng/. Mu.l, and stored at-20 ℃.

5. Amplification of Cκ -pelB

(1) Cκ -pelB and CL-pelB were amplified using vectors pCκ and pCL (vectors purchased from add gene) (100 ng/. Mu.l) as templates

For example, amplified C.kappa. -pelB is shown in Table 6.

TABLE 6

pCκ	10μl
		HCK (see the aforementioned references for sequences)	60μl
Pelb (see the aforementioned references for sequences)	60μl
		ddH₂O	370μl
PrimerSTAR	500μl
		Total	1000μl

(2) The reaction conditions are shown in Table 7

TABLE 7

(3) Mu.l of the product was taken and electrophoretically detected, and the band size was about 400bp.

(4) All amplified Cκ -pelB were mixed, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol, mixed well and left at-20℃overnight.

(5) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis using 1% gel.

(7) The purified C kappa-pelB was diluted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.

(8) Amplifying CL-pelB, and performing the operation according to the steps (1) - (7), wherein the amplified template pCκ is replaced by pCL.

6. Human V kappa/Human C kappa/CL/Human VH or Human V lambda/Human CL/Human VH fusion is exemplified by a Human V kappa/Human C kappa/CL/Human VH fusion

(1) The different amounts of VL, VH, ck-pelB were mixed as in Table 8 below

VL concentration 100 ng/. Mu.l, VH concentration 100 ng/. Mu.l, C kappa-pelB concentration 100 ng/. Mu.l

TABLE 8

VL	10μl
		VH	10μl
Ck-pelB	10μl
		Primerstar Mix	500μl
H₂O	470μl
		Total	1000μl

(2) The reaction conditions are shown in Table 9

TABLE 9

Step1	98℃	15s
			Step2	98℃	10s
Step3	50℃	5s
			Step4	72℃	5s
Step5		Go to Step 2x10
			Step6	72℃	1m 30s
Step7	4℃	Forever

(3) Amplification of human VL/human C kappa/human VH, see Table 10

Table 10

Splice product	50μl
		C-5' SFIVL (see the abovementioned references for sequences)	4μl
C-3' sfivh (see the aforementioned references for sequences)	4μl
		ddH₂O	17μl
PrimerSTAR	25μl
		Total	100μl

(4) Amplification conditions are shown in Table 11

TABLE 11

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s(Go to Step2 x 39)
Step5	72℃	1m 30s
			Step6	4℃	Forever

(5) Electrophoresis detection

10 Mu l of PCR product is taken, electrophoresis detection is carried out, and the band size of the target gene after fusion is 1.2Kb. (results see FIG. 4)

(6) All amplified human VL/human C kappa/human VH were mixed by adding 0.1 volumes of 3M sodium acetate and 2.2 volumes of ethanol, and left overnight at-20 ℃.

(7) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis using 1% gel.

(8) The 1.2Kb size band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was determined.

(9) The Human VL/h. Mu. Man C.kappa./Human VH purified nucleic acid was diluted to a final concentration of 150 ng/. Mu.l and stored at-20 ℃.

(10) The fusion of the human V.lambda./human CL/human VH is carried out according to steps (1) to (9).

7. SfiI-cut human V kappa/human C kappa/CL/human VH and human V lambda/human CL/human VH

Take human V kappa/human C kappa/CL/human VH cleavage as an example

(1) The enzyme digestion system is shown in Table 12

Table 12

human Vκ/human Cκ/CL/human VH(150ng/μl)	200μl
		10Xbuffer	30μl
H₂O	60μl
		SfiI 40u/μl	10μl

(2) The reaction is carried out for 3 hours in a water bath at 50 ℃.

(3) Electrophoresis was performed using a 1% gel, and a 1.2 kb-sized band was excised and recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706).

(4) After SfiI digestion is detected, the human VL/human Ck/human VH fragments are recovered, the concentration is adjusted to 50 ng/. Mu.l, and the mixture is preserved at-20 ℃.

(5) The cleavage of the Human V.lambda./Human CL/Human VH is carried out according to steps (1) to (4).

8. SfiI cleavage pC3C (vector from add gene)

(1) The enzyme digestion system is shown in Table 13

TABLE 13

pC3C 1μg/μl	50μl
		10Xbuffer	30μl
H₂O	208μl
		SfiI 40u/μl	12μl

(2) The reaction is carried out for 3 hours in a water bath at 50 ℃.

(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment.

(Results see FIG. 5)

(4) 3.5Kb of vector backbone was recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706).

(5) The concentration of the recovered vector backbone after Sfi cleavage was adjusted to 100 ng/. Mu.l.

9. Connecting pC3C (SfiI) and Human VL/Human Cκ/Human VH (SfiI) and Human V λ/Human CL/Human VH

Taking pC3C (SfiI) and human VL/human C kappa/human VH (SfiI) connections as examples

(1) The connection system is shown in Table 14

TABLE 14

PC3C after cleavage of 100 ng/. Mu.l SfiI	1.5μl
		50 Ng/. Mu.l SfiI post-digestion human VL/human C kappa/human VH	2μl
10XT4 DNA ligase buffer	2μl
		H₂O	13.5μl
T4 DNA ligase 2000u/μl	1μl

The control group is shown in Table 15

TABLE 15

PC3C after cleavage of 100 ng/. Mu.l SfiI	1.5μl
		10XT4 DNA ligase buffer	2μl
H₂O	15.5μl
		T4 DNA ligase 2000u/μl	1μl

(2) The reaction was carried out at 16℃overnight.

(3) All ligation reactions were combined and 1/10 volume of 3M sodium acetate solution was added.

(4) Adding 2.2 times of pre-cooled absolute ethyl alcohol, uniformly mixing up and down, and precipitating at-20 ℃ overnight.

(5) 16000G, centrifuged at 4℃for 30min and the supernatant carefully discarded.

(6) The precipitate was gently washed with 70% ethanol, 16000g, centrifuged at 4℃for 5min and the supernatant discarded.

(7) And (5) drying at room temperature.

(8) The reaction mixture is dissolved in an appropriate amount of water, and usually 1 ligation reaction is performed by dissolving 1. Mu.l of ddH ₂ O in volume and storing it at-20 ℃.

(9) The connection pC3C (SfiI) and the Human V lambda/Human CL/Human VH were operated according to steps (1) - (8).

10. Ligation product conversion XL1-Blue

(1) XL1-Blue electric shock competent ice was placed for 10min for dissolution.

(2) Taking 19 μl of the precipitated and concentrated connection product, adding into a 1.5ml centrifuge tube, ice-bathing, adding 300 μl of XL1-Blue electric shock competence, mixing, rapidly transferring into a 2mm electric shock cup, and standing on ice for 1min.

(3) Electric shock conditions: 2.5KV,4ms

(4) After the completion of the electrotransfer, 5ml (1 ml+2ml+2ml) of SOC medium was rapidly added, and the mixture was transferred to a 50ml tip centrifuge tube, and incubated at 37℃and 250rpm for 1 hour. Mu.l of the bacterial liquid was added to 198. Mu.l of LB and simultaneously uniformly spread on LB plates containing 100. Mu.g/. Mu.l of carbenicillin, and incubated overnight at 37℃for calculation of transformation efficiency and colony PCR identification positive rate. The PCR results are shown in FIG. 6.

(5) 10Ml SB medium, 3. Mu.l 100. Mu.g/. Mu.l carbenicillin, and 30. Mu.l 5. Mu.g/. Mu.l tetracycline were added. The culture was carried out at 37℃and 250rpm for 1 hour.

(6) Add 4.5. Mu.l 100. Mu.g/. Mu.l carbenicillin and continue at 37℃at 250rpm for 1-4h.

(7) The cultured bacteria were transferred to a 500ml flask, 84ml SB medium, 42.5. Mu.l 100. Mu.g/. Mu.l carbenicillin, 170. Mu.l 5. Mu.g/. Mu.l tetracycline, and 1ml VCM 13 helper phage (10 ¹¹-10¹² pfu/ml), 37℃at 275rpm,90min were added.

(8) Mu.l of 50. Mu.g/. Mu.l kanamycin was added, incubated at 37℃and 275rpm overnight.

(9) 3000G, and centrifuged at 4℃for 15min.

(10) Precipitation phage

The supernatant (200 ml) was transferred to a 500ml clean centrifuge tube, 8g PEG-8000 and 6g NaCl were added, and the mixture was placed at 37℃at 300rpm for 5min to promote dissolution. Placing on ice for 30min-1h.15000g, centrifugation at 4℃for 15min. The supernatant was discarded, the centrifuge flask was placed upside down on filter paper and left to dry for 10min, taking care to remove excess liquid. Phage were resuspended in 2ml TBS (pipetted up and down) with 1% BSA, centrifuged at 16000g at 4℃for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. The product can be stored directly on ice in a short time or stored at-20deg.C by adding 0.01 times of 2% sodium azide in volume and adding 1 time of glycerol in a long time.

2. Phage antibody library screening

Taking RBD phage antibody screening as an example

5 Rounds of screening were performed using a 96-well plate coated with RBD protein, and then monoclonal antibodies were selected for identification of RBD-specific antibodies.

1. Antigen coating

(1) RBD was added to a carbonic acid buffer at a concentration of 2. Mu.g/ml,

(2) Kang Ninggao protein adsorption ELISA 96-well plates, 50 mu lRBD is added to each well, and the wells are left at 4 ℃ overnight;

(3) Discarding the supernatant, adding 200 μl/well of 5% skimmed milk powder, and standing at 37deg.C for 1 hr;

(4) The supernatant was discarded, and washed 5 times with 0.05% TBST.

2. RBD antibody screening

(1) Phage antibody library was diluted 10-fold with 2% nonfat milk powder, 2 wells coated with RBD antigen were taken, 100 μl of diluted phage was added, and left at 37deg.C for 1h.

(2) The supernatant was discarded, washed 5 times with 0.05% TBST, 100. Mu.l of 100mM glycine was added, and the mixture was left at 37℃for 15 minutes, during which time the blow was continued, and 9. Mu.l of 1M Tris was added.

(3) The phage eluted in step (2) was added to 2ml XL1-Blue bacteria and left at room temperature for 15min.

(4) 6Ml of SB medium was added, 1.6. Mu.l of 100. Mu.g/. Mu.l of carbenicillin, 12. Mu.g of 5. Mu.g/. Mu.l of tetracycline were added, and the mixture was incubated at 37℃for 1 hour at 250 rpm.

(5) 2.4. Mu.l of 100. Mu.g/. Mu.l of carbenicillin were added and the culture was continued for 1h;

(6) 1ml of helper phage VCSM13 (10 ¹¹-10¹² pfu/ml) was added, transferred to a 500ml flask, 91ml SB medium was added, 46. Mu.l 100. Mu.g/. Mu.l carbenicillin was added, 184. Mu.l 5. Mu.g/. Mu.l tetracycline was added, incubated at 37℃for 1.5h at 250rpm, 140. Mu.l 50. Mu.g/. Mu.l kanamycin was added, and incubated overnight at 37℃at 250 rpm.

(7) Phage antibody concentration: centrifuging the bacterial liquid in the last step to remove bacterial bodies, and collecting supernatant (100 ml); 4g PEG-8000 and 3g NaCl were added and the mixture was placed at 37℃at 300rpm for 5min to promote dissolution. Placing on ice for 30min-1h.15000g, centrifugation at 4℃for 15min. The supernatant was discarded, the centrifuge flask was placed upside down on filter paper and left to dry for 10min, taking care to remove excess liquid. Phage were resuspended in 2ml TBS (pipetted up and down) with 1% BSA, centrifuged at 16000g at 4℃for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. The product can be stored directly on ice in a short time or stored at-20deg.C by adding 0.01 times of 2% sodium azide in volume and adding 1 time of glycerol in a long time.

(8) Taking phage of step (7), and carrying out 5 rounds of screening according to steps (1) - (7).

(9) ELISA identification of antibody libraries from five rounds of screening:

1) ELISA 96-well plates, coated with 100ng RBD per well;

2) 100 μl phage (diluted 10-fold with 2% skimmed milk powder) was added and incubated at 37deg.C for 1h;

3) 0.05% TBST wash 5 times;

4) 100 μl of anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000-fold with 5% skimmed milk powder was added and incubated at 37deg.C for 1h;

5) 0.05% TBST wash 5 times;

6) Adding 100 μl TMB, and developing for 5min in dark;

7) The reaction was stopped by adding 50. Mu.l of 1M H ₂SO₄.

8) Absorbance was measured at 450nm using a microplate reader.

The results are shown in FIG. 7.

3. RBD monoclonal antibody identification

(1) Taking RBD phage selected in the 5 th round, diluting 10 ^-7, adding 1 μl to 200 μl XL1-Blue bacteria, and standing at room temperature for 15min;

(2) Coating all the bacteria in the step (1) on a carbenicillin LB individual culture medium containing 100 mug/ml, and culturing overnight at 37 ℃;

(3) 200 single clones were selected and added to 6ml of liquid LB medium containing 10. Mu.g/ml tetracycline and 100. Mu.g/ml carbenicillin, and cultured at 37℃and 250rpm for XY4 hours, and a small amount of bacterial liquid was taken for PCR identification of bacterial liquid, and clones with a band size of 1200bp were selected (see FIG. 8 for the results).

Colony PCR amplification system is shown in Table 16:

Table 16

Bacterial liquid	0.5μl
		VHSEQ (see the aforementioned references for sequences)	0.2μl
Vlseq (see the aforementioned references for sequences)	0.2μl
		ddH₂O	4.1μl
PrimerSTAR	5μl
		Total	10μl

Colony PCR reaction conditions are shown in Table 17:

TABLE 17

Step1	98℃	3min
			Step2	98℃	10s
Step3	50℃	5s
			Step4	72℃	15s
Step5		Go to Step 2x34
			Step6	72℃	1m 30s
Step7	4℃	Forever

(4) Uniformly dividing the positive clone bacterial liquid in the step (3) into 2 parts, adding 3 μl of helper phage VCSM13 (10 ¹¹-10¹² pfu/ml) into one part, standing at room temperature for 20min, adding 2.1 μl of 50 μg/μl of kanamycin, and culturing the other part overnight at 37 ℃ without adding any substance at 250 rpm;

(5) Centrifuging the bacteria added with the auxiliary phage at 4000rpm for 10min, transferring the supernatant into a clean centrifuge tube, diluting the supernatant by 10 times, taking 100 mu l of the supernatant to be added into an enzyme-linked immunosorbent assay plate coated with RBD, incubating at 37 ℃ for 1H, washing with 0.05% TBST for 5 times, adding 2000 times of diluted anti-phage antibody with HRP (purchased from Beijing-Yi-Qianshen Biotechnology Co., ltd.), incubating at 37 ℃ for 1H, washing with 0.05% TBST for 5 times, adding 100 mu lTMB, developing for 2min, adding 50 mu l of 1M H ₂SO₄, stopping the reaction, measuring absorbance at 450nm by using an enzyme-labeled instrument, and obtaining positive clones with absorbance higher than 2.1 times of the control.

(6) Selecting positive clones in the step (5), extracting plasmids without adding bacteria of auxiliary phage, and sequencing to obtain heavy chain variable region and light chain variable region sequences.

4. S1 phage antibody screening was performed according to steps 1-3.

3. Expression and Activity characterization of fully humanized antibodies

1. Fully humanized vector construction

(1) Designing corresponding primers according to the obtained antibody heavy chain variable region or light chain variable region sequence, respectively amplifying, adding secretion signal peptides at the N ends of the heavy chain and the light chain, and adding an Fc fragment of IgG1 at the C end of the heavy chain;

(2) The heavy chain variable region and the light chain variable region were each homologously recombined into the pcDNA3.4 vector. (pcDNA3.4 vector was purchased from Wuhan vast Biotechnology Co., ltd.)

2. Expression of fully humanized antibodies

(1) Extracting the corresponding heavy chain expression vector and light chain expression vector respectively by using endotoxin removal kit (purchased from OMEGA);

(2) Transfection with PEI was performed when 293T cells (purchased from the Living technologies Co., ltd.) reached 80% confluence;

(3) 12h after transfection, changing into serum-free protein expression culture medium, and culturing at 37 ℃ for 7 days by 5% CO ₂;

(4) Antibodies were purified using protein A/G packing. The results are shown in FIG. 9.

3. Identification of fully humanized antibody Activity

Taking RBD antibody Activity assay as an example

(1) ELISA identification

The obtained antibody was adjusted to a concentration of 1mg/ml, diluted 2000-fold with 5% nonfat dry milk, added to an ELISA well coated with RBD antigen, incubated at 37℃for 1H, washed 5 times with 0.05% TBST, diluted 2000-fold with 5% nonfat dry milk anti-human Fab-HRP secondary antibody (available from Beijing Soy Biotechnology Co., ltd.), incubated at 37℃for 1H, washed 5 times with 0.05% TBST, added 100. Mu. lTMB, developed for 2min, terminated with 50. Mu.l of 1M H ₂SO₄, and absorbance was measured at 450nm using a microplate reader. The results are shown in FIG. 10.

(2) Fully humanized antibody pseudovirus neutralization detection

1) Mixing 200 TCIDs 50 of pseudovirus expressing SARS-Cov-2S protein with a series of antibodies with different dilution concentrations in equal quantity, adding 100 μl of the total volume into a 96-well cell culture plate, incubating for 1h at 37 ℃, and adding 100 μl of 293T cells containing 20000 over-expressed ACE 2; the control was not added with antibody and an equivalent amount of pseudovirus was mixed with 293T over-expressed ACE 2; mixing background equivalent pseudoviruses with 293T cells; after leaving 5% CO ₂ at 37℃for 48 hours, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) ×100%. The results are shown in FIG. 11.

2) The antibody stack neutralization effect was detected using a combination of fully humanized antibodies XY1, XY4, XY7, XY10 as follows: mixing 200 TCIDs 50 of pseudovirus expressing SARS-Cov-2S protein with a series of antibodies with different dilution concentrations in equal volume (single antibody concentration 0.009766 mug/ml; equal mixing when two antibodies are combined, total concentration 0.009766 mug/ml), adding 100 mul total volume into 96-well cell culture plate, incubating for 1h at 37 ℃, adding 100 mul 293T cells over-expressing ACE2 of 20000; the control was not added with antibody and an equivalent amount of pseudovirus was mixed with 293T over-expressed ACE 2; mixing background equivalent pseudoviruses with 293T cells; after leaving 5% CO ₂ at 37℃for 48 hours, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) ×100%.

The results are shown in FIG. 12.

(3) Affinity assay for fully humanized antibodies

Determination of antibody affinity was performed using BIAcore 8k (BIAcore, cytiva), antibody was diluted to a concentration of 20. Mu.g/ml, injected at a flow rate of 10. Mu.l/min for 20s, yielding a 1000 Rm response. The binding affinity for SARS-CoV-2RBD protein was measured at a flow rate of 50. Mu.L/min and an injection time of 2 minutes. The affinity results are shown in Table 18.

Table 18 antibody affinity assay

(4) RBD detection for detecting SARS-Cov-2 by antibody combination

ELISA sandwich method is adopted for detection, monoclonal fully humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 are coated on ELISA plates, SARS-Cov-2 antigen RBD is added, XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 phage antibodies are added, different combinations are carried out, then antibodies with HRP marked anti-M13 are added, detection color development is carried out, the deeper the color shows that the capturing capacity of the antibody on SARS-Cov-2 antigen is stronger, and meanwhile the antibody is suitable for detection of SARS-Cov antigen. The results are shown in Table 19.

The specific operation is as follows:

1) ELISA 96-well plates, coated with 100ng of fully humanized antibody per well;

2) Mu.l of antigen (10 pg/ml) was added and incubated for 1h at 37 ℃;

3) 0.05% TBST wash 5 times;

4) Phage antibodies diluted 10-fold with 2% skimmed milk powder were added and incubated for 1h at 37 ℃;

5) 0.05% TBST wash 5 times;

6) anti-M13-HRP antibody (available from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000-fold with 5% nonfat milk powder was added and incubated at 37℃for 1h;

7) Adding 100 μl TMB, and developing for 5min in dark;

8) The reaction was stopped by adding 50. Mu.l of 1M H ₂SO₄.

9) Absorbance was measured at 450nm using a microplate reader.

10 The detection of S1 antigen is performed according to steps (1) - (9).

In Table 19 below, the monoclonal fully humanized antibodies are listed in the columns and phage antibodies are listed in the rows, and phage antibodies and the full antibody heavy chain variable region and light chain variable region are identical, except for the different forms of the same antibody, and the combination of the underlined data in the table below shows more excellent results.

Table 19 detection of RBD proteins by cross-combining different monoclonal antibodies

	XY1	XY2	XY3	XY4	XY5	XY6	XY7	XY8	XY9	XY10	XY11	Control
													XY1		0.8848	0.7326	3.497333	0.7735	0.475067	1.022033	1.0165	1.800633	3.002033	1.066633	0.07912
XY2	0.6635		0.7934	3.561467	0.551133	0.2924	0.631633	0.847033	1.0656	2.019467	0.958733	0.05423
													XY3	0.3361	0.59015		3.0677	0.33515	0.17445	0.369	0.4125	0.5762	1.27265	0.64975	0.06881
XY4	0.8688	1.1878	0.90005		0.60735	0.3471	0.9918	1.03855	0.83135	2.4142	0.90515	0.05987
													XY5	0.56505	0.79005	0.7313	2.83085		0.24635	0.69735	0.52915	0.7721	1.7483	0.6084	0.07762
XY6	0.42205	0.55845	0.54215	2.7278	0.4206		0.5086	0.48525	0.7785	1.3035	0.702	0.06931
													XY7	0.398	0.73475	0.9844	3.1126	0.4679	0.1885		0.41515	0.92155	1.0476	0.6443	0.05542
XY8	0.37835	0.46405	0.5455	3.0873	0.50125	0.24975	0.4681		0.7979	1.71195	0.68565	0.08339
													XY9	0.5695	0.5438	0.4639	2.3447	0.3912	0.206	0.6099	0.45535		1.32155	0.52985	0.06871
XY10	0.7053	0.75605	0.75415	3.2243	0.47525	0.17805	0.54015	0.5249	0.45935		0.5754	0.05324
													XY11	0.27535	0.4699	0.4894	2.7212	0.27635	0.13865	0.3255	0.4718	0.69185	0.92985		0.04996

The column is coated antibody, the row is detected antibody, no phage antibody is added to the blank, but helper phage VCSM13 and anti-M13-HRP antibody are added.

Through a series of activity identification, 11 novel coronavirus antibodies of monoclonal fully humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 are obtained.

Sequence listing

<110> Xiangya Hospital at university of south China

<120> Novel coronavirus monoclonal antibody XY1 and use thereof

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<170> SIPOSequenceListing 1.0

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<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 2

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 2

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 3

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 3

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 4

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 4

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

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Gln Gly Ile Arg Asn Ser

1 5

<210> 6

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

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Asp Ala Ser

1

<210> 7

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<212> PRT

<213> Homo sapiens (Homo sapiens)

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Gln His Tyr Phe Gly Thr Pro Leu Thr

1 5

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Gly Ser Thr Gly Asp Ala Glu Ile Val Met Thr Gln Ser Pro Ser Ser

1 5 10 15

Leu Ser Ala Ser Glu Gly Asp Arg Val Ile Ile Thr Cys Arg Ala Ser

20 25 30

Gln Gly Ile Arg Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Leu Tyr Asp Ala Ser Lys Leu Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr

65 70 75 80

Ile Asp Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His

85 90 95

Tyr Phe Gly Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 9

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

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Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 10

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 10

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 11

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 11

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 12

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 12

Gly Ser Thr Gly Asp Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 13

Gln Thr Ile Ser Lys Tyr

1 5

<210> 14

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 14

Glu Ala Ser

1

<210> 15

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 15

Gln Gln Ser Tyr Ser Ser Arg Phe Thr

1 5

<210> 16

<211> 113

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 16

Gly Ser Thr Gly Asp Ala Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser

1 5 10 15

Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Arg Ala Ser

20 25 30

Gln Thr Ile Ser Lys Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Glu

35 40 45

Ala Pro Lys Leu Leu Ile Ser Glu Ala Ser Thr Phe Gln Gly Gly Val

50 55 60

Ser Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Tyr Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Ser Arg Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 17

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 17

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 18

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 18

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 19

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 19

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 20

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 20

Gly Ser Thr Gly Asp Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 21

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 21

Gln Gly Ile Ser Ser Trp

1 5

<210> 22

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 22

Ala Ala Ser

1

<210> 23

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 23

Gln Gln Ala Asn Ser Phe Pro Leu Thr

1 5

<210> 24

<211> 113

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 24

Gly Ser Thr Gly Asp Ala Ala Ile Gln Met Thr Gln Ser Pro Ser Ser

1 5 10 15

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25 30

Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Ala Asn Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 25

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 25

Gly Gly Thr Phe Ser Ser Ile Ala

1 5

<210> 26

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 26

Ile Ile Pro Ile Phe Gly Thr Ala

1 5

<210> 27

<211> 14

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 27

Ala Arg Asp Val Ile Glu Ala Thr Ile Tyr Gly Met Asp Val

1 5 10

<210> 28

<211> 126

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 28

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly

20 25 30

Thr Phe Ser Ser Ile Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Ala Trp Met Gly Lys Ile Ile Pro Ile Phe Gly Thr Ala Asn

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Ala Asp Glu Ser

65 70 75 80

Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Asp Val Ile Glu Ala Thr Ile Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 29

Gln Ser Ile Ser Thr Tyr

1 5

<210> 30

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 30

Gly Ala Ser

1

<210> 31

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 31

Gln Gln Ser Tyr Ser Ala Pro Tyr Thr

1 5

<210> 32

<211> 113

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 32

Gly Ser Thr Gly Asp Ala Asp Ile Val Met Thr Gln Ser Pro Ser Ser

1 5 10 15

Leu Pro Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser

20 25 30

Gln Ser Ile Ser Thr Tyr Val Asn Trp Tyr Gln Gln Lys Ser Gly Asn

35 40 45

Ala Pro Glu Leu Leu Met Tyr Gly Ala Ser Ile Leu Gln Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Ala Pro Tyr Thr Phe Ala Gln Gly Thr Lys Leu Glu Ile

100 105 110

Arg

<210> 33

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 33

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 34

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 34

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 35

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 35

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 36

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 36

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 37

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 37

Gln Gly Val Ser Asn Tyr

1 5

<210> 38

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 38

Ala Ala Ser

1

<210> 39

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 39

Gln His Tyr Asp Ser Pro Pro Tyr Thr

1 5

<210> 40

<211> 113

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 40

Gly Ser Thr Gly Asp Ala Val Ile Trp Met Thr Gln Ser Pro Ser Ser

1 5 10 15

Leu Ser Ala Ser Met Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25 30

Gln Gly Val Ser Asn Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Lys

35 40 45

Ala Pro Glu Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Ala Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His

85 90 95

Tyr Asp Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Val

100 105 110

Lys

<210> 41

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 41

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 42

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 42

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 43

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 43

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 44

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 44

Gly Ser Thr Gly Asp Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 45

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 45

Ala Leu Ala Lys His Phe

1 5

<210> 46

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 46

Lys Asp Thr

1

<210> 47

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 47

Gln Ser Pro Asp Thr Thr Gly Arg Ile

1 5

<210> 48

<211> 117

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 48

Gly Ser Thr Gly Asp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val

1 5 10 15

Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala

20 25 30

Leu Ala Lys His Phe Gly His Trp Tyr Gln Gln Arg Pro Gly Gln Ala

35 40 45

Pro Val Leu Val Ile Tyr Lys Asp Thr Glu Arg Pro Leu Gly Ile Pro

50 55 60

Glu Arg Phe Ser Gly Ser Ser Ser Gly Ala Thr Val Thr Leu Thr Ile

65 70 75 80

Ser Ala Val Glu Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Pro

85 90 95

Asp Thr Thr Gly Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu

100 105 110

Gly Gln Pro Lys Ala

115

<210> 49

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 49

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 50

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 50

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 51

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 51

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 52

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 52

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 53

Gln Gly Ile Ser Ser Trp

1 5

<210> 54

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 54

Ala Ala Ser

1

<210> 55

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 55

Gln Gln Ser Tyr Ser Ile Pro Arg Thr

1 5

<210> 56

<211> 113

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 56

Gly Ser Thr Gly Asp Ala Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser

1 5 10 15

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25 30

Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 57

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 57

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 58

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 58

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 59

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 59

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 60

<211> 122

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp Thr Phe Thr Ser His

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 61

<211> 6

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 61

Gln Asp Ile Ser Asp Trp

1 5

<210> 62

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 62

Arg Ala Val

1

<210> 63

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 63

Gln Gln Thr Asn Thr Phe Pro Ile Thr

1 5

<210> 64

<211> 120

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 64

Gly Ser Thr Gly Asp Ala Val Ile Trp Met Thr Gln Ser Pro Ile Gln

1 5 10 15

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Tyr Val Gly Asp Arg Val

20 25 30

Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asp Trp Leu Ala Trp

35 40 45

Tyr Gln Gln Ala Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala

50 55 60

Val Thr Leu Gln Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

65 70 75 80

Gly Thr Asp Phe Ser Leu Thr Ile Thr Gly Leu Gln Arg Glu Asp Phe

85 90 95

Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Thr Phe Pro Ile Thr Phe Gly

100 105 110

His Gly Thr Arg Leu Glu Ile Lys

115 120

<210> 65

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 65

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 66

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 66

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 67

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 67

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 68

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 68

Gly Ser Thr Gly Asp Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 69

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 69

Ser Ser Asp Val Gly Ser Tyr Asn Leu

1 5

<210> 70

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 70

Glu Val Thr

1

<210> 71

<211> 10

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 71

Ile Ser Tyr Ala Gly Asn Asn Asn Leu Val

1 5 10

<210> 72

<211> 121

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 72

Gly Ser Thr Gly Asp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Val

1 5 10 15

Ser Gly Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Thr Ser

20 25 30

Ser Asp Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His Pro

35 40 45

Gly Lys Ala Pro Lys Leu Ile Ile Ile Glu Val Thr Lys Arg Pro Pro

50 55 60

Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

65 70 75 80

Leu Thr Val Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr His Cys

85 90 95

Ile Ser Tyr Ala Gly Asn Asn Asn Leu Val Phe Gly Gly Gly Thr Gln

100 105 110

Leu Thr Val Leu Gly Gln Pro Lys Ala

115 120

<210> 73

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 73

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 74

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 74

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 75

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 75

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 76

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 76

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 77

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 77

Ser Ser Asp Ile Gly Arg Ser Ser

1 5

<210> 78

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 78

Arg Asn Asn

1

<210> 79

<211> 11

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 79

Ala Ala Trp Asp Asn Thr Leu Arg Gly Tyr Val

1 5 10

<210> 80

<211> 121

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 80

Gly Ser Thr Gly Asp Ala Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala

1 5 10 15

Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser

20 25 30

Ser Asp Ile Gly Arg Ser Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly

35 40 45

Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly

50 55 60

Val Pro Asp Arg Leu Ser Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu

65 70 75 80

Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala

85 90 95

Ala Trp Asp Asn Thr Leu Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys

100 105 110

Val Thr Val Leu Gly Gln Pro Lys Ala

115 120

<210> 81

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 81

Glu Asp Thr Phe Thr Ser His Tyr

1 5

<210> 82

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 82

Ile Asn Pro Thr Gly Gly Ser Ile

1 5

<210> 83

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 83

Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val

1 5 10 15

<210> 84

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 84

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

1 5 10 15

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp

20 25 30

Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser

50 55 60

Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser

65 70 75 80

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala

100 105 110

Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 85

<211> 12

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 85

Gln Ser Val Leu Phe Ser Pro Asn Asn Lys Asn Tyr

1 5 10

<210> 86

<211> 3

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 86

Trp Ala Ser

1

<210> 87

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 87

Gln Gln Tyr Asp Ser Ser Pro Trp Thr

1 5

<210> 88

<211> 119

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 88

Gly Ser Thr Gly Asp Ala Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser

1 5 10 15

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

20 25 30

Gln Ser Val Leu Phe Ser Pro Asn Asn Lys Asn Tyr Leu Ala Trp Tyr

35 40 45

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser

50 55 60

Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

65 70 75 80

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

85 90 95

Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Trp Thr Phe Gly Gln

100 105 110

Gly Thr Lys Val Glu Ile Lys

115

Claims

1. The novel coronavirus antibody is characterized by being XY1,

The XY1 antibody heavy chain variable region comprises:

CDRH1 amino acid sequence: EDTFTSHY A

CDRH2 amino acid sequence: INPTGGSI A

CDRH3 amino acid sequence: ARGGFTPDTSAPMDV;

the XY1 antibody light chain variable region comprises:

CDRL1 amino acid sequence: QGIRNS A

CDRL2 amino acid sequence: DAS (DAS)

CDRL3 amino acid sequence: QHYFGTPLT.

2. The novel coronavirus antibody of claim 1, wherein XY1 antibody heavy chain variable region amino acid sequence:

XY1 antibody light chain variable region amino acid sequence:

GSTGDAEIVMTQSPSSLSASEGDRVIITCRASQGIRNSLAWYQQKPGKAPKLLLYDASKLESGVPSRFSGSGSGTHFTLTIDSLQPEDFATYYCQHYFGTPLTFGGGTKVEIK.

3. the novel coronavirus antibody of claim 1, wherein XY1 is an antibody for novel coronavirus therapy.

4. The antibody of any one of claims 1-3, wherein the antibody type is Fab, fab '-SH, fv, scFv, (Fab') 2 fragment.

5. Any nucleic acid capable of expressing the novel coronavirus antibody of any one of claims 1-3.

6. A formulation for the detection, prevention or treatment of a novel coronavirus comprising the novel coronavirus antibody of any one of claims 1-3.

7. Use of a novel coronavirus antibody according to any one of claims 1 to 3, comprising any one or more of the following;

(1) For preparing novel coronavirus detection formulations;