CN113461812A - Humanized new coronavirus neutralizing antibody nCoV-00D and application thereof - Google Patents

Abstract

The invention discloses a humanized novel coronavirus neutralizing antibody nCoV-00D and application thereof. The invention utilizes the immune hybridoma technology to obtain a humanized neutralizing antibody nCoV-00D with specificity aiming at the surface antigen of the novel coronavirus through sequencing and optimizing a base sequence, can efficiently prevent the novel coronavirus from infecting host cells in-vitro tests, has higher affinity and good inhibitory activity to mutant strains, and is expected to be prepared into specific antibody drugs for clinically preventing and treating the novel coronavirus pneumonia.

Description

Humanized new coronavirus neutralizing antibody nCoV-00D and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a humanized novel coronavirus neutralizing antibody nCoV-00D and application thereof.

Background

The novel coronavirus SARS-CoV-2 is a new virus discovered in 2019, and can cause human viral pneumonia or lung infection. The genome of the coronavirus sequentially encodes structural proteins such as spinous process protein, envelope protein, membrane protein, nucleocapsid protein and the like. Among them, Spike protein (Spike) is the most important surface membrane protein of coronavirus, and contains two subunits, S1 and S2, wherein S1 mainly comprises Receptor Binding Domain (RBD), which is responsible for recognizing cell receptor and is the main epitope, and mutation sites of various variant strains circulating in the world are concentrated in the RBD region. The new coronavirus spike protein interacts with human angiotensin converting enzyme 2(ACE2) to infect human respiratory epithelial cells.

Therapeutic antibodies are artificial substances that are capable of binding to specific proteins on the surface of the virus. Most therapeutic antibodies are monoclonal antibodies, meaning that they are antibodies produced by a single immune cell clone. Each monoclonal antibody binds to only one antigen. Both of these properties are very important when these antibodies are used for therapeutic purposes. The therapeutic antibody has two main characteristics of specific combination and accurate administration, so that the therapeutic antibody has good treatment effect. Monoclonal antibodies can specifically target virtually any substance; this ability is very useful in detecting and targeting drug delivery there are many different types of therapeutic antibodies that can be generated in a number of different ways.

At present, there is still a need to develop an antibody which can specifically bind to SARS-CoV-2 virus with high affinity and which has an effective inhibitory effect on the mutant strain.

Disclosure of Invention

The invention aims to provide a humanized novel coronavirus neutralizing antibody nCoV-00D and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides a humanized novel coronavirus neutralizing antibody nCoV-00D, which is characterized by the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NOS: 1-3; the amino acid sequences of the hypervariable regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO. 4-6.

The amino acid sequences of the light and heavy chain variable regions of antibody nCoV-00D are shown in SEQ ID NOS: 7 and 8, respectively.

In a second aspect, the invention provides nucleic acid molecules encoding the antibody nCoV-00D. Wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are shown in SEQ ID NOS: 9 and 10, respectively.

In a third aspect, the present invention provides biological materials containing the above-described nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

In a fourth aspect, the invention provides a single chain antibody or a full antibody engineered from the antibody nCoV-00D.

In a fifth aspect, the invention provides a medicament for preventing or treating infection by a novel coronavirus and related diseases caused by the infection, wherein the effective component of the medicament is an antibody nCoV-00D or a single-chain antibody or a full antibody obtained by modifying the antibody nCoV-00D.

In one embodiment of the present invention, the amino acid sequences of the light and heavy chains of a whole antibody IgG are shown in SEQ ID NOS: 11 and 12, respectively, and the nucleotide sequences encoding the light and heavy chains are shown in SEQ ID NOS: 13-14, respectively.

In a sixth aspect, the invention provides a novel reagent for detecting coronaviruses, which comprises an antibody nCoV-00D or a single-chain antibody or a full antibody obtained by modifying the antibody nCoV-00D.

In a seventh aspect, the invention provides any of the following uses of an antibody nCoV-00D or a single chain antibody or a full antibody engineered from an antibody nCoV-00D:

1) for the preparation of a medicament for the prevention or treatment of infections with new coronaviruses and associated diseases caused by infections therewith;

2) is used for preparing a new coronavirus detection reagent or a new coronavirus detection kit.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention successfully obtains a hybridoma cell capable of secreting the antibody by utilizing an immune hybridoma technology, obtains a humanized neutralizing antibody nCoV-00D specific to the surface antigen of the novel coronavirus through sequencing and base optimization, has a neutralizing function of preventing the novel coronavirus from infecting sensitive cells in vitro, has high affinity to the antigen, and has good inhibitory activity to mutant strains. The humanized neutralizing anti-new type coronavirus surface antigen gene engineering antibody variable region gene obtained by the method and the whole antibody gene under the characteristics of each antibody gene are expressed and produced in a eukaryotic cell system or any other gene containing the antibody gene after reconstruction based on the antibody, so that an antibody product with the effect of neutralizing new type coronavirus infection is obtained, and the antibody product can be prepared into a specific antibody medicament for clinically preventing and treating new type coronavirus pneumonia.

Drawings

FIG. 1 is a diagram of a purified immunoblot of the neutralizing antibody nCoV-00D of human origin in a preferred embodiment of the invention.

FIG. 2 is a graph showing the affinity assay of antibody nCoV-00D for various wild-type RBDs and mutant RBDs.

FIG. 3 shows the results of the analysis of the neutralizing novel coronavirus (pseudovirus) of antibody nCoV-00D.

FIG. 4 shows the result of flow cytometry on the binding activity of the antibody nCoV-00D and the new coronavirus RBD protein.

FIG. 5 is a graph of indirect immunofluorescence to detect the effect of nCoV-00D on SARS-nCoV infected host cells.

FIG. 6 is a diagram showing the immunoblotting technique to detect the effect of nCoV-00D on SARS-nCoV infected host cells.

FIG. 7 is a graph showing the effect of real-time quantitative PCR detection of nCoV-00D on SARS-nCoV infected host cells.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples were run under conventional experimental conditions, such as those suggested by the manufacturer's instructions.

Example 1 preparation and function of humanized novel coronavirus neutralizing antibody nCoV-00D

Materials (I) and (II)

1. The pcDNA-3.1 vector was stored in this laboratory. Strain Top 10 was purchased from Tiangen reagent, China.

2. Antigen: the novel coronavirus RBD protein is expressed by the laboratory

3. Expi293F cells, 293T cells, Huh7 cells were all stored in this laboratory.

Second, method and results

1. Hybridoma cell construction and screening for monoclonal antibodies to RBD

1.1 RBD animal immunization and identification of immunization status

BALB/c mice 8 weeks old were immunized 4 times with the expressed and purified RBD protein. The RBD protein is uniformly mixed with an equivalent Freund's complete adjuvant and then is immunized in the first immunization, the RBD protein is uniformly mixed with an equivalent Freund's incomplete adjuvant and then is immunized in the second immunization after 2 weeks, the RBD protein is uniformly mixed with an equivalent Freund's incomplete adjuvant and then is immunized in the third immunization after 2 weeks, and the RBD protein is uniformly mixed with an equivalent Freund's incomplete adjuvant and then is immunized in the 4 th immunization after one month. The immunization routes are abdominal subcutaneous immunization, and the immunization dose is 0.5 mug/mouse. 3 days before fusion, the RBD protein without adjuvant is injected into abdominal cavity, 5 mg/mouse. Collecting the mouse serum before and after immunization, and searching the conditions of optimal antigen coating concentration, optimal coating condition, optimal sealing condition, antibody working concentration, optimal reaction time and the like to establish an indirect ELISA method and detect the immune antibody level of the mouse by the method. The results show that the antibody titers after immunization are higher, as shown in table 1.

TABLE 1

Animal(s) production	Titer of the product
		1	1:51200
2	1:51200
		3	>1:51200
4	1:3200
		5	1:6400

1.2 obtaining and screening of hybridoma cells

The non-immune BALB/c feeder cells are taken, diluted properly and then distributed on 5-10 96-well cell plates with 100 mu L/well. The spleens which have been immunized with BALB/c are fully ground, and cell suspensions are counted. Spleen cells and SP2/0 myeloma cells are fused according to the proportion of 5-10: 1 and then inoculated into a cell plate paved with feeder cells. The antibodies secreted by the cells into the supernatant were detected by indirect ELISA. The total number of tests before subcloning was 3. A hybridoma cell capable of secreting antibody is obtained and named nCoV-00D.

2. Nucleic acid sequence analysis and humanization modification of antibody variable region genes

Selected hybridoma cell RNAs were extracted and reverse transcribed using Trizol, and the amplified products were ligated to T-vectors (purchased from takara) and sequenced. After base optimization, sequences are synthesized, primers (heavy chain upstream cctgtgggttttgctgCTCTGGGTTCCAGGTTCCACTCAGGTGCAGCTGCAGCAGA, heavy chain downstream gctggctgaggagacggtGGCAGACACTGTCACCAGGG, light chain upstream cctgtgggttttgctgCTCTGGGTTCCAGGTTCCACTGATATCGTGCTGACACAGTCTCC and light chain downstream gtgcagccacagttcgCTTGATCTCCAGCTTTGTTCCTC) are designed to amplify a humanized antibody light-heavy chain variable region gene fragment, BamH I/Xho I enzyme cutting sites are introduced at the 5 'end and the 3' end of a kappa chain and the 5 'end and the 3' end of a heavy chain, and the light and heavy chains are respectively cloned to a full antibody expression vector pcDNA3.1 vector to obtain heavy chain and light chain recombinant vectors which are named pcDNA3.1-nCoV-00D VH and pcDNA3.1-nCoV-00D VK.

3. Construction and expression purification of whole antibody recombinant expression plasmid

Mixing the constructed expression plasmid with 1mg/mL Polyethyleneimine (PEI) solution according to a ratio of 1:5(w/v), and then carrying out instantaneous transfection with the density of 2 × 10⁶The cells of Expi293F (Invitrogen) were incubated at 37 ℃ and shaken at 120rpm for 4 days, and then the supernatants were collected by purification using a Protein-A affinity column from GE. The purity of the antibody was analyzed by SDS-polyacrylamide gel (SDS-PAGE), and further purified by using a 30 kDa cut-off concentration tube (produced by Millipore) and a GE molecular sieve. As a result, it was found that nCoV-00D antibody was expressed and purified at a constant concentration and purity (FIG. 1).

4. Functional assay for antibody nCoV-00D

4.1 measurement of antibody affinity by IBIAcore method

Surface Plasmon Resonance (SPR) is an optical physical phenomenon. When a beam of polarized light is incident on the end face of the prism within a certain angle range, surface plasma waves are generated at the interface between the prism and the metal film. When the propagation constant of the incident light wave matches the propagation constant of the surface plasmon wave, free electrons in the metal film are caused to resonate, i.e., surface plasmon resonance. By using the SPR technology, the intermolecular interaction can be obtained in real time without marking. When in analysis, the conjugate is firstly coupled to the chip, then the analyte flows through the surface of the chip, if the conjugate and the analyte have binding activity, the refractive index of the surface of the metal film is changed, and finally the SPR angle is changed, and the information such as the kinetic constants (the binding rate constant ka, the dissociation rate constant KD and the affinity constant KD) and the specificity of the wild-type and mutant RBD and nCoV-00D is obtained by detecting the SPR angle change through the response signal value RU of the Biacore.

Data results processing raw data was output using Biacore instrumentation Evaluation program software and subjected to kinetic fitting analysis in a 1:1 Binding mode. From the fitting results, the association rate constant (ka) and dissociation rate constant (KD) and affinity constant (KD) were obtained. As shown in Table 2, nCoV-00D has an affinity of 5.32X 10 against wild type of the RBD protein of the novel coronavirus^-10M; affinity for RBD mutant (N501Y) was 2.46X 10^-13M; the affinity to RBD mutant (K417T E484K N501Y) is 5.30X 10^-13M; the affinity to RBD mutant (K417N E484K N501Y) is 7.91X 10^-12M (FIG. 2).

TABLE 2

RBD type	MAbs	ka（1/Ms）	kd（1/s）	KD（M）
					WT	54.4	2.51e+05	1.33e-04	5.32e-10
N501Y	55.9	3.87e+05	9.51e-08	2.46e-13
					K417T E484K N501Y	123.9	3.74e+04	1.98e-08	5.30e-13
K417N E484K N501Y	100.0	2.97e+04	2.35e-07	7.91e-12

4.2 flow cytometry detection of the binding Activity of anti-human novel coronavirus surface antigen genetically engineered antibody and novel coronavirus RBD protein

Taking GFP-ACE2 protein expression plasmid and 1mg/mL Polyethyleneimine (PEI) solution according to the proportion of 1:5(w/v)After mixing, Expi293T cells confluent to 80% were transiently transfected and incubated at 37 ℃ CO₂And (5) incubating for 36 h. Digesting the cells, washing the cells for 3 times by using PBS, adding Alexa Fluor 647-labeled RBD protein, and detecting the binding capacity of RBD and ACE 2; alexa Fluor 647-labeled RBD protein 500ng and 2.5 mg nCoV-00D antibody were incubated at room temperature for 30min and then added to the digested cells. Wash 3 times with PBS. After washing 3 times with PBS, the cells were resuspended in 200ul PBS and then tested on the machine (BD FACSAria)^TMII). The results are shown in FIG. 3, which shows the fluorescence intensity of GFP-ACE2 on the abscissa. Compared with a control group only added with the RBD protein, the percentage of Alexa Fluor-647 cell population is obviously reduced in an experimental group mixed with the antibody and the RBD protein, which indicates that the nCoV-00D antibody can be combined with the novel coronavirus RBD protein to inhibit the combination of the RBD protein and the host cell ACE 2.

4.3 neutralization assay of the antibody nCoV-00D against the novel coronavirus (pseudovirus)

Firstly, pseudovirus is coated, and the steps are simplified as follows: 293T (ATCC) was plated in six well plates at 1X106 cells per well. The cells were then transfected according to the corresponding ratio (pEZT-S: psPSX 2: plvx-Luciferase = 0.75: 1.5: 2), and after 6-8 hours the medium was changed to complete medium containing 1.5ml of 10% serum. And collecting virus supernatant after 48-72 h, and adding fresh culture medium again. Centrifuging the virus at 4000 rpm for 10min, measuring the toxic valence, and packaging. Then, Huh7 cells were seeded uniformly in a 96-well plate, and a pseudotoxic antibody neutralization experiment was performed after the cells were attached. From the highest final concentration of 1ug/ml, the solution is diluted to 0.0003ug/ml by a serum-free DMEM medium three-time gradient; diluting the pseudotoxin by serum-free DMEM medium, taking 50ul of antibody diluent and 50ul of pseudotoxin diluent (/ hole), mixing uniformly in a vortex manner, standing and incubating for 1h at 37 ℃, finally adding corresponding pseudotoxin antibody neutralizing liquid, and detecting the luciferase value after 24 h. The results show that the antibody nCoV-00D can be efficiently combined with pseudovirus, and can block the pseudovirus from combining with host cells and IC₅₀The value was about 9 ng/ml (FIG. 4), indicating that the nCoV-00D antibody had a strong neutralizing activity.

4.4 Indirect immunofluorescence assay for the Effect of nCoV-00D on host cell infection with New coronavirus

The Huh7 cells are paved in a confocal dish, antibodies with certain concentration and viruses are incubated for 1h at 37 degrees (a control group with virus infection cells is arranged at the same time), then the cells are inoculated, after infection for 16h, the cells are fixed by 4% PFA for 30min, membrane rupture permeation treatment is carried out by 0.5% TritonX-100/PBS for 25min, after the cells are sealed for 1h at 37 ℃ by 1% BSA/PBS, anti-SARS-CoV-2 Spike primary antibody is added for incubation for 2h at 37 ℃, and then fluorescent secondary antibody and DAPI staining nuclei are added. After PBST cleaning, sealing the PBST by using an anti-fluorescence quencher, keeping the PBST at 4 ℃ in a dark place, and finally photographing and observing the PBST by using a fluorescence confocal microscope. The results showed that both the fluorescence intensity and the amount of the S protein in the test group were significantly reduced compared to the control group (FIG. 5), indicating that the nCoV-00D antibody was able to inhibit the infection of host cells by the novel coronavirus.

4.5 immunoblotting to examine the Effect of nCoV-00D on host cell infection with New coronavirus

The Huh7 cells are paved in a 12-well plate, antibodies with certain concentration and viruses are incubated for 1h at 37 ℃ (a control group with virus infected cells is arranged at the same time), then the cells are inoculated, after infection for 16h, the cells are lysed by RIPA lysate, and the expression condition of virus N protein is detected by an immunoblotting method. The results showed that intracellular N protein expression was significantly reduced in the antibody test group compared to the control group (fig. 6), indicating that nCoV-00D antibody was able to inhibit infection of host cells by the novel coronavirus.

4.6 real-time quantitative PCR detection of the Effect of nCoV-00D on host cells infected with New coronavirus

The Huh7 cells are paved in a 12-well plate, antibodies with certain concentration and viruses are incubated for 1h at 37 ℃ (a control group of virus infected cells is arranged at the same time), then the cells are inoculated, after infection for 16h, supernatant of the infected cells is collected and RNA is extracted, and the mRNA expression level of virus ORF1 is detected by a one-step fluorescence quantification method. The results showed a significant reduction in viral mRNA in the cell supernatant of the antibody group compared to the control group (fig. 7), indicating that the nCoV-00D antibody was able to inhibit infection of host cells by the novel coronavirus.

Sequence listing

<110> Beijing aigret flying biotechnology

<120> humanized novel coronavirus neutralizing antibody nCoV-00D and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Tyr Thr Phe Thr Arg Tyr Tyr

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ile Asn Pro Asn Asn Gly Gly Thr

1 5

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Thr Ile Phe Ile Thr Thr Val Ile Ala Arg Asp Tyr

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Ser Val Glu Tyr Ser Gly Thr Thr Leu

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Ala Ser

1

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

His Gln Ser Arg Glu Val Pro Trp Thr

1 5

<210> 7

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Ser

20 25 30

Gly Thr Thr Leu Met Gln Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Asp Asp Val Ala Met Tyr Phe Cys His Gln Ser Arg

85 90 95

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

100 105 110

Thr Val Ser Ala

115

<210> 8

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Asn Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ile Phe Ile Thr Thr Val Ile Ala Arg Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu

<210> 9

<211> 332

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gatatcgtgc tgacacagtc tccagcaagc ctggccgtga gcctgggaca gcgggccaca 60

atctcctgca gagccagcga gtccgtggag tactccggca ccacactgat gcagtggttc 120

cagcagaagc ctggccagcc ccctaagctg ctgatctatg ccgcctccaa cgtggagtct 180

ggagtgcctg caaggttctc tggaagcgga tccgaaccga cttttccctg aatatccacc 240

cagtggagga ggacgatgtg gccatgtact tctgtcacca gtctagggag gtgccatgga 300

cctttggcgg aggaacaaag ctggagatca ag 332

<210> 10

<211> 356

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggtgcagc tgcagcagag cggagcagag ctggtgaagc caggagcctc tgtgaagctg 60

agctgcaaga cctccggcta cacctttaca cggtactata tctattgggt gaagcagcgg 120

cccggacagg gcctggagtg gatcggcgag atcaacccta acaatggcgg cacaaacttc 180

aatgagaagt ttaagtccaa ggccaccctg acatggacaa gagctccaat accgcctaca 240

tgcagctgtc tagcctgaca agcgaggatt ccgccgtgta ctattgtacc atcttcatca 300

ccacagtgat cgcaagggac tattggggac agggcaccct ggtgacagtg tctgcc 356

<210> 11

<211> 238

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

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

35 40 45

Glu Tyr Ser Gly Thr Thr Leu Met Gln Trp Phe Gln Gln Lys Pro Gly

50 55 60

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

65 70 75 80

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

85 90 95

Asn Ile His Pro Val Glu Glu Asp Asp Val Ala Met Tyr Phe Cys His

100 105 110

Gln Ser Arg Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

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

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ser

225 230 235

<210> 12

<211> 472

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Asn Asn Gly Gly Thr Asn Phe Asn

65 70 75 80

Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Ile Phe Ile Thr Thr Val Ile Ala Arg Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

145 150 155 160

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

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

210 215 220

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280 285

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

340 345 350

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

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

370 375 380

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

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

420 425 430

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

435 440 445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

450 455 460

Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 13

<211> 717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggagacgg atacgctgct cctgtgggtt ttgctgctct gggttccagg ttccactgat 60

atcgtgctga cacagtctcc agcaagcctg gccgtgagcc tgggacagcg ggccacaatc 120

tcctgcagag ccagcgagtc cgtggagtac tccggcacca cactgatgca gtggttccag 180

cagaagcctg gccagccccc taagctgctg atctatgccg cctccaacgt ggagtctgga 240

gtgcctgcaa ggttctctgg aagcggatcc ggaaccgact tttccctgaa tatccaccca 300

gtggaggagg acgatgtggc catgtacttc tgtcaccagt ctagggaggt gccatggacc 360

tttggcggag gaacaaagct ggagatcaag cgaactgtgg ctgcaccaag cgtgtttatc 420

ttccctccca gcgacgagca gctgaagagc ggcaccgcca gcgtggtctg tctcctgaac 480

aacttctatc ccagggaggc caaggtccag tggaaagtgg acaacgccct gcaaagcggc 540

aatagccagg agtccgtcac agagcaggac agcaaggaca gcacctacag cctgtccagc 600

accctgaccc tcagcaaggc cgactacgag aagcacaagg tgtacgcttg cgaggtgacc 660

catcagggcc tgtccagccc cgtgaccaag tccttcaaca ggggcgaatg cagctaa 717

<210> 14

<211> 1418

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atggagacgg atacgctgct cctgtgggtt ttgctgctct gggttccagg ttccactcag 60

gtgcagctgc agcagagcgg agcagagctg gtgaagccag gagcctctgt gaagctgagc 120

tgcaagacct ccggctacac ctttacacgg tactatatct attgggtgaa gcagcggccc 180

ggacagggcc tggagtggat cggcgagatc aaccctaaca atggcggcac aaacttcaat 240

gagaagttta agtccaaggc caccctgaca gtggacaaga gctccaatac cgcctacatg 300

cagctgtcta gcctgacaag cgaggattcc gccgtgtact attgtaccat cttcatcacc 360

acagtgatcg caagggacta ttggggacag ggcaccctgg tgacagtgtc tgccaccgtc 420

tcctcagcca gcaccaaagg cccgagcgtg tttccgctgg cgccgagcag caaaagcacc 480

agcggcggca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc 540

gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag 600

agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc 660

cagacctata tttgcaacgt gaaccataaa ccgagcaaca ccaaagtgga taaacgcgtg 720

agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct gaactcctgg 780

ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga 840

cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca 900

actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt 960

acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg 1020

gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc gagaaaacta 1080

tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg 1140

atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg 1200

acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc 1260

ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg gacaagagca 1320

ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact 1380

acacgcagaa gagcctctcc ctgtctccgg gtaaatga 1418

Claims (9)

1. Humanized new coronavirus neutralizing antibody nCoV-00D, which is characterized in that the amino acid sequences of a heavy chain hypervariable region CDR1, CDR2 and CDR3 are shown as SEQ ID NO 1-3; the amino acid sequences of the hypervariable regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO. 4-6.

2. The neutralizing antibody of claim 1, further comprising light and heavy chain variable regions having the amino acid sequences shown in SEQ ID NO 7 and 8, respectively.

3. A nucleic acid molecule encoding the neutralizing antibody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are set forth in SEQ ID NOS: 9 and 10, respectively.

5. A biological material comprising a nucleic acid molecule according to claim 3 or 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

6. A single chain antibody or a full antibody modified from the neutralizing antibody of claim 1 or 2.

7. A medicament for preventing or treating infection by a novel coronavirus or a disease associated with the infection, which comprises the neutralizing antibody according to claim 1 or 2 or the single-chain antibody or the whole antibody according to claim 6 as an active ingredient.

8. A reagent for detecting a novel coronavirus, which comprises the neutralizing antibody according to claim 1 or 2 or the single-chain antibody or the full-length antibody according to claim 6.

9. Use of the neutralizing antibody of claim 1 or 2 or the single chain antibody or full antibody of claim 6 for any one of the following:

1) for the preparation of a medicament for the prevention or treatment of infections with new coronaviruses and associated diseases caused by infections therewith;

2) is used for preparing a new coronavirus detection reagent or a new coronavirus detection kit.

Images