CN113801223A

CN113801223A - Neutralizing antibody against SARS-COV-2 of severe acute respiratory syndrome type II coronavirus

Info

Publication number: CN113801223A
Application number: CN202111222859.3A
Authority: CN
Inventors: 刘密; 王义鹏; 刁璐; 章铭辉; 马琳; 镇学初; 龙亚秋
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-11-25
Filing date: 2021-10-20
Publication date: 2021-12-17
Anticipated expiration: 2041-10-20
Also published as: WO2022110962A1; CN116396967A; CN116254270B; CN116003584B; CN116396967B; CN116254270A; CN116003584A; CN117467668A; CN112442120A; CN113801223B; CN117467668B

Abstract

The invention discloses a neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biological medicine. The invention extracts peripheral immune cells from the blood of a patient recovering from COVID-19, screens B cells capable of combining with a new coronavirus antigen protein-spike protein from the peripheral immune cells, and analyzes single B cells for producing antibodies at a single cell level to obtain gene sequences of heavy chains and light chains of variable regions of neutralizing antibodies in the B cells. The sequences can be used for reconstructing in vitro and expressing a neutralizing antibody capable of neutralizing the new coronavirus, and are expected to be used for treating and preventing diseases such as pneumonia caused by the new coronavirus.

Description

Neutralizing antibody against SARS-COV-2 of severe acute respiratory syndrome type II coronavirus

Technical Field

The invention relates to a neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biological medicine.

Background

Pneumonia (COVID-19) caused by infection of severe acute respiratory syndrome type II coronavirus (SARS-CoV 2) is a serious infectious disease and causes serious influence on the global scale. It is an urgent need to find an effective treatment for this virus.

The neutralizing antibody is an antibody capable of eliminating the ability of virus infection after binding to a virus, is a corresponding antibody produced by B lymphocytes when pathogenic microorganisms invade the body, and is a soluble protein secreted by adaptive immune response cells. Invasion of cells by pathogenic microorganisms requires the binding of specific molecules expressed by the pathogen itself to receptors on the cells in order to infect and further amplify the cells. The neutralizing antibody can bind to an antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to a target cell receptor and invading cells. After the virus invades the human body, B cells secrete neutralizing antibodies into the blood, and the antibodies are combined with virus particles in the blood to prevent the virus from infecting the cells and destroying the virus particles, so that the virus is neutralized. It follows that neutralizing antibodies play a major role in killing extracellular free virus.

Certain specific neutralizing antibodies derived from the blood of a patient cured of a viral infection have a virus-neutralizing effect and are therefore useful in the treatment of infectious diseases, rendering the virus non-pathogenic. With the progress of antibody production technology, therapeutic antibodies have gradually exerted excellent effects in the treatment of various diseases. Existing vaccines, such as measles vaccine, polio vaccine, hepatitis b vaccine, and hepatitis a vaccine, all provide neutralizing antibodies to vaccinees to prevent viral infection. Since neutralizing antibodies can destroy viruses before they enter cells and can scavenge free viruses outside cells in vivo, neutralizing antibodies in the blood of patients who recover from viral infection can be used for the treatment of viral infections.

Disclosure of Invention

In order to solve the technical problem, the invention extracts peripheral immune cells from the blood of a COVID-19 rehabilitation patient, screens B cells capable of being combined with a new coronavirus antigen protein-spike protein from the peripheral immune cells, and analyzes single B cells for producing antibodies at a single cell level to obtain gene sequences coding heavy chains and light chains of variable regions of neutralizing antibodies in the B cells. The sequences can be used for reconstructing in vitro and expressing a neutralizing antibody capable of neutralizing the new coronavirus, and are expected to be used for treating and preventing diseases such as pneumonia caused by the new coronavirus.

The first object of the present invention is to provide a neutralizing antibody against SARS-COV-2, a severe acute respiratory syndrome type II coronavirus, comprising: a light chain variable region DNA sequence, and a heavy chain variable region DNA sequence;

the nucleotide sequence of the light chain variable region DNA sequence is one or more combinations of SEQ ID NO. 1-5;

the nucleotide sequence of the heavy chain variable region DNA sequence is one or more of SEQ ID NO. 6-8.

Further, the neutralizing antibody is a whole antibody comprising a constant region and a variable region, a partial antibody comprising only a variable region, or a chimeric antibody comprising only a variable region.

The second purpose of the invention is to provide a gene encoding the neutralizing antibody.

The third purpose of the invention is to provide an expression vector carrying the gene.

It is a fourth object of the invention to provide a recombinant cell expressing said neutralizing antibody.

The fifth purpose of the invention is to provide the application of the neutralizing antibody in preparing a medicament for treating pneumonia COVID-19.

The sixth purpose of the invention is to provide a kit for treating pneumonia COVID-19, wherein the kit contains the neutralizing antibody.

The invention has the beneficial effects that:

the invention extracts peripheral immune cells from the blood of a patient recovering from COVID-19, screens B cells capable of combining with a new coronavirus antigen protein-spike protein from the peripheral immune cells, and analyzes single B cells for producing antibodies at a single cell level to obtain gene sequences of heavy chains and light chains of variable regions of neutralizing antibodies in the B cells. The sequences can be used for reconstructing in vitro and expressing a neutralizing antibody capable of neutralizing the new coronavirus, and are expected to be used for treating and preventing diseases such as pneumonia caused by the new coronavirus.

Drawings

FIG. 1 is a schematic diagram of a procedure for screening for neutralizing antibodies using individual B cells in the blood of a convalescent patient with COVID-19;

FIG. 2 shows the results of an antibody experiment after expression purification by SDS-PAGE analysis;

FIG. 3 shows a negative control antibody Pmab (no neutralizing effect) and a neutralizing antibody HC5K VH/B5K VL (IC)₅₀0.1273 mug/mL) pseudovirus neutralization experiment result;

FIG. 4 shows the neutralization experiment results (IC) of the neutralizing antibody D5K VH/B5K VL pseudovirus₅₀～0.00033μg/mL)；

FIG. 5 shows the neutralizing antibody HC5K VH/D4K VL (IC)₅₀About 0.00084. mu.g/mL) and the neutralizing antibody HF4L VH/D5KVL (IC)₅₀About 0.0046 mug/mL) pseudovirus neutralization assay results;

FIG. 6 shows the neutralizing antibody HC5K VH/D5K VL (IC)₅₀～0.0066μg/mL) and neutralizing antibody HD5K VH/A5KVL (IC)₅₀About 0.0017. mu.g/mL) pseudovirus neutralization assay results;

FIG. 7 shows the neutralizing antibody HF4L VH/A5K VL (IC)₅₀0.6049 ug/mL) and neutralizing antibody HF4L VH/B5K VL (IC)₅₀0.6077 mug/mL) pseudovirus neutralization experiment result;

FIG. 8 shows the neutralizing antibody HD5K VH/D4K VL (IC)₅₀0.1996 ug/mL) pseudovirus neutralization assay results.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1: separation and extraction of single B cell capable of combining with SARS-COV-2

About 15mL of blood of a plurality of patients with the new coronavirus pneumonia is extracted, Peripheral Blood Mononuclear Cells (PBMC) in the blood of each patient are separated and extracted by a Ficoll gradient method, and the obtained PBMC are washed twice for standby.

Fc block is added into each PBMC sample, after 15 minutes of action, APC-H7 labeled anti-human CD3 antibody, BV421 labeled anti-human CD19 antibody, BB700 labeled anti-human CD27 antibody, Biotin labeled SARS-COV-2Spike protein and Biotin labeled SARS-COV-2Spike RBD are added in sequence. Followed by addition of Streptavidin-APC and action for 30 min. Antibody-labeled PBMC samples were then collected using FACS AriaTMIII flow cytometer loading analysis. CD3 is a specific surface marker of T cells, CD19 is a specific surface marker of B cells, CD27 is a specific surface marker of memory B cells, two antigens marked by Biotin can be specifically combined with Streptavidin marked by APC, so the cells positive to APC are cells capable of specifically combining with SARS-COV-2. Selecting CD3^-CD19⁺CD27⁺APC⁺The cells are the memory B cells which are searched for and can be specifically combined with SARS-COV-2. Separating single B cell with flow cytometer, pumping into 96-well plate with cell lysate, sealing membrane, and culturingPlacing on dry ice for quick freezing, and then placing at-80 ℃ for storage for later use. Thus, a single B cell that specifically binds to SARS-COV-2 can be isolated.

Example 2: amplification and sequencing of variable region light and heavy chains in single B cells

Since B cells are antibody-secreting cells, the sequence information of antibodies is stored in B cells that bind to SARS-COV-2. The single B cell lysates from 96-well plates that had been lysed were divided into 3 aliquots. One portion was used for Kappa light chain sequence analysis, one portion was used for lamda light chain sequence analysis, and the other portion was used for heavy chain sequence analysis.

In this part of the experiment, a cDNA library was obtained by reverse transcription using RT-PCR, and then the DNA sequence in individual cells was amplified using nested PCR. Since the primers used in the experiments were specific for either the light chain variable region or the heavy chain variable region, the resulting sequences were the sequences of the light and heavy chain portions of the antibody variable regions in that single B cell. And carrying out DNA sequencing analysis on the amplified sequence to obtain the sequence information of the light chain and the heavy chain of the antibody variable region contained in a single B cell.

RT-PCR amplification of single B cell antibody light and heavy chain variable region gene

RT-PCR primers (SEQ ID NO.9-25) were designed as shown in Table 1:

TABLE 1

Single B cell RT-PCR was performed using the selected single B cell as a template. The PCR system configuration and sample adding are completed in a biological safety cabinet and are operated on ice, and an RT-PCR reaction system and reaction conditions are as follows:

and (3) PCR reaction system:

TABLE 2

Composition (I)	System of	Final concentration
			RNase-free water	-	-
5 × RT-PCR buffer	10.0μl	1×
			dNTP mixture	2.0μl	dNTP 400. mu.M each
Primer A	-	0.6μM
			B primer	-	0.6μM
Mixed enzyme	2.0μl
			RNase inhibitors	-	5-10 units
Template RNA	-	1pg–2μg
			General System	50.0μl	-

And (3) PCR reaction conditions:

nest type PCR amplification single B cell antibody light and heavy chain variable region gene

Nested PCR primers (SEQ ID NO.26-38) were designed as shown in Table 3:

TABLE 3

Establishing three PCR reaction systems by taking RT-PCR products as templates, respectively amplifying variable region genes of an H chain, a kappa chain and a lambda chain of the antibody, wherein each reaction system respectively uses a mixed primer corresponding to the reaction system, and the nested PCR reaction system and the reaction conditions are as follows:

and (3) PCR reaction system:

TABLE 4

PCR reagent	Volume (. mu.l) of each sample (25. mu.l system)	Final concentration
			Taq DNA polymerase	0.25	50U ml^-1
10 Xbuffer	2.5	1×
			dNTPs(10mM)	0.5	200μM
Forward primers VH3a and VH3b or PanV kappa	0.5	1.2μM
			Reverse primer PW-Cgma or CK494-516	0.5	1.2μM
RNase-free water	17.25-19.25 (complement system to 24 μ l)	-
			Form panel	1.0	-

And (3) PCR reaction conditions:

example 3: results of the experiment

The application can obtain 5 light chain variable region sequences and 3 heavy chain variable region sequences in the B cells of the recovered patients combined with the spike protein of the new coronavirus. The 5 light chain variable region sequences and 3 heavy chain variable region sequences can be freely combined into an antibody, namely the neutralizing antibody of the new coronavirus SARS-COV-2.

Wherein, 5 light chain variable regions are respectively named as A5K VL (SEQ ID NO.1), B5K VL (SEQ ID NO.2), D4K VL (SEQ ID NO.3), D5K VL (SEQ ID NO.4) and D7K VL (SEQ ID NO.5), and 3 heavy chain variable regions are respectively named as HD5K VH (SEQ ID NO.6), HF4L VH (SEQ ID NO.7) and HC5K VH (SEQ ID NO. 8).

The expression and purification of the antibody were carried out as follows:

in this example, 9 antibodies were constructed, which were: HC5K VH/B5K VL, HD5K VH/D4K VL, HF4L VH/A5K VL, HF4L VH/B5K VL, HD5K VH/B5K VL, HC5K VH/D4K VL, HF4L VH/D5K VL, HC5K VH/D5K VL and HD5K VH/A5K VL.

1. Vector construction

Primers were designed to amplify the corresponding VH and VL sequences according to the corresponding antibody light and heavy chains. The amplified VH and VL sequences were recombined onto antibody expression vector backbone plasmids using recombinase (Cloneexpress II One Step Cloning Kit, C112-01/02, Vazyme), and the bacterial solutions were sequenced for validation.

2. Plasmid extraction

The correctly confirmed clonotypes were inoculated into LB (Amp 100ug/ml) medium, cultured overnight at 37 ℃, and the next day, the plasmid was extracted with a plasmid macrokit and the concentration was measured.

3. Transient expression of cells

The extracted plasmid was transiently transferred to 293F cells as follows:

1) the 293F cells in logarithmic growth phase were counted and the cells were resuspended in fresh 293 medium to a density of 2 x 10⁶Perml, total volume 100 mL.

2) 100ug of plasmid was diluted with medium.

3) PEI (1. mu.g/. mu.L) was diluted with the medium, and the diluted PEI was added to the diluted plasmid DNA. Mix by rotating and/or inverting the tube or gently pipetting 2 to 3 times. The complex was incubated at room temperature for about 20 minutes.

4) plasmid/PEI mixtures added to cellsIn suspension, shake flask gently during addition, and place at 37 deg.C, 8% CO₂Culturing at 125 rpm.

5) 5% (v/v) of the feed was added to the flask 16-22 hours after transfection, the flask was gently shaken during the addition and returned to the 37 ℃ incubator.

6) The supernatant was harvested on day 6.

4. Antibody purification

1) Harvesting the supernatant

Centrifuging by using a centrifuge, and harvesting the supernatant. The supernatant was filtered through a 0.4um filter membrane and the column was docked.

2) Protein purification

Selecting and using Protein A affinity filler Mabselect SureTM of GE company, removing culture medium components, and enriching target Protein.

5. Purification of the resulting protein by SDS-PAGE analysis

The resulting antibodies were purified by SDS-PAGE electrophoretic analysis. The concentration of the separation gel used during the analysis was 4-20%. Reducing the sample, adding the reduced sample into a Loading Buffer, boiling for 10min at 70 ℃, and then Loading for analysis. The results are shown in FIG. 2.

Example 4: pseudovirus neutralization assay

This experiment was performed to evaluate new corona neutralizing antibodies against pseudoviruses carrying the spike protein of new corona viruses. The virus system takes HIV-1 carrying luciferase reporter gene as a virus framework, simultaneously expresses new coronavirus Spike protein on a virus shell, forms pseudovirus particles to infect an exogenous cell line with high expression of ACE2, highly simulates the invasion process of the new coronavirus to target cells through Spike-ACE2, and the degree of infection of the target cells by the pseudovirus particles is positively correlated with the luminous value of luciferase and negatively correlated with the neutralizing activity of antibody.

The neutralizing antibody is combined with S protein of virus outer shell in vitro, and the combined site of the S protein and ACE2 is closed, so that the S protein cannot be combined with ACE2, and the virus cannot invade cells; on the contrary, the antibody without neutralizing activity cannot interfere the combination of the S protein and ACE2 on the cell surface, the virus entering the cell expresses the Fluc protein, and the luminous value of the virus is detected by an enzyme-labeling instrument after the virus reacts with a luminous substrate.

The experimental steps are as follows:

1. sample preparation: the pseudovirus is pre-thawed in a refrigerator or ice at-80 deg.C to 4 deg.C, and diluted to 1-2 × 10 with 10% FBS-containing DMEM before use⁴TCID₅₀mL (if the infected ACE2 overexpressing cell line was purchased by non-self, then the best TCID50 needed to be self-groped).

2. A new 96-well cell culture plate was taken for sample dilution.

3. 135. mu.L of the test sample was added to column 2 of the 96-well cell culture plate.

4. Columns 3-10 were filled with 90. mu.l serum-free DMEM, then 45. mu.l of the diluent was added to column 3 by a line gun until diluted to column 9, and the excess 45. mu.l of the liquid in the last column was discarded. Then 90. mu.l of the diluted pseudovirus solution was added to each well. 90. mu.L of pseudovirus was added to the virus control group (VC), 180. mu.L of serum-containing DMEM medium was added to the cell control group (CC), and the 96-well cell culture plate was incubated at 37 ℃ for 1 hour.

5. The sample-pseudovirus mixture in the 96-well cell culture plate and the two sets of controls were divided into three transfers to a 96-well white plate at 50. mu.L/well (3 replicates).

6. Immediately after 50. mu.l of the solution had a density of 0.4X 10⁶cells/ml (number 2X 10)⁴cells/well) 293-ACE2 cells were plated into 96-well plates (without medium change) and incubated in a 37 ℃ incubator.

7. After 20-24h incubation, 25 μ L of DMEM medium containing 10% FBS, pre-warmed at 37 deg.C, was added to each well.

8. And continuously culturing for 48h, taking out a 96-hole white plate, balancing to room temperature, adding 125 mu l of room-temperature-balanced Bio-Lite reporter gene detection reagent into each hole, vibrating the plate for 2min, standing at room temperature for 5min, and detecting a chemiluminescence value (RLU) by using an enzyme-labeling instrument.

9. Data processing: log values of antibody concentrations and corresponding RLUs were taken into graph pad software, and IC50 values were calculated and compared.

The results of the experiment are shown in table 5 and fig. 3 to 8: the neutralizing antibody of the invention has certain neutralizing capacity to the new coronavirus. However, the neutralizing capacity of each antibody to the new coronavirus is different. The lower the IC50 value in the virus neutralization experiment, the better, indicating that only few antibodies are needed to neutralize the virus. Neutralizing antibodies against the novel coronaviruses have been reported to have IC50 values in virus neutralization experiments in excess of 0.5. mu.g/mL to 0.0012. mu.g/mL. In the novel coronavirus neutralizing antibody, some antibodies such as HD5K VH/B5K VL and HC5K VH/D4K VL achieve 0.00033 mu g/mL and 0.00084 mu g/mL of IC50 in a virus neutralizing experiment, and have stronger virus neutralizing capacity than most of novel coronavirus neutralizing antibodies.

TABLE 5 neutralizing antibody virus neutralizing ability

Antibodies	Virus neutralization assay IC₅₀(μg/mL)
		HC5K VH/B5K VL	0.1273
HD5K VH/D4K VL	0.1996
		HF4L VH/A5K VL	0.6049
HF4L VH/B5K VL	0.6077
		HD5K VH/B5K VL	0.00033
HC5K VH/D4K VL	0.00084
		HF4L VH/D5K VL	0.0046
HC5K VH/D5K VL	0.0066
		HD5K VH/A5K VL	0.0017

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention, such as base substitution of less than 3% for the heavy chain or light chain sequence of the antibody variable region, or amino acid substitution of less than 3% for the amino acid sequence of the portion of the antibody expressed by the heavy chain or light chain sequence of the antibody variable region, are within the scope of the present invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> Suzhou university

<120> neutralizing antibody against SARS-COV-2, a type II coronavirus of Severe acute respiratory syndrome

<160> 38

<170> PatentIn version 3.3

<210> 1

<211> 501

<212> DNA

<213> (Artificial sequence)

<400> 1

gtcctgctct gtgacactct cctgggagtt acccgattgg agggcgttat ccaccttcca 60

ctgtactttg gcctctctgg gatagaagtt attcagcagg cacacaacag aggcagttcc 120

agatttcaac tgctcatcag atggcgggaa gatgaagaca gatggtgcag ccacagttcg 180

tttgatctcc agcttggtcc cctggccaaa agtgtacaaa ctattatact gttggcagta 240

ataagttgca aaatcatcag gctgcaggct gctgatggtg agagtgaatt ctgtcccaga 300

tccactgccg ctgaaccttg atgggaccac atcttctaaa ctagacgcct tatagatcaa 360

gaccttaggg gctttccctg gtttctgctg ataccaggcc aaccaggtac caatatcttg 420

actggcccgg caagtgatgg tgactctgtc tcctacagat gcagacaggg agaatgggct 480

ggggtcataa cagagcagcg g 501

<210> 2

<211> 503

<212> DNA

<213> (Artificial sequence)

<400> 2

ccctgctgtc ctgctctgtg acactctcct gggagttacc cgattggagg gcgttatcca 60

ccttccactg tactttggcc tctctgggat agaagttatt cagcaggcac acaacagagg 120

cagttccaga tttcaactgc tcatcagatg gcgggaagat gaagacagat ggtgcagcca 180

cagttcgttt gatctccagc ttggtcccct ggccaaaagt gtaaggagtt tgtagagctt 240

gcatgcagta ataaacccca acatcctcag cctccactct gctgattttc agtgtgaaat 300

ctgtgcctga tccactgcca ctgaacctgt cagggacccc ggaggcccga tgagaattca 360

aatagatcag gagctgtgga gactgccctg gcttctgcag gtaccaatcc aaatagttgt 420

atccatcact aagcaggagg ctctgactag acctgcagga gatggaggcc ggctctccag 480

gggtgacggg cagggagaat gga 503

<210> 3

<211> 502

<212> DNA

<213> (Artificial sequence)

<400> 3

ctccattctc cctgcccgtc acccctggag agccggcctc catctcctgc aggtctagtc 60

agaggctcct gcatagtaat ggatacaact atttggattg gtacctgcag aagccagggc 120

agtctccaca gctcctgatc tatttgggtt ctaatcgggc ctccggggtc cctgacaggt 180

tcagtggcag tggatcaggc acagatttta cactgaaaat cagcagagtg gaggctgagg 240

atgttggggt ttattactgc atgcaagctc tgcaaactcc gtggacgttc ggccaaggga 300

ccaaggtgga aatcaaacga actgtggctg caccatctgt cttcatcttc ccgccatctg 360

atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca 420

gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac tcccaggaga 480

gtgtcacaga gcaggacagc ag 502

<210> 4

<211> 484

<212> DNA

<213> (Artificial sequence)

<400> 4

cattctccct gtctgcatct gtaggagaca gagtcaccat cacttgccgg gccagtcaga 60

atattaatac ctggttggcc tggtatcagc agaaaccagg gcaagcccct aaggtcctga 120

tccataaggc gtctacctta gaaagtgggg tcccatcaag gatcagcggc agtggatctg 180

ggacagaatt cactctcacc atcagcagcc tgcagcctga tgattttgca acttattact 240

gccaacagta tgattattat cctcacactt ttggccaggg gaccaaagta gatatcaaac 300

gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 360

gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 420

ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 480

gcag 484

<210> 5

<211> 471

<212> DNA

<213> (Artificial sequence)

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, or t

<400> 5

cgatnggagg gcgttatcca ccttttccac ttgtactttt ggccttctcc tgggatagaa 60

gttattcagc aggcacacaa cagaggcaag atccagattt caactgctca tcagatggcg 120

ggaagatgaa gacagatggt gcagccacag ttcgtttgat ctccacctta gtccctccgc 180

cgaaagtgag gggccacctg tcacgctgct gacagtaata aactgcaaaa tcttcaggct 240

ctaggccgcg gatggtgaga gtgaagtctg tcccagaccc actgccactg aacctggcgg 300

ggatgccaga ggccctgacg gacgcatcat agatgaggag cctgggagtc tggccaggtt 360

tgtgttggta ccaggttaag tagaacctaa cactctgact ggtcctacag gagacggtgg 420

ctctgtcccc tgcagacaaa gacagggaga atggagcctg ggttcataat a 471

<210> 6

<211> 466

<212> DNA

<213> (Artificial sequence)

<400> 6

tgaggtgcca cctgttggag tctgggggag gcttggtcca gcctgggggg tccctgagac 60

tctcctgtgc agcctctgga ttcaccttta ataactattg gatgacctgg gtccgccagg 120

ctccagggaa ggggctggaa tgggtggcca gcataaagca agatggaggt gagaaatact 180

ttgtggactc tgtgaagggc cgattcacca tctccagaga caacgccagg aactcactgt 240

atctgcaaat ggacaacctg agagccgacg acacggctct gtattactgt gcgagcctta 300

atgtaattcg atattttgac tggacgacca cgggatactt tgactactgg ggccagggaa 360

ccctggtcac cgtcgcctca gcctccacca agggcccatc ggtcttcccc ctggcgccct 420

gctccaggag cacctccgag agcacagcgg ccctgggctg cctggt 466

<210> 7

<211> 431

<212> DNA

<213> (Artificial sequence)

<400> 7

caggcagccc agggccgctg tgcccccaga ggtgctcttg gaggagggtg ccagggggaa 60

gaccgatggg cccttggtgg aggctgagga gacaatgacc agggttccct ggccccactg 120

ggcagcgtaa ttactgtagg tcccacccca tctcgcacag taatacatgg cggtgtccga 180

ggccttcagg ctgctccact gcaggtaggc ggtgttgatg gacctgtcgg ctgagatggt 240

gacctggcct tgaaaggacg gactgtattt ggtatcagag tcacgaagat agatgattcc 300

caaccactcc aggcccttcc cgggcatctg gcggacccaa ccgatccagt agttgggaaa 360

gttgtttccg aaagccttac aggaaatctt cagagactcc ccgggctttt tcacctctac 420

tccagactcc a 431

<210> 8

<211> 446

<212> DNA

<213> (Artificial sequence)

<400> 8

tgttggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc tcctgtgcag 60

cgtctggatt cagcttcagt agctatggca tgcactgggt ccgccaggct ccaggcaagg 120

ggctggagtg ggtggcagtt atttggtttg atggaagtaa taaatactat gcagactccg 180

tgaagggccg attcaccatc tccagagaca attccaagaa caccctgttt ctgcaaatga 240

gcggcctgag agccgacgac acggctgtct attactgtgc gacagaggtc gtcggccagg 300

gcttctccgg tgacaacggg ggggaccatt ggggccaggg aaccctggtc accgtctcct 360

cagcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag agcacctctg 420

ggggcacagc ggccctgggc tgcctg 446

<210> 9

<211> 25

<212> DNA

<213> (Artificial sequence)

<400> 9

atgaggstcc cygctcagct gctgg 25

<210> 10

<211> 28

<212> DNA

<213> (Artificial sequence)

<400> 10

ctcttcctcc tgctactctg gctcccag 28

<210> 11

<211> 25

<212> DNA

<213> (Artificial sequence)

<400> 11

atttctctgt tgctctggag ctctg 25

<210> 12

<211> 25

<212> DNA

<213> (Artificial sequence)

<400> 12

agtagtcctt caccaggcag cccag 25

<210> 13

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 13

ggtcctgggc ccagtctgtg ctg 23

<210> 14

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 14

ggtcctgggc ccagtctgcc ctg 23

<210> 15

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 15

gctctgtgac ctcctatgag ctg 23

<210> 16

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 16

ggtctctctc scagcytgtg ctg 23

<210> 17

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 17

gttcttgggc caattttatg ctg 23

<210> 18

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 18

ggtccaattc ycaggctgtg gtg 23

<210> 19

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 19

gagtggattc tcagactgtg gtg 23

<210> 20

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 20

caccagtgtg gccttgttgg cttg 24

<210> 21

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 21

acaggtgccc actcccaggt gcag 24

<210> 22

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 22

aaggtgtcca gtgtgargtg cag 23

<210> 23

<211> 27

<212> DNA

<213> (Artificial sequence)

<400> 23

cccagatggg tcctgtccca ggtgcag 27

<210> 24

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 24

caaggagtct gttccgaggt gcag 24

<210> 25

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 25

tcttgtccac cttggtgttg ct 22

<210> 26

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 26

atgacccagw ctccabycwc cctg 24

<210> 27

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 27

gtgctgtcct tgctgtcctg ct 22

<210> 28

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 28

ctgctaccgg ttcctgggcc cagtctgtgc tgackcag 38

<210> 29

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 29

ctgctaccgg ttcctgggcc cagtctgccc tgactcag 38

<210> 30

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 30

ctgctaccgg ttctgtgacc tcctatgagc tgacwcag 38

<210> 31

<211> 37

<212> DNA

<213> (Artificial sequence)

<400> 31

ctgctaccgg ttctctctcs cagcytgtgc tgactca 37

<210> 32

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 32

ctgctaccgg ttcttgggcc aattttatgc tgactcag 38

<210> 33

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 33

ctgctaccgg ttccaattcy cagrctgtgg tgacycag 38

<210> 34

<211> 28

<212> DNA

<213> (Artificial sequence)

<400> 34

ctcctcactc gagggyggga acagagtg 28

<210> 35

<211> 18

<212> DNA

<213> (Artificial sequence)

<400> 35

sargtgcagc tcgtggag 18

<210> 36

<211> 18

<212> DNA

<213> (Artificial sequence)

<400> 36

gaggtgcagc tgttggag 18

<210> 37

<211> 20

<212> DNA

<213> (Artificial sequence)

<400> 37

ggaattctca caggagacga 20

<210> 38

<211> 25

<212> DNA

<213> (Artificial sequence)

<400> 38

agtagtcctt gaccaggcag cccag 25

Claims

1. A neutralizing antibody against SARS-COV-2, a severe acute respiratory syndrome type II coronavirus comprising: a light chain variable region DNA sequence, and a heavy chain variable region DNA sequence;

2. The neutralizing antibody of claim 1, wherein said neutralizing antibody is a whole antibody comprising constant and variable regions, a partial antibody comprising only variable regions, or a chimeric antibody comprising only variable regions.

3. A gene encoding the neutralizing antibody of claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. A recombinant cell expressing the neutralizing antibody of claim 1 or 2.

6. Use of a neutralising antibody as claimed in claim 1 or claim 2 in the manufacture of a medicament for the treatment of pneumonia COVID-19.

7. A kit for treating pneumonia COVID-19, wherein the kit contains the neutralizing antibody of claim 1 or 2.