阅读说明：本技术 抗严重急性呼吸系统综合征ii型冠状病毒sars-cov-2的中和抗体 (Neutralizing antibody against SARS-COV-2 of severe acute respiratory syndrome type II coronavirus ) 是由 刘密 王义鹏 章铭辉 刁璐 马琳 镇学初 龙亚秋 于 2020-11-25 设计创作，主要内容包括：本发明公开了一种抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体,属于生物医药技术领域。本发明从COVID-19康复患者血液中提取外周免疫细胞,并从中筛选可以和新冠病毒抗原蛋白-刺突蛋白结合的B细胞,然后对以生产抗体的单个B细胞在单细胞水平上进行分析,获得了B细胞内编码中和抗体可变区重链和轻链的基因序列。这些序列即可用来体外重新构建和表达可中和新冠病毒的中和抗体,有望用于新冠病毒所引起的肺炎等疾病的治疗和预防。(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.)

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;

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.

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.

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.

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-2 Spike protein and Biotin labeled SARS-COV-2 Spike 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, and two antigens marked by Biotin can be marked with APCThe binding specificity of Streptavidin is indicated, so that the APC-positive cells are those that specifically bind to 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 film, freezing on dry ice, and storing at-80 deg.c. 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.

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. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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