阅读说明：本技术 免疫检测用的抗新型冠状病毒的抗体 (Antibodies against novel coronaviruses for immunodetection ) 是由 张黎 郑滨洋 高行素 贾斯月 储凯 陶虹 胡月梅 朱凤才 王祥喜 朱玲 孙瑶 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种免疫检测用的抗新型冠状病毒的抗体,其包含抗体轻链可变区的抗原互补决定区CDR1,CDR2和CDR3分别为SEQ ID NO：5,SEQ ID NO：6及SEQ ID NO：7的氨基酸序列；抗体重链可变区的抗原互补决定区CDR1,CDR2和CDR3分别为SEQ ID NO：1,SEQ ID NO：2及SEQ ID NO：3的氨基酸序列。本发明还公开了该抗体的制备过程及该抗体重链可变区与轻链可变区氨基酸序列。(The invention discloses an antibody for resisting novel coronavirus for immunodetection, which comprises an antigen complementarity determining region CDR1 of an antibody light chain variable region, wherein CDR2 and CDR3 are respectively SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the antibody heavy chain variable region are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The invention also discloses the preparation process of the antibody and the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody.)

1. A monoclonal antibody or a derivative thereof against a novel coronavirus, comprising a first variable region and a second variable region, wherein the first variable region is an antibody light chain variable region comprising the antigen complementarity determining region CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences set forth in SEQ ID NOs: 5, SEQ ID NO: 6 and SEQ ID NO: 7; wherein the second variable region is an antibody heavy chain variable region having the antigen complementarity determining regions CDR1, CDR2 and CDR3 of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;

preferably, the first variable region is an antibody light chain variable region that is SEQ ID NO: 8; wherein said second variable region is an antibody heavy chain variable region represented by SEQ id no: 4.

2. The monoclonal antibody or derivative thereof according to claim 1, characterized in that it comprises the antibody light chain variable region and human antibody light chain constant region, and the hinge, CH1, CH 2 and CH3 regions of the antibody heavy chain variable region and human antibody heavy chain constant region;

preferably, the human antibody light chain constant region is from a human antibody kappa chain or antibody lamda chain and the human antibody heavy chain constant region is from a human IgG1, IgG2, IgG3 or IgG4 subtype.

3. A DNA molecule or gene encoding the monoclonal antibody or derivative thereof of claim 1 or 2; preferably, the antibody light chain variable region is SEQ ID NO: 18, and the variable region of the antibody heavy chain is SEQ ID NO: 17.

4. An expression vector comprising the DNA molecule sequence of claim 3 operably linked to an expression control sequence.

5. A recombinant host cell transformed with the expression vector of claim 4; preferably, the recombinant host cell or progeny thereof expresses the monoclonal antibody or derivative thereof of claim 1 or 2.

6. A method of preparing the monoclonal antibody or derivative thereof of claim 1 or 2, comprising the steps of:

a) providing an expression vector comprising the DNA molecule sequence of claim 3 and an expression control sequence operably linked to said sequence;

b) transforming a host cell with the expression vector of step a);

c) culturing the host cell obtained in step b) under suitable conditions: and

d) and (3) separating and purifying the monoclonal antibody or the derivative thereof from the culture solution of the host cell.

7. A composition or kit comprising the monoclonal antibody or derivative thereof of claim 1 or 2; the composition or kit further comprises a second monoclonal antibody or derivative thereof comprising heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, light chain variable region CDR 3; wherein the content of the first and second substances,

heavy chain variable region CDR1 comprises SEQ ID NO: 9;

heavy chain variable region CDR2 comprises SEQ ID NO: 10;

heavy chain variable region CDR3 comprises SEQ ID NO: 11;

light chain variable region CDR1 contains SEQ ID NO: 13;

light chain variable region CDR2 contains SEQ ID NO: 14;

light chain variable region CDR3 contains SEQ ID NO: 15;

more preferably, the second monoclonal antibody or derivative thereof comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises SEQ ID NO: 12 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16.

8. Use of the monoclonal antibody or derivative thereof according to claim 1 or 2 for the preparation of a novel coronavirus detection product.

9. Use of the monoclonal antibody or derivative thereof according to claim 1 or 2 for the preparation of a novel diagnostic product for coronavirus infection.

10. Use of the composition of claim 7 for the preparation of a product for the detection of novel coronaviruses.

Technical Field

The invention belongs to the fields of cellular immunology and molecular biology, and relates to an antibody for immunoassay against a novel coronavirus.

Background

The international committee for viral classification named the novel coronavirus SARS-CoV-2 and the world health organization named the pneumonia caused by infection with this virus COVID-19. The virus has strong infectivity and wide transmission path. The virus can adapt to the environment of human body rapidly, has transmission capability in latent period after infection, and reports by some asymptomatic infectors that virus nucleic acid is detected even in various animals. These factors complicate the control of the virus and no effective therapeutic drugs and vaccines are currently on the market.

SARS-CoV-2 belongs to the genus Coronavirus, is a single-stranded positive-strand RNA virus, has a size of about 30kb, has a similarity of 79% to SARS-CoV, and has a similarity of up to about 88% to a Coronavirus (CoV) isolated from Bats. SARS-CoV-2 has typical coronavirus characteristics, and the virus envelope has typical spinous processes, which are shaped like coronages. The Nucleocapsid is of a spiral symmetrical type, the main structural protein is Nucleocapsid Protein (NP), and the total length of the NP is 420 amino acids. The NP has the most content in virus structural protein, is expressed in a large amount in the early stage of host infection, has stronger immunogenicity, and can cause strong immune response of a host. Thus, NP can be used as the main target antigen for serological diagnosis of SARS-CoV-2 infection.

Because specific therapeutic drugs and effective vaccines are not developed successfully, early diagnosis becomes an important measure for preventing and controlling epidemic situations, and early nucleic acid diagnosis and clinical diagnosis become important basis for accurate diagnosis. Although the nucleic acid diagnosis speed is high, the influence of the quality of the sampling is large, false positive and false negative exist, and the implementation of the prevention and control measures is influenced. Nucleic acid detection of part of asymptomatic infected persons is negative in the late stage of the disease process, and missed diagnosis is easy to occur only by nucleic acid detection. Serological diagnosis is to detect the immune response of an organism after pathogen infection, the duration is long, the immune response is stable, and the immune response shows a dynamic change trend along with the progress of the disease course. Serodiagnosis is therefore also an important tool for early diagnosis and assessment of the current state of infection.

Disclosure of Invention

One of the technical problems to be solved by the present invention is to provide a monoclonal antibody or its derivatives such as antibody Fab fragment, single chain antibody, etc. against novel coronavirus.

The second technical problem to be solved by the present invention is to provide a DNA molecule or gene encoding the above antibody.

The third technical problem to be solved by the present invention is to provide a method for preparing the above antibody.

In order to solve the technical problems, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a monoclonal antibody against a novel coronavirus, or a derivative thereof, comprising a first variable region and a second variable region, wherein the first variable region is an antibody light chain variable region comprising the antigen complementarity determining region CDR1, CDR2 and CDR3 respectively comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; wherein the second variable region is an antibody heavy chain variable region having the antigen complementarity determining regions CDR1, CDR2 and CDR3 of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;

the derivatives of the monoclonal antibody include antibody Fab fragments, single chain antibodies, bispecific antibodies (bi-specific), and the like.

As a preferred embodiment of the present invention, the first variable region is an antibody light chain variable region which is represented by SEQ id no: 8; wherein said second variable region is an antibody heavy chain variable region represented by SEQ id no: 4.

As a preferred embodiment of the present invention, it comprises the antibody light chain variable region and the human antibody light chain constant region, and the hinge region, CH1 region, CH 2 region and CH3 region of the antibody heavy chain variable region and the human antibody heavy chain constant region.

As a preferred embodiment of the present invention, the human antibody light chain constant region is derived from a human antibody kappa chain or antibody lamda chain, and the human antibody heavy chain constant region is derived from a human IgG1, IgG2, IgG3 or IgG4 subtype.

In a second aspect of the invention, there is provided a DNA molecule or gene nucleotide sequence encoding the monoclonal antibody or derivative thereof as hereinbefore described.

As a preferred embodiment of the present invention, the variable region of the antibody light chain is SEQ ID NO: 18, and the variable region of the antibody heavy chain is SEQ ID NO: 17.

In addition, the invention includes sequences that specifically hybridize to any of the nucleotide sequences provided herein. The term "specifically hybridizes" refers to the ability of a nucleotide sequence to hybridize to at least 12, 15, 20, 25, 30, 35, 40, 45, 50, or 100 linked nucleotides of a sequence provided herein or of a sequence complement thereto, such that it has less than 15%, preferably less than 10%, and more preferably less than 5% background hybridization to a control nucleic acid (e.g., non-specific DNA or DNA other than the specific antibody sequence provided herein). Various hybridization conditions can be used to detect specific hybridization, and stringency is largely determined by the washing steps of the hybridization assay. In general, high temperatures and low salt concentrations produce high stringency, while low temperatures and high salt concentrations produce low stringency. Low stringency hybridization is achieved by washing at 50 ℃ in, for example, about 2.0 XSSC, and high stringency is achieved with about 0.2 XSSC at 50 ℃.

The nucleotides encoding the antibodies of the invention or derivatives thereof may contain leader or signal sequences. The leader and signal sequences may be varied and may be substituted with alternative leader sequences, and it will be understood that in certain embodiments, the antibodies of the invention contain sequences without leader sequences. Any suitable alternative preamble or signal sequence may be used.

In a third aspect of the invention, there is provided an expression vector comprising the DNA molecule/gene nucleotide sequence set forth above and expression control sequences operatively linked to the sequence.

In a fourth aspect of the invention, there is provided a recombinant host cell transformed with an expression vector as hereinbefore described.

The recombinant host cell or progeny thereof expresses the monoclonal antibody or derivative thereof as described above.

In a fifth aspect of the invention, there is provided a method of preparing a monoclonal antibody or derivative thereof as hereinbefore described, the method comprising the steps of:

a) providing an expression vector comprising the DNA molecule sequence as described above and an expression control sequence operably linked to the sequence;

b) transforming a host cell with the expression vector of step a);

c) culturing the host cell obtained in step b) under suitable conditions: and

d) and (3) separating and purifying the monoclonal antibody or the derivative thereof from the culture solution of the host cell.

In a sixth aspect of the invention, there is provided a composition comprising a monoclonal antibody or derivative thereof as hereinbefore described.

In a seventh aspect of the invention, there is provided a kit comprising a monoclonal antibody or derivative thereof as hereinbefore described. The kit also includes a diagnostic agent.

The diagnostic agent includes a detectable substance, examples of which include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally US4,741,900. suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosidase, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin, and biotin, suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin, suitable luminescent materials include luminol, suitable bioluminescent materials include luciferase, luciferin, and aequorin, radioisotopes such as, for example, luciferase, luciferin, and aequorin¹²⁵I、¹³¹I、¹¹¹In and⁹⁰Y、Lu¹⁷⁷bismuth, bismuth²¹³Californium²⁵²Iridium (III)¹⁹²And tungsten¹⁸⁸Rhenium¹⁸⁸、²¹¹Astatine, astatine,⁹⁹Tc。

The kits of the invention also include a label and instructions for administration.

The composition or kit of the invention as described above may further comprise a second monoclonal antibody or derivative thereof comprising heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, light chain variable region CDR 3; wherein the content of the first and second substances,

heavy chain variable region CDR1 comprises SEQ ID NO: 9;

heavy chain variable region CDR2 comprises SEQ ID NO: 10;

heavy chain variable region CDR3 comprises SEQ ID NO: 11;

light chain variable region CDR1 contains SEQ ID NO: 13;

light chain variable region CDR2 contains SEQ ID NO: 14;

light chain variable region CDR3 contains SEQ ID NO: 15;

preferably, the second monoclonal antibody or derivative thereof comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises SEQ ID NO: 12 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16.

In the eighth aspect of the invention, the application of the monoclonal antibody or the derivative thereof in preparing a novel coronavirus detection product is provided.

In the ninth aspect of the invention, the application of the monoclonal antibody or the derivative thereof in preparing a novel coronavirus infection diagnosis product is provided.

In a tenth aspect of the invention, there is provided the use of a composition as hereinbefore described in the manufacture of a product for the detection of a novel coronavirus.

The term "monoclonal antibody (mab)" as used herein refers to an immunoglobulin derived from a pure line of cells, having the same structural and chemical properties, and being specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody being directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are obtained by hybridoma or recombinant engineered cell culture, and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The terms "antibody" and "immunoglobulin" as used herein are heterotetrameric proteins of about 150000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end. Followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the light and heavy chain variable regions that become Complementarity Determining Regions (CDRs) or hypervariable regions.

The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of the heavy and light chains of an antibody each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs which form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held closely together by the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Ka ba t et al, NIH Pu bl. No.91-3242, Vol. 1, 647, 669 (1991)). Antibody constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated toxicity (CDC) of an antibody.

The term "expression control sequence" as used herein generally refers to a sequence involved in controlling the expression of a gene. Expression control sequences include a promoter and a termination signal operably linked to a gene of interest. The gene (DNA) sequence encoding the antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the protein sequence according to the present disclosure or amplification by PCR. The resulting DNA fragment may then be inserted into a suitable expression vector by a variety of methods well known in the art. The expression vector used in the present invention may be a commercially available expression vector known to those skilled in the art, such as pCDNA3.1 expression vector of Invitrogen corporation.

Suitable host cells for transformation with the host cells to which the expression vectors are administered generally include prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.

After culturing the host cells transformed with the expression vector under suitable conditions (e.g., adherent or suspension culture in a cell culture flask or bioreactor in serum-free medium), the culture supernatant is harvested and then purified by conventional separation procedures or means well known to those skilled in the art, including protein-A affinity chromatography, ion exchange chromatography, filter sterilization, etc., to obtain the antibody of the present invention.

The purified antibody of the present invention can be dissolved in a suitable solvent such as a sterile physiological saline solution, and the solubility can be prepared to be between 0.01 and 100mg/ml, and the desired final solubility can be prepared to be between 1 and 20 mg/ml.

Drawings

FIG. 1 shows a SDS-PAGE pattern of the recombinant SARS-CoV 2NP protein of the present invention;

FIG. 2 is a graph showing the results of detection of antibody titer by indirect ELISA;

FIG. 3 is a graph showing the results of detecting the binding of an antibody to an antigen using WB;

FIG. 4 shows the results of the affinity activity of JS01 detected by SPR;

FIG. 5 shows the results of the affinity activity of JS02 detected by SPR;

FIG. 6 is a graph showing the results of detecting the affinity activity of JS03 using SPR;

FIG. 7 is a graph showing the results of detecting the affinity activity of JS04 using SPR;

FIG. 8 is a graph showing the results of detecting the affinity activity of JS05 using SPR;

FIG. 9 is a graph showing the results of detecting the affinity activity of JS06 using SPR;

FIG. 10 is a graph showing the results of detecting the affinity activity of JS07 using SPR;

FIG. 11 is a graph showing the results of detecting the affinity activity of JS08 using SPR;

FIG. 12 is a graph showing the results of detecting the affinity activity of JS09 using SPR;

FIG. 13 is a graph showing the results of detecting the affinity activity of JS10 using SPR;

FIG. 14 is a graph showing the results of detecting the affinity activity of JS11 using SPR;

FIG. 15 is a graph showing the results of detecting the affinity activity of JS12 using SPR;

FIG. 16 is a graph showing the results of detecting the affinity activity of JS13 using SPR;

FIG. 17 is a graph showing the results of detecting the affinity activity of JS14 using SPR;

FIG. 18 is a graph showing the results of detecting the affinity activity of JS15 using SPR;

FIG. 19 is a graph showing the results of detecting the affinity activity of JS16 using SPR;

FIG. 20 is a graph showing the results of measuring the antibody coating concentration by the double antibody sandwich method;

FIG. 21 is a graph showing the results of detection sensitivity of antibodies by the double antibody sandwich method;

FIG. 22 is a graph showing the detection effect of the antigen detection chromatographic strip of the present invention.

Detailed Description

The invention is further illustrated by the figures and examples. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.