CN113980099A

CN113980099A - N antigen specific epitope of new coronavirus and application thereof

Info

Publication number: CN113980099A
Application number: CN202110331945.1A
Authority: CN
Inventors: 于晓波; 梁特; 张家辉; 王红叶
Original assignee: Institute Of Life Sciences Academy Of Military Medicine Academy Of Military Sciences
Current assignee: Institute Of Life Sciences Academy Of Military Medicine Academy Of Military Sciences
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2022-01-28

Abstract

The invention relates to a N antigen specific epitope of a new coronavirus SARS-CoV-2 and application thereof, and provides a polypeptide, a kit containing the polypeptide and application thereof in preparation of products for SARS-CoV-2 detection, diagnosis or drug screening. The invention also provides a method for detecting SARS-CoV-2 and a method for screening drugs. By adopting the polypeptide to design or screen the antibody, the advantages of low detection limit, high specificity and high sensitivity can be obtained.

Description

N antigen specific epitope of new coronavirus and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a method for detecting and diagnosing 2019 novel coronavirus (SARS-CoV-2) and screening medicaments, and application of polypeptide or an antibody aiming at the polypeptide in preparation of products for detecting and diagnosing 2019 novel coronavirus (SARS-CoV-2) and screening medicaments.

Background

New coronary pneumonia (COVID-19) caused by the novel coronavirus-2 (SARS-CoV-2) has become a worldwide epidemic. The antibody is an important material for resisting the pandemic diseases, and also plays an important role in helping people to research pathogenesis of new coronary pneumonia, diagnose and detect, and develop a treatment method for neutralizing SARS-CoV-2 virus activity. However, the amino acid sequence ("epitope", "epitops") by which an antibody specifically recognizes and binds an antigen is unknown to most new coronavirus antibodies.

The novel coronavirus (SARS-CoV-2) has four major structural proteins: spike protein (S protein), Nucleocapsid protein (N protein), Membrane protein (M protein), Envelope protein (E protein). Among them, the S protein is abundant in coronavirus, and RBD in the S protein contains a highly similar core structure and a greatly different Receptor Binding Motif (RBM), which is a main structure recognizing angiotensin converting enzyme 2(ACE2), and plays a crucial role in screening antibodies, preparing vaccines, and the like. The N antigen is used as a structural protein and can also be used for detection or diagnosis.

At present, nucleic acid and serum antibody detection is still the main technology for diagnosing the new coronary pneumonia. For example, patent document CN112409462A discloses a SARS-CoV-2 specific antigen and SARS-CoV-2 immunoglobulin detection kit, which comprises S protein and N protein as antigens for detection and has a lower false positive and false negative rate. Patent document CN112485425A discloses a kit for detecting N-S antigen of novel salivary coronavirus and an application method thereof, wherein the method is convenient to operate, short in detection period and easy to interpret. Patent document CN111999496A discloses a kit for joint detection of SARS-CoV-2 antigen and antibody and a preparation method thereof, which adopts a kit for joint detection of antigen and antibody, wherein the kit comprises a reaction plate coated with S antigen. However, the above prior arts do not disclose a specific epitope targeted by an antibody, and the antibody is designed only for the full-length S protein or the full-length N protein, and has limited specificity and sensitivity.

Therefore, the application provides a polypeptide which can be used for designing or screening antibodies, achieves high specificity and sensitivity, and has no report on specific recognition epitopes of antibody pairs at present.

Disclosure of Invention

In a first aspect of the invention, there is provided a use of a polypeptide in the preparation of a product for detecting, diagnosing or drug screening SARS-CoV-2, said polypeptide comprising the amino acid sequence of SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

In a second aspect of the invention, there is provided a use of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

Preferably, the polypeptide comprises:

A) SEQ ID NO: 1, and SEQ ID NO: 2 and/or 3; or,

B) SEQ ID NO: 1, and SEQ ID NO: 4 to 15, or a combination of two or more thereof.

In a third aspect of the invention, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

Preferably, the polypeptide comprises:

A) SEQ ID NO: 1, and SEQ ID NO: 2 and/or 3; or,

In a fourth aspect of the invention, there is provided a kit for the detection or diagnosis of SARS-CoV-2, which kit comprises the polypeptide as defined above.

Preferably, the kit further comprises a substrate on which the polypeptides can be distributed in an array. Further preferably, the substrate includes, but is not limited to, glass, silicon wafer, ceramic, mica, metal, plastic, high polymer film, etc., preferably glass, silicon wafer and ceramic.

The polypeptide of the present invention may be an amino acid sequence or a combination of amino acid sequences.

In a fifth aspect of the invention, there is provided a method for screening or verifying the function of a drug, said method comprising contacting the polypeptide with a sample to be screened.

Preferably, the method comprises the following steps:

converting a polypeptide comprising SEQ ID NO: 1, and then adding a polypeptide comprising SEQ ID NO: 2 and/or 3; alternatively, a polypeptide comprising SEQ ID NO: 1, and then adding a polypeptide comprising SEQ ID NO: 4-15, or a combination of two or more thereof.

In a sixth aspect of the invention, there is provided a method for detecting SARS-CoV-2, comprising contacting a sample to be detected with a capture antibody, and adding a detection antibody, wherein the capture antibody recognizes the amino acid sequence of SEQ ID NO: 1, and the detection antibody recognizes the polypeptide shown in SEQ ID NO: 2-15 or a combination of two or more thereof.

Preferably, the detection method comprises coating the capture antibody on a carrier, and blocking; adding a sample to be detected, and incubating; adding a detection antibody, and incubating; adding a labeled antibody, and incubating; the reaction was terminated.

In a seventh aspect of the invention, there is provided an antibody pair for use in the manufacture of a product for detecting or diagnosing SARS-CoV-2, said antibody pair comprising a capture antibody and a detection antibody, said capture antibody recognizing an epitope comprising the amino acid sequence of SEQ ID NO: 1, and the detection antibody recognition epitope comprises a) an epitope shown in SEQ ID NO: 2 and SEQ ID NO: 3, and/or b) the epitope set forth in SEQ ID NO: 4-15 or a combination of two or more of the epitopes.

In an eighth aspect of the invention, there is provided the use of an antibody pair for detecting SARS-CoV-2, said antibody pair comprising a capture antibody and a detection antibody, said capture antibody recognizing an epitope comprising SEQ ID NO: 1, and the detection antibody recognition epitope comprises a) an epitope shown in SEQ ID NO: 2 and SEQ ID NO: 3, and/or b) the epitope set forth in SEQ ID NO: 4-15 or a combination of two or more of the epitopes.

Preferably, the antibody pair targets ORF1ab polyprotein, Sglycoprotein, ORF3a protein, envelope protein, membrane glycoprotein, ORF6 protein, ORF7a protein, ORF8 protein, nucleocapesid phosphoprotein and/or ORF10 protein of SARS-CoV-2, as shown in Table 1.

In one embodiment of the invention, the antibody targets a specific epitope of the N protein of SARS-CoV-2 (NP-828858.1).

TABLE 1 SARS-CoV-2 coronavirus encoded proteins

Specifically, the epitope recognized by the capture antibody and the epitope recognized by the detection antibody are shown in table 2:

TABLE 2

Preferably, the product may be a kit, a chip, or the like.

The ninth aspect of the invention provides the application of the antibody to the recognition epitope or polypeptide as a drug target or a therapeutic target in the preparation of drugs for treating diseases caused by the new coronavirus.

In a tenth aspect of the invention, there is provided an antibody pair comprising a capture antibody and a detection antibody, wherein the capture antibody recognizes an epitope comprising the amino acid sequence of SEQ ID NO: 1, and the detection antibody recognition epitope comprises a) an epitope shown in SEQ ID NO: 2 and SEQ ID NO: 3, and/or b) the epitope set forth in SEQ ID NO: 4-15 or a combination of two or more of the epitopes.

In the eleventh aspect of the invention, a kit for detecting or diagnosing SARS-CoV-2 is provided, wherein the kit comprises the above-mentioned antibody pair recognition epitope or polypeptide or the above-mentioned antibody pair. Preferably, the kit further comprises a carrier, a washing solution, a blocking solution, a labeled antibody, a developing solution and/or a termination reaction solution.

Further preferably, the carrier may be any container or plate capable of supporting an antigen-antibody reaction. More preferably, a 96-well plate is used.

Further preferably, the wash solution may be PBST or a solution comprising PBST.

Further preferably, the blocking liquid can be PBST and/or milk. Further preferably 0.2% PBST + 5% skim milk.

Further preferably, the labeled antibody can be a horseradish peroxidase (HRP) labeled goat anti-rabbit IgG antibody.

More preferably, the color-developing solution may be 3 ', 3', 5 ', 5' -Tetramethylbenzidine (TMB).

Further preferably, the reaction terminating solution may be sulfuric acid, hydrochloric acid or NaOH.

In the twelfth aspect of the invention, a method for detecting SARS-CoV-2 is provided, which comprises contacting a sample to be detected with a capture antibody, and adding a detection antibody.

Further preferably, the vector may be a 96-well plate.

Further preferably, the blocking is performed by using a blocking liquid (preferably 0.2% PBST + 5% skim milk) at 30-40 ℃ (preferably 37 ℃).

In one embodiment of the present invention, the sample to be detected can be any sample containing SARS-CoV-2 protein (including S protein, N protein, M protein or E protein, preferably N protein). Specifically, the sample can be human or non-human animal blood, serum, cells, tissues or organs (such as nasopharyngeal swab, deep expectoration, alveolar lavage fluid, lung tissue biopsy specimen), or can be isolated blood, serum, cells, tissues or organs (such as nasopharyngeal swab, deep expectoration, alveolar lavage fluid, lung tissue biopsy specimen) of human or non-human animal, or can be any living goods, buildings and other articles which can be attached with SARS-CoV-2, such as ice cream, meat, seafood, food package, stair handles, table tops and the like.

Based on the difference between the above samples to be tested, the SARS-CoV-2 detection method described in this application can be used for therapeutic purposes or non-therapeutic purposes.

Preferably, the incubation temperature is 30-40, more preferably 37 ℃.

Preferably, the incubation time is 0.5-2h, more preferably 1 h.

Preferably, the labeled antibody can be a horseradish peroxidase (HRP) labeled goat anti-rabbit IgG antibody.

In one embodiment of the invention, the capture antibody is coated in a 96-well plate overnight at 4 ℃, washed and then blocked in 0.2% PBST + 5% skim milk for 1h at 37 ℃; after being cleaned by 0.2% PBST, a sample to be detected is added, and the mixture is incubated for 1h at 37 ℃; adding a detection antibody after cleaning, and incubating for 1h at 37 ℃; after PBST is washed, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG antibody is added, and incubation is carried out for 1h at 37 ℃; after PBST washing, 3 ', 5 ', 5 ' -Tetramethylbenzidine (TMB) was added, followed by addition of the terminating reaction solution.

Preferably, the method further comprises the step of detecting the absorbance at 450 nm.

The capture antibody or detection antibody of the present invention may be in the form of an antibody, or an antigen-binding fragment. Of course, a single-chain antibody or a single-domain antibody may be used. Either monoclonal or polyclonal. Also encompassed are fragments such as Fab, Fab '-SH, Fv, scFv, (Fab') 2, single domain antibodies, diabodies (dAbs), or linear antibodies.

The "detection" of the present invention is to determine whether a sample to be detected contains a novel coronavirus-2 (SARS-CoV-2) or whether an autoantibody is contained.

The "diagnosis" of the present invention is to confirm whether an individual is infected with SARS-CoV-2 and causes new coronary pneumonia (COVID-19)

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: the antibody of the N antigen-antibody pair of the neocoronaviruse recognizes an epitope, wherein a is #14 recognition epitope, b is #12 recognition epitope, and c is #13 recognition epitope, and Z-score ═ (x- μ)/σ where x is an observed value, μ is an average value of the group of data, and σ is a standard deviation of the group of data.

FIG. 2: as a result of detecting SARS-CoV-2 recombinant N protein by ELISA method, a represents pairing 1, and b represents pairing 2.

FIG. 3: ELISA method for detecting SARS-CoV-2 virus infected VERO cell culture liquid N protein result, wherein, a represents pairing 1, b represents pairing 2.

Detailed Description

The following will specifically explain the embodiments of the present invention with reference to the examples.

Sources of antibodies, cells and reagents used in the present application:

antibody #12, purchased from nano Biological Inc, cat # 40143-R001, antibody type MAb, host Rabbit;

antibody #13, purchased from nano Biological Inc, cat # 40143-RP01, antibody type PAb, host Rabbit;

antibody #14, purchased from nano Biological Inc, cat # 40143-MM05, antibody type MAb, host Mouse;

recombinant New coronavirus N protein, purchased from Sino Biological, Inc., Cat 40588-V08B.

Example 1 determination of the recognition of epitopes by antibodies for detecting SARS-CoV-2

According to the sequence of all the encoding proteins of SARS-CoV-2 extracted from NCBI data described in CN202010215184.9 (application date: 2020-03-24), SARS-CoV-2 virus proteome polypeptide chip is designed and prepared to realize panoramic scan of all SARS-CoV-2 virus antibodies in blood of patient infected by new type pneumonia virus, and all the contents of the invention can be used as the contents of the invention.

The epitope of the antibody specifically reacting with the polypeptide chip is shown in FIG. 1.

EXAMPLE 2 detection of SARS-CoV-2 nucleocapsid protein by enzyme Linked immunosorbent assay

The experimental steps are as follows: antibody #14 was coated into 96-well plates (Corning, USA) overnight at 4 ℃ and blocked in 0.2% PBST + 5% skim milk for 1h at 37 ℃ after washing. After washing with 0.2% PBST, diluted recombinant new coronavirus N protein or SARS-CoV-2IME-BJ01 strain was added to a 96-well plate and incubated at 37 ℃ for 1h, followed by addition of antibody #12 or #13 after infection of the inactivated VERO cell culture 2-3 days (reference Escapza, Thomas J et al, "High affinity nanobodies block SARS-CoV-2spike receptor binding domain interaction with human angiotensin converting enzyme. After incubation for 1h at 37 ℃, PBST was washed. Horse Radish Peroxidase (HRP) -labeled goat anti-rabbit IgG antibody (CoWin Biosciences, China) was added to a 96-well plate, incubated at 37 ℃ for 1h, washed with PBST, and then 3 ', 3', 5 ', 5' -Tetramethylbenzidine (TMB) (CoWin Biosciences, China) was added thereto, and an enzyme-linked immunosorbent assay termination reaction solution (Solarbio, China) was added thereto. After a short shaking of the plate, the absorbance at 450nm was read.

Wherein the capture antibody is: # 14; the detection antibody was (#12, #13) and represents two antibody pairs (pair 1: #14 and #12, pair 2: #14 and # 13).

Statistical analysis: statistical analysis was performed using GraphPad Prism software and Microsoft Excel

The experimental results are as follows: the ELISA method detects SARS-CoV-2 recombinant N protein, the detection limit of pairing 1 and pairing 2 is 1.425ng/mL and 1.03ng/mL respectively (FIG. 2a and FIG. 2 b); the limit LOD of detection of N protein in VERO cell culture fluid for detecting SARS-CoV-2 virus infection by ELISA method is 1.119 x 10 respectively⁵PFU/mL (mate 1, FIG. 3a) and 1.615 x 10⁴PFU/mL (paired)2, fig. 3 b). That is, ELISA methods based on antibody pairs (#14 and #12, #14 and #13) have great potential in clinical detection of SARS-CoV-2.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

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Claims

1. The application of the polypeptide in preparing products for detecting, diagnosing or screening drugs of SARS-CoV-2 is characterized in that the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

2. Use of a polypeptide for detecting SARS-CoV-2, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

3. The use of claim 1 or 2, wherein said polypeptide comprises:

A) SEQ ID NO: 1, and SEQ ID NO: 2 and/or 3; or,

4. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1-15 or a combination of two or more of the amino acids shown.

5. The polypeptide of claim 4, wherein said polypeptide comprises:

A) SEQ ID NO: 1, and SEQ ID NO: 2 and/or 3; or,

6. A kit for the detection or diagnosis of SARS-CoV-2, comprising the polypeptide of claim 5.

7. A method of drug screening comprising contacting a sample to be screened with a polypeptide of any one of claims 4 to 5.

8. The method of claim 7, wherein the method comprises:

9. A method for detecting SARS-CoV-2, which comprises contacting a capture antibody with a sample to be detected, and adding a detection antibody, wherein the capture antibody recognizes the amino acid sequence of SEQ ID NO: 1, and the detection antibody recognizes the polypeptide shown in SEQ ID NO: 2-15 or a combination of two or more thereof.

10. The detection method according to claim 9, wherein the detection method comprises coating a capture antibody on a carrier, blocking; adding a sample to be detected, and incubating; adding a detection antibody, and incubating; adding a labeled antibody, and incubating; the reaction was terminated.