CN111978377A - COVID-19 antigen, preparation method and application - Google Patents
COVID-19 antigen, preparation method and application Download PDFInfo
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- CN111978377A CN111978377A CN202010657369.5A CN202010657369A CN111978377A CN 111978377 A CN111978377 A CN 111978377A CN 202010657369 A CN202010657369 A CN 202010657369A CN 111978377 A CN111978377 A CN 111978377A
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses important antigen epitopes SEQ ID:01 and SEQ ID:02 for COVID-19 antibody detection, and recombinant proteins containing the antigen epitopes can be used for preparation of antibodies such as IgM and IgG resisting COVID-19. The antibody detection reagent for resisting COVID-19 is an effective supplement for detecting the nucleic acid of the new coronavirus, and can be used for screening contacts of the new coronavirus and crowds who acquire immunity. The invention overcomes the problems of insufficient single S protein epitope, insufficient detection sensitivity of the IgM antibody resisting COVID-19 and the like, and the anti-neocorolla antibody detection reagent prepared by the recombinant protein containing the SEQ01-04 neoepitope has the advantages of high sensitivity, stable performance, accurate qualitative determination and the like.
Description
Technical Field
The invention relates to the field of immunology, in particular to a COVID-19 antigen, a preparation method and application.
Background
The causative agent of the novel coronavirus (COVID-19) has recently been identified as a positive-strand RNA virus whose genome is a single-stranded RNA containing Poly (A) and has a total length of approximately 30 kb. The surface of the virus has a rod-shaped structure of about 20nm and is in a crown shape. The pathogen has strong infectivity and infectivity, and after the virus is subjected to an early replication process in a patient body, large-scale inflammatory reaction is caused in the lung, and finally lung tissue damage is caused. Because of the lack of effective treatment drugs in clinic, the isolation treatment of infected patients is the most effective prevention and treatment means at present.
Diagnostic methods that have been developed and used include:
1. RT-PCR was used to check the virus. Can check the patient with poison in time, and shows negative after recovery. However, due to the limitation of instruments and equipment, many false negatives and false positives are easy to generate, which leads to missed diagnosis or misdiagnosis.
2. The diagnosis is carried out by comprehensive evaluation of clinical symptoms. This method cannot be used for accurate diagnosis in the early stage of the disease because the symptoms are not obvious. In the late stages of the disease, although accurate diagnosis is possible, effective treatment of the disease is delayed.
3. Serum immune test virus antibodies (fluorescence immunoassay or ELISA). Antibody production requires at least 14 days after onset, and therefore, this approach does not allow for early diagnosis. However, immunologically, the production of pathogen-specific antibodies IgM by the body is relatively early and the selection of an appropriate antigen to bind to it can also be used as a diagnostic reference.
4. Most of the methods currently in use are the first three methods, and the methods cannot simply, quickly and accurately judge and distinguish patients from suspected cases in the clinical use process.
4. Antigen detection (ELISA). The method for detecting the pathogen in the blood or tissue of the patient by preparing the pathogen specific antibody (monoclonal antibody and polyclonal antibody) has the advantages of high accuracy, simple and convenient operation and suitability for clinical popularization. But currently lack pathogen-specific antibodies.
Therefore, there is an urgent need in the art to develop new more effective methods and kits for early diagnosis of novel coronaries.
Disclosure of Invention
In order to solve the problems of the prior art, in one aspect, the invention provides a COVID-19 antigen, the amino acid sequence of which comprises any one of SEQ ID 01: VSEETGTLIVNSVLLFL, or SEQ ID 02: YVYSRVKNLNSSRVPDLLV, or the two amino acid sequences of SEQ ID 01 and SEQ ID 02 in tandem in a reasonable manner.
As a further improvement of the embodiment of the invention, the COVID-19 antigen is prepared by an amino acid synthesis method, a cysteine is added at the N end or the C end, and the cysteine is coupled with the human serum albumin to generate human serum albumin conjugates I and II.
In another aspect, the present invention provides a coding gene of a COVID-19 antigen, the nucleotide sequence of which comprises SEQ ID 03: GTTT CGGAAGAGACAGGTACGTTA ATAGTTAATA GCGTACTTCT TTTTCTT or SEQ ID 04: TAC GTTTA CTCTCGTT AAAAATCTGA ATTCTTCTAG AGTTCCTGAT CTTCTGGTC or the two nucleotide sequences of SEQ ID 03 and SEQ ID 04 are connected in series in a reasonable manner; wherein, the SEQ ID 03 is the coding gene of the SEQ ID 01, and the SEQ ID 04 is the coding gene of the SEQ ID 02.
In another aspect, the invention discloses a recombinant expression plasmid, wherein the recombinant expression plasmid is constructed by directionally cloning the nucleotide sequence into corresponding sites of a vector to obtain a recombinant expression vector, and further transforming the recombinant expression vector into BL21(DE3) competent cells.
In another aspect, the invention further discloses a recombinant expression plasmid, wherein the recombinant expression plasmid is formed by connecting the nucleotide sequences SEQ ID 03 and SEQ ID 04 with the N end and the C end of the S1 gene respectively, constructing an expression vector as described above, and transfecting escherichia coli or cells.
In another aspect, the invention discloses an application of the COVID-19 antigen in preparing a detection kit for detecting an anti-COVID-19 antibody.
On the other hand, the invention discloses a detection kit for detecting an anti-COVID-19 antibody, which comprises a solid phase carrier coated or marked with a COVID-19 antigen, anti-human IgM \ IgG or IgA and a sample diluent.
The COVID-19 antigen comprises the amino acid sequence or the coding gene of the COVID-19 antigen comprises the nucleotide sequence.
As a further improvement of the embodiment of the invention, the COVID-19 antigen comprises the amino acid sequence or the coding gene of the COVID-19 antigen comprises the nucleotide sequence or is obtained by the recombinant expression plasmid.
As a further improvement of the embodiment of the invention, the solid phase carrier comprises a microporous plate, colloidal gold microspheres, fluorescent microspheres and an NC membrane.
In another aspect, the invention discloses a method for using the above-mentioned antibody detection kit against COVID-19, comprising the steps of:
(a) contacting the sample with the above-described COVID-19 antigen;
(b) detection of formation of antigen-antibody complexes: the formation of a complex indicates the presence of anti-COVID-19 antibodies in the sample.
In another aspect, the invention discloses an application of the COVID-19 antigen in the early diagnosis of the COVID-19.
In another aspect, the invention discloses an application of the COVID-19 antigen in preparation of a COVID-19 epitope vaccine.
In another aspect, the invention discloses an application of the COVID-19 antigen in preparing a medicament for treating or preventing novel coronavirus.
The invention has the following beneficial effects:
1. the invention finds an amino acid sequence with strong antigenicity by analyzing the sequence of the latent antigen protein of the new coronavirus, synthesizes the target sites of the latent antigens by synthesizing polypeptides, and then obtains an antibody by immunizing the polypeptides, thereby developing an enzyme-linked immunosorbent assay method and a kit for diagnosing the new coronavirus at early stage in clinic by detecting the new coronavirus. The invention can obviously improve the diagnosis efficiency, and is simple and convenient, and has high accuracy;
2. the invention overcomes the problems of insufficient single S protein epitope, insufficient detection sensitivity of the IgM antibody resisting COVID-19 and the like, and the anti-neocorolla antibody detection reagent prepared by the recombinant protein containing the SEQ01-04 neoepitope has the advantages of high sensitivity, stable performance, accurate qualitative determination and the like;
3. the detection kit has high detection speed and high accuracy, and overcomes the problem of high false positive rate in the prior art;
4. because the affinity of the specific polypeptide and the polypeptide conjugate of the new coronavirus and the anti-new coronavirus antibody is high, and the affinity of the antibody and the new coronavirus is high, the detection method is sensitive, simple and convenient and has high accuracy;
5. the new coronavirus specific polypeptide can compete with viruses for receptors in vivo, prevent the fusion of viruses and membranes and inhibit the viruses from infecting normal cells, so the new coronavirus specific polypeptide has high clinical application value.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram showing the comparison of the detection results of the novel corona detection kit provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
The invention provides a COVID-19 antigen, the amino acid sequence of which comprises any one sequence of SEQ ID 01: VSEETGTLIVNSVLLFL or SEQ ID 02: YVYSRVKNLNSSRVPDLLV or the two amino acid sequences of SEQ ID 01 and SEQ ID 02 are connected in series in a reasonable mode.
In the embodiment of the present invention, the specific tandem mode includes, but is not limited to, asymmetric sticky end complementary construction, that is, the target gene is cut at two side sites by enzyme digestion to generate asymmetric sticky ends, and the target gene is inserted to obtain the amino acid of the target sequence.
In other optional embodiments, the series mode can also be selected as follows: non-cohesive end complementary construction, joint connection, isocaudarner construction, multi-copy expression and the like.
In the present examples, coupling of a single amino acid sequence, e.g., coupling of SEQ ID 01, coupling of SEQ ID 02; there is no coupling between the two amino acids.
The COVID-19 antigen is prepared by an amino acid synthesis method, specifically, a cysteine is added at the N end or the C end, and the cysteine is coupled with the human serum albumin to generate human serum albumin conjugates I and II. Can also be prepared in other reasonable ways in a polypeptide synthesizer, and the polypeptide synthesis means is common knowledge in the field and is not specifically described here.
Example 2
The invention provides a coding gene of a COVID-19 antigen, and the nucleotide sequence of the coding gene comprises the nucleotide sequence shown in SEQ ID 03: GTTT CGGAAGAGACAGGTACGTTA ATAGTTAATA GCGTACTTCT TTTTCTT or SEQ ID 04: TAC GTTTA CTCTCGTT AAAAATCTGA ATTCTTCTAG AGTTCCTGAT CTTCTGGTC or the two nucleotide sequences of SEQ ID 03 and SEQ ID 04 are connected in series in a reasonable manner; wherein, the SEQ ID 03 is the coding gene of the SEQ ID 01, and the SEQ ID 04 is the coding gene of the SEQ ID 02.
In the embodiment of the invention, the method for establishing the recombinant expression plasmid is adopted for SEQ ID 03, SEQ ID 04 and the method for establishing the series connection of SEQ ID 03 and SEQ ID 04 in a reasonable manner; specifically, the recombinant vector is constructed by directionally cloning SEQ ID 03 or SEQ ID 04 to corresponding sites on the same plasmid and transforming the recombinant vector into competent cells.
The invention also discloses a recombinant expression plasmid, which is constructed by directionally cloning nucleotide sequences SEQ ID 03 and SEQ ID 04 between corresponding sites of a vector to obtain a recombinant expression vector, and further transforming the recombinant expression vector into BL21(DE3) competent cells.
The invention further discloses a recombinant expression plasmid, wherein the nucleotide sequences SEQ ID 03 and SEQ ID 04 are respectively connected with the N end and the C end of the S1 gene, an expression vector is constructed as above, and escherichia coli or cells are transfected.
Example 3
The embodiment of the invention discloses application of the COVID-19 antigen in preparation of a detection kit for detecting an anti-COVID-19 antibody.
Specifically, the detection kit for detecting the anti-COVID-19 antibody comprises a solid phase carrier coated or marked with a COVID-19 antigen, anti-human IgM \ IgG or IgA, and a sample diluent;
wherein, the COVID-19 antigen comprises the amino acid sequence (the amino acid sequence comprises any sequence of SEQ ID 01: VSEETGTLIVNSVLLFL or SEQ ID 02: YVYSRVKNLNSSRVPDLLV or the two amino acid sequences of SEQ ID 01 and SEQ ID 02 are connected in series in a reasonable mode) or the coding gene of the COVID-19 antigen comprises the nucleotide sequence, namely the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID 03: GTTT CGGAAGAGACAGGTACGTTA ATAGTTAATA GCGTACTTCT TTTTCTT or SEQ ID 04: TAC GTTTA CTCTCGTT AAAAATCTGA ATTCTTCTAG AGTTCCTGAT CTTCTGGTC or the two nucleotide sequences of SEQ ID 03 and SEQ ID 04 are connected in series in a reasonable manner; wherein, the SEQ ID 03 is the coding gene of the SEQ ID 01, and the SEQ ID 04 is the coding gene of the SEQ ID 02.
Wherein, the COVID-19 antigen is obtained by the recombinant expression plasmid.
Specifically, the solid phase carrier comprises a microporous plate, a colloidal gold microsphere, a fluorescent microsphere and an NC membrane.
The preparation and operation of the anti-COVID-19 antibody detection kit (colloidal gold immunochromatography) are as follows:
1. preparing colloidal gold:
preparing the colloidal gold by a trisodium citrate reduction method.
200mL of process water was added to a 250mL round bottom flask and placed in a constant temperature magnetic stirrer and heated to boiling. Carefully pouring out the boiling water, adding 200mL of process water into a round-bottom flask, adding 300 μ L of 25% chloroauric acid, heating to boil, rapidly adding 840 μ L of 25% trisodium citrate, continuing to boil for 15min after the solution color does not change. After cooling, the flask was transferred to a volumetric flask and brought to a capacity of 200mL with process water. Transferring to a clean reagent bottle, and keeping at 4 ℃ for later use.
2. Preparing a colloidal gold-labeled mouse anti-human IgM antibody:
and (3) taking the required colloidal gold, adjusting the pH value with a labeling buffer solution, adding a labeled anti-human IgM antibody, reacting for 30min, adding BSA (bovine serum albumin) for sealing reaction for 30min, centrifuging, discarding the supernatant, and re-dissolving the precipitate with a heavy suspension to obtain the colloidal gold labeled anti-human IgM antibody.
3. Preparation of a novel coronavirus (2019-nCoV) antigen labeled with colloidal gold.
And (3) taking the required colloidal gold, adjusting the pH value with a labeling buffer solution, adding a novel coronavirus (2019-nCoV) antigen for labeling, reacting for 30min, adding BSA (bovine serum albumin) for sealing reaction for 30min, centrifuging, discarding supernatant, and re-dissolving a precipitate with a heavy suspension to obtain the novel coronavirus (2019-nCoV) antigen labeled with the colloidal gold.
In this step, the novel coronavirus (2019-nCoV) antigen labeled with colloidal gold used in the present invention is prepared by labeling the antigen SEQ ID 01 or SEQ ID 02 with a single antigen according to the above method and then spraying gold, or labeling the antigen prepared by tandem connection of SEQ ID 01 or SEQ ID 02 according to the colloidal gold labeling method.
4. The kit comprises the following components:
detecting a card: 25 parts/box and 50 parts/box
Drying agent: 25 bags and 50 bags
Sample diluent: 10 mL. times.2 bottles, 20 mL. times.2 bottles
The specification is as follows: 1 part of
The invention further discloses a method for detecting whether the anti-COVID-19 antibody exists in a sample in vitro by using the detection kit for detecting the anti-COVID-19 antibody, which comprises the following steps:
(a) contacting the sample with the COVID-19 antigen according to the invention;
(b) detection of formation of antigen-antibody complexes: the formation of a complex indicates the presence of anti-COVID-19 antibodies in the sample.
As a further improvement of the embodiment of the present invention, the detection method in step (b) is carried out using the above-mentioned anti-COVID-19 antibody detection kit.
Specifically, the operation steps of the novel crown kit related to the embodiment of the invention are as follows:
before use, the reagent card, the sample diluent and the sample are returned to room temperature (the temperature is preferably 20-25 ℃) and numbered. The test card should be used as soon as possible within 2 hours, especially in highly humid environments and at room temperature above 30 c, from the originally packaged reagent bag.
And secondly, adding 20 mu L or 1 drop of sample into the sample adding hole of the detection card, and then adding 60 mu L or 3 drops of diluent (which can not be added dropwise first) for timing for 10 minutes.
And thirdly, observing the color development condition of the detection card.
The results were determined as shown in FIG. 1, and analyzed specifically as follows:
the 1 control line (line C) must be developed and the results are valid (see Figures 1A, 1B and 1C). The C line is used for functional quality detection and cannot be used for explaining the T line.
2 if C line color development:
2.1, only C line is developed, and two T lines are not developed (as shown in Figure 1A), judging that the IgM/total antibody of the new coronavirus (2019-nCoV) in the sample is negative;
2.2C line color development, T line color development of IgM card, T line color development of total antibody card (as Figure 1B), judging whether the new coronavirus (2019-nCoV) IgM antibody in the sample is negative, and the total antibody is positive;
2.3C line color development, and if both T lines color (as Figure 1C), judging that the new coronavirus (2019-nCoV) IgM/total antibody in the sample is positive;
3 if the line C does not develop color, no matter whether the line T develops color or not (as shown in Figures 2A, 2B and 2C), the detection result is invalid, and the new detection card is required to be used for re-detection.
The invention discloses an application of the detection kit for the antibody against COVID-19 in the early diagnosis of COVID-19.
In order to highlight the detection effect and verify the accuracy, a nucleic acid detection contrast experiment and an S protein antigen are set as a control experiment in the embodiment of the invention; specifically, the determination of the new crown detection kit according to the embodiment of the present invention is as follows:
(1) the results of the new crown detection kit prepared by marking the single antigen of SEQ ID 01 according to the method are as follows:
| categories | Number of positive results | Number of negative results | Positive rate | Quality control product CV |
| Reagent box in this embodiment (1) | 15 | 5 | 75％ | 8.2％ |
| S protein antigens | 13 | 7 | 65％ | 7.8％ |
| Results of nucleic acid detection | 18 | 2 | 90％ | / |
20 clinically confirmed positive samples of the novel coronavirus are taken, and the detection results of the samples and the control nucleic acid are shown in the table below, wherein the positive rates are 75%, 65% and 90% respectively. The nucleic acid detection of the 18 and 20 samples is negative and is not detected, but the clinical imaging CT detects that the lesion meets the clinical standard of the new coronary pneumonia, so the antibody detection can be used as an auxiliary means of the nucleic acid detection.
Comparison of results of novel coronavirus detection kit prepared from single antigen of SEQ ID 01 and nucleic acid detection
| Serial number | Reagent kit in this embodiment | Nucleic acid detection | S protein antigens |
| 1 | + | + | + |
| 2 | + | + | + |
| 3 | + | + | + |
| 4 | - | + | - |
| 5 | + | + | - |
| 6 | + | + | + |
| 7 | - | + | - |
| 8 | + | + | + |
| 9 | + | + | + |
| 10 | - | + | - |
| 11 | + | + | + |
| 12 | + | + | + |
| 13 | + | + | + |
| 14 | + | + | + |
| 15 | - | + | - |
| 16 | - | + | - |
| 17 | + | + | + |
| 18 | + | - | - |
| 19 | + | + | + |
| 20 | + | - | + |
| Number of positive results | 15 | 18 | 13 |
| Number of negative results | 5 | 2 | 7 |
| Positive rate | 75％ | 90％ | 65％ |
(2) The result of the new crown detection kit prepared by marking the single antigen of SEQ ID 02 according to the method is as follows:
| categories | Number of positive results | Number of negative results | Positive rate | Quality control product CV |
| Reagent box in this embodiment (2) | 15 | 5 | 75％ | 8.0％ |
| S protein antigens | 14 | 6 | 70％ | 8.0％ |
| Results of nucleic acid detection | 18 | 2 | 90％ | / |
20 clinically confirmed positive samples of the novel coronavirus are taken, and the detection results of the samples and the control nucleic acid are shown in the table below, wherein the positive rates are 75%, 70% and 90% respectively. The No. 13 and No. 20 sample nucleic acid detection is negative and is not detected, but the clinical imaging CT detects that the lesion meets the clinical standard of the new coronary pneumonia, so the antibody detection can be used as an auxiliary means of nucleic acid detection.
Comparison of results of novel coronavirus detection kit prepared from single antigen of SEQ ID 02 and nucleic acid detection
| Serial number | Reagent kit in this embodiment | Nucleic acid detection | S protein antigens |
| 1 | + | + | + |
| 2 | + | + | + |
| 3 | + | + | + |
| 4 | + | + | - |
| 5 | + | + | - |
| 6 | + | + | + |
| 7 | - | + | - |
| 8 | + | + | + |
| 9 | + | + | + |
| 10 | - | + | - |
| 11 | + | + | + |
| 12 | + | + | + |
| 13 | - | - | - |
| 14 | + | + | + |
| 15 | - | + | - |
| 16 | + | + | + |
| 17 | + | + | + |
| 18 | + | + | + |
| 19 | + | + | + |
| 20 | - | - | + |
| Number of positive results | 15 | 18 | 14 |
| Number of negative results | 5 | 2 | 6 |
| Positive rate | 75％ | 90％ | 70％ |
(3) The result of the new crown detection kit prepared by labeling the antigen formed by connecting SEQ ID 01 and SEQ ID 02 in series according to the method is as follows:
in the embodiment of the present invention, the specific tandem mode is an asymmetric sticky end complementary construction, that is, the target gene is cut at two side sites by enzyme digestion to generate an asymmetric sticky end, and the target gene is inserted to obtain the amino acid of the target sequence.
In other optional embodiments, the series mode can also be selected as follows: non-cohesive end complementary construction, joint connection, isocaudarner construction, multi-copy expression and the like.
20 clinically confirmed positive samples of the novel coronavirus are taken, and the detection results of the samples and the control nucleic acid are shown in the table below, wherein the positive rates are 85%, 70% and 90% respectively. The nucleic acid detection of the No. 3 and No. 20 samples is negative and is not detected, but the clinical imaging CT detects that the lesion meets the clinical standard of the new coronary pneumonia, so the antibody detection can be used as an auxiliary means of the nucleic acid detection.
Comparison of the results of nucleic acid detection and the novel coronavirus detection kit prepared from the antigen formed by the tandem connection of SEQ ID 01 and SEQ ID 02
The invention further discloses an application of the detection kit for the antibody against COVID-19 in the aspect of evaluating the effectiveness of the COVID-19 vaccine.
In other alternative embodiments, the COVID-19 antigen of the present invention may be used in the preparation of a medicament for treating or preventing a novel coronavirus.
The invention has the following beneficial effects:
1. the invention finds an amino acid sequence with strong antigenicity by analyzing the sequence of the latent antigen protein of the new coronavirus, synthesizes the target sites of the latent antigens by synthesizing polypeptides, and then obtains an antibody by immunizing the polypeptides, thereby developing an enzyme-linked immunosorbent assay method and a kit for diagnosing the new coronavirus at early stage in clinic by detecting the new coronavirus. The invention can obviously improve the diagnosis efficiency, and is simple and convenient, and has high accuracy;
2. the invention overcomes the problems of insufficient single S protein epitope, insufficient detection sensitivity of the IgM antibody resisting COVID-19 and the like, and the anti-neocorolla antibody detection reagent prepared by the recombinant protein containing the SEQ01-04 neoepitope has the advantages of high sensitivity, stable performance, accurate qualitative determination and the like;
3. the detection kit has high detection speed and high accuracy, and overcomes the problem of high false positive rate in the prior art;
4. because the affinity of the specific polypeptide and the polypeptide conjugate of the new coronavirus and the anti-new coronavirus antibody is high, and the affinity of the antibody and the new coronavirus is high, the detection method is sensitive, simple and convenient and has high accuracy;
5. the new coronavirus specific polypeptide can compete with viruses for receptors in vivo, prevent the fusion of viruses and membranes and inhibit the viruses from infecting normal cells, so the new coronavirus specific polypeptide has high clinical application value.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<120> COVID-19 antigen, preparation method and application
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Claims (11)
1. COVID-19 antigen characterized in that the amino acid sequence thereof comprises any one sequence of SEQ ID 01: VSEETGTLIVNSVLLFL or SEQ ID 02: YVYSRVKNLNSSRVPDLLV or the two amino acid sequences of SEQ ID 01 and SEQ ID 02 are connected in series in a reasonable manner.
2. The coding gene of the COVID-19 antigen is characterized in that the nucleotide sequence of the coding gene comprises the nucleotide sequence shown in SEQ ID 03: GTTT CGGAAGAGACAGGTACGTTA ATAGTTAATA GCGTACTTCTTTTTCTT or SEQ ID 04: TAC GTTTA CTCTCGTT AAAAATCTGA ATTCTTCTAG AGTTCCTGAT CTTCTGGTC or the two nucleotide sequences of SEQ ID 03 and SEQ ID 04 are connected in series in a reasonable manner; the SEQ ID 03 is the coding gene of SEQ ID 01 as claimed in claim 1, and the SEQ ID 04 is the coding gene of SEQ ID 02 as claimed in claim 1.
3. A recombinant expression plasmid, wherein the recombinant expression plasmid is constructed by directionally cloning the nucleotide sequence of claim 2 into the corresponding sites of a vector to obtain a recombinant expression vector, and further transforming the recombinant expression vector into BL21(DE3) competent cells.
4. Use of the COVID-19 antigen of claim 1 in the preparation of a detection kit for detecting an anti-COVID-19 antibody.
5. The use of the COVID-19 antigen of claim 4 in the preparation of an anti-COVID-19 antibody detection kit, wherein the anti-COVID-19 antibody detection kit comprises a solid phase carrier coated or labeled with the COVID-19 antigen, an anti-human IgM \ IgG or IgA, a sample diluent;
the COVID-19 antigen comprising the amino acid sequence of claim 1 or the gene encoding the COVID-19 antigen comprising the nucleotide sequence of claim 2.
6. The use of the COVID-19 antigen of claim 4 in the preparation of a detection kit for detecting an anti-COVID-19 antibody, wherein the COVID-19 antigen comprises the amino acid sequence of claim 1 or the gene encoding the COVID-19 antigen comprises the nucleotide sequence of claim 2 or is obtained from the recombinant expression plasmid of claim 3.
7. The use of the COVID-19 antigen of claim 5 in the preparation of a detection kit for detecting an anti-COVID-19 antibody, wherein the solid support comprises a microplate, colloidal gold microspheres, fluorescent microspheres, and NC membranes.
8. A method of using an anti-COVID-19 antibody detection kit as claimed in any one of claims 5 to 7, comprising the steps of:
(a) contacting the sample with the codv-19 antigen of claim 1;
(b) detection of formation of antigen-antibody complexes: the formation of a complex indicates the presence of anti-COVID-19 antibodies in the sample.
9. Use of a detection kit for detecting an anti-COVID-19 antibody according to claim 5 in the early diagnosis of COVID-19.
10. Use of the COVID-19 antigen of claim 1 in the preparation of a COVID-19 epitope vaccine.
11. Use of the COVID-19 antigen of claim 1 in the manufacture of a medicament for the treatment or prevention of a novel coronavirus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11547673B1 (en) | 2020-04-22 | 2023-01-10 | BioNTech SE | Coronavirus vaccine |
| US11732030B2 (en) | 2020-04-02 | 2023-08-22 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
| US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468962A (en) * | 2003-06-05 | 2004-01-21 | 中国科学院上海药物研究所 | Expression and purification of small envelope protein of soluble SARS virus |
| CN111239394A (en) * | 2020-03-09 | 2020-06-05 | 四川省人民医院 | Novel coronavirus antibody rapid detection kit based on mixed antigens |
| CN111624349A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection test strip |
-
2020
- 2020-07-09 CN CN202010657369.5A patent/CN111978377B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468962A (en) * | 2003-06-05 | 2004-01-21 | 中国科学院上海药物研究所 | Expression and purification of small envelope protein of soluble SARS virus |
| CN111239394A (en) * | 2020-03-09 | 2020-06-05 | 四川省人民医院 | Novel coronavirus antibody rapid detection kit based on mixed antigens |
| CN111624349A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection test strip |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11732030B2 (en) | 2020-04-02 | 2023-08-22 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
| US11547673B1 (en) | 2020-04-22 | 2023-01-10 | BioNTech SE | Coronavirus vaccine |
| US11925694B2 (en) | 2020-04-22 | 2024-03-12 | BioNTech SE | Coronavirus vaccine |
| US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
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| CN111978377B (en) | 2022-05-31 |
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