CN112034174B - Polypeptide chip and application thereof in virus detection - Google Patents
Polypeptide chip and application thereof in virus detection Download PDFInfo
- Publication number
- CN112034174B CN112034174B CN202010215184.9A CN202010215184A CN112034174B CN 112034174 B CN112034174 B CN 112034174B CN 202010215184 A CN202010215184 A CN 202010215184A CN 112034174 B CN112034174 B CN 112034174B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- chip
- protein
- cov
- sars
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 83
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 83
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 83
- 238000001514 detection method Methods 0.000 title claims description 24
- 241000700605 Viruses Species 0.000 title description 8
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 39
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- 108010067390 Viral Proteins Proteins 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims description 17
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 14
- 235000013336 milk Nutrition 0.000 claims description 13
- 239000008267 milk Substances 0.000 claims description 13
- 210000004080 milk Anatomy 0.000 claims description 13
- 239000007850 fluorescent dye Substances 0.000 claims description 10
- 108700010904 coronavirus proteins Proteins 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 101710193592 ORF3a protein Proteins 0.000 claims description 5
- 101710087110 ORF6 protein Proteins 0.000 claims description 5
- 101710128341 ORF7a protein Proteins 0.000 claims description 5
- 108010089430 Phosphoproteins Proteins 0.000 claims description 5
- 102000007982 Phosphoproteins Human genes 0.000 claims description 5
- 101710193546 Tegument protein VP16 homolog Proteins 0.000 claims description 5
- 101710135104 Uncharacterized protein p6 Proteins 0.000 claims description 5
- 101710091045 Envelope protein Proteins 0.000 claims description 4
- 101710188315 Protein X Proteins 0.000 claims description 4
- 101710172711 Structural protein Proteins 0.000 claims description 4
- 102100021696 Syncytin-1 Human genes 0.000 claims description 4
- 239000003550 marker Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 108010076039 Polyproteins Proteins 0.000 claims description 3
- -1 S glycioprotein Proteins 0.000 claims description 2
- 239000000975 dye Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 241000711902 Pneumovirus Species 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 5
- 208000025721 COVID-19 Diseases 0.000 abstract description 3
- 108010026552 Proteome Proteins 0.000 abstract description 2
- 230000009897 systematic effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 13
- 101710141454 Nucleoprotein Proteins 0.000 description 7
- 238000011282 treatment Methods 0.000 description 6
- 239000011521 glass Substances 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000012154 double-distilled water Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 206010035737 Pneumonia viral Diseases 0.000 description 2
- 101710151619 Replicase polyprotein 1ab Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 208000009421 viral pneumonia Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 208000034657 Convalescence Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 208000001528 Coronaviridae Infections Diseases 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 101800001632 Envelope protein E Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- 101710096370 ORF8 protein Proteins 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 108091005774 SARS-CoV-2 proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- JPBBNLWRCVBGJS-KCAUTNRHSA-N glucosaminylmuramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O JPBBNLWRCVBGJS-KCAUTNRHSA-N 0.000 description 1
- 229950001715 glucosaminylmuramyl dipeptide Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
-
- 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a polypeptide chip, which comprises a substrate and n polypeptides distributed on the substrate in an array mode, wherein each polypeptide sequence has 10-20 amino acids, the group consisting of sequences from a first polypeptide to an n polypeptide covers at least 95% of a viral protein sequence, and adjacent polypeptides have overlapping of 3-8 amino acids, and n is 810-1370. The invention adopts proteomics and systematic biology strategies to extract all coding protein sequences of the COVID-19 from NCBI data, designs and prepares SARS-CoV-2 virus proteome polypeptide chips, and realizes panoramic scanning of all SARS-CoV-2 virus antibodies in blood of a novel pneumovirus infected patient.
Description
Technical Field
The invention belongs to the technical field of biomedicine, relates to a polypeptide chip, and in particular relates to a polypeptide chip, a preparation method thereof and application thereof in the field of 2019 novel coronavirus (SARS-CoV-2) detection.
Background
The biochip generally refers to a biological information molecule (such as a gene fragment, a cDNA fragment or a polypeptide, a protein, a saccharide, etc.) which is fixed on a support medium in a high density according to a preset sequence, and the accurate, rapid and high-throughput detection of biological components is realized according to the principle of specific binding between molecules. The existing common biological chips mainly comprise a gene chip, a polypeptide chip, a chip laboratory and the like, wherein the polypeptide chip is more and more widely focused and applied due to the advantages of being capable of directly analyzing crude biological samples (serum, urine, body fluid and the like), being wide in functions and the like. The polypeptide chip is also called a protein chip or a protein microarray, is firstly proposed by Roger Ekin in the last century 80, is an accurate, rapid and high-throughput protein analysis technology, and can be used for researching protein-protein interaction, even DNA-protein and RNA-protein interaction, screening protein targets of drug action, protein expression profile analysis and the like.
Since 2019, 12 months later, a plurality of cases of viral pneumonia are found worldwide, and the diagnosis results are all viral pneumonia. The world health organization formally named the novel coronavirus as 2019 novel coronavirus (SARS-CoV-2) on 11 months of 2020 has a similarity of 82% with SARS virus. However, the novel virus still has no effective vaccine, strong infectivity, wide spreading range and long hiding time, and few virus infected patients have no disease symptoms or have lighter symptoms, and people who are in quick infection contact are not controlled in time, so that secondary and even tertiary transmission can be induced. In addition, if no effective vaccine is developed, new pneumoviruses may be associated with the long-term presence of humans.
The most important clinical detection means for SARS-CoV-2 is nucleic acid detection, and its sensitivity and specificity are very high. However, some patients with clinical symptoms were found to be nucleic acid negative, probably because RNA instability is prone to degradation during sample processing and the human immune system would clear a portion of viral nucleic acid. In addition, nucleic acid detection does not reflect the patient's immune response to viral infection and its changes in severity and before and after treatment. Finally, nucleic acid detection has high requirements on experimental conditions, instruments and levels of detection personnel, and cannot meet the huge requirements of small hospitals and communities on screening and diagnosing novel suspected cases of coronavirus infection.
Viruses are rapidly recognized by the human immune system after invading the human body, and specific IgM, igG and IgA (mucosal infection) antibodies appear in the human blood within 1-7 days, and remain at relatively high levels for a period of time. For some convalescence patients, neutralizing antibodies can be generated, and successful identification of the neutralizing antibodies can block virus and target cell receptors and prevent invasion of cells, so that the aim of treatment is fulfilled. Existing studies indicate that only a small fraction of the proteins of the virus can produce antibodies, and that most of the proteins are not immunogenic, demonstrating that the detection of antibodies in the blood of patients infected with the patient from a proteomic scale is critical for the discovery of antibody marker molecules with high specificity and sensitivity. Therefore, the marker molecules for the early detection, differential diagnosis and treatment rehabilitation guidance of the novel pneumovirus of the COVID-19 can be identified by comprehensively detecting the antibody level change rules of patients infected by the novel pneumovirus of the COVID-19 in blood with different severity (asymptomatic, mild and severe) and before and after treatment, and further the development of a rapid diagnosis reagent and a neutralizing antibody medicament hopefully used for clinical treatment have very important significance for the prevention and control and treatment of the novel pneumovirus for a long time.
The invention adopts proteomics and systematic biology strategies to extract all coding protein sequences of SARS-CoV-2 from NCBI data, designs and prepares SARS-CoV-2 virus proteome polypeptide chip, and realizes panoramic scanning of all SARS-CoV-2 virus antibodies in blood of patients infected by novel pneumovirus.
Disclosure of Invention
In one aspect, the invention provides a polypeptide chip comprising a substrate and n polypeptides distributed on the substrate in an array, wherein each polypeptide sequence has 10-20 amino acids, the group consisting of the sequences of the first polypeptide to the n polypeptide covers at least 95% of the viral protein sequence, and the adjacent polypeptides have an overlap of 3-8 amino acids, wherein n is 810-1370.
Preferably, each of said polypeptide sequences has 10-18 amino acids.
Preferably, each of said polypeptide sequences has 15 amino acids.
Preferably, the virus is SARS-CoV-2 coronavirus and the viral protein is SARS-CoV-2 coronavirus protein.
Preferably, the SARS-CoV-2 coronavirus protein is selected from the group consisting of ORF1ab polyprotein, S glycinogen, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8protein, N nucleocapsid phosphoprotein and ORF10 protein.
More preferably, the SARS-CoV-2 coronavirus protein is an rf1ab polyprotein, an S glycioprotein, an ORF3a protein, E envelope protein, M structural protein, an ORF6 protein, an ORF7a protein, an ORF8protein, N nucleocapsid phosphoprotein, or an ORF10 protein.
Preferably, the sequence of the first to nth polypeptides is selected from the group consisting of sequences covering at least 98% of the SARS-CoV-2 coronavirus protein sequence.
More preferably, the sequence of the first to nth polypeptides is selected from the group consisting of sequences covering at least 99% of the SARS-CoV-2 coronavirus protein sequence.
Particularly preferably, the group consisting of the sequences of the first to nth polypeptides covers the complete sequence of SARS-CoV-2 coronavirus protein.
In a specific embodiment of the present invention, the set of sequences of the first to nth polypeptides covers the complete sequence of SARS-CoV-2 coronavirus rf1ab polyprotein, S glycopin, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8protein, N nucleocapsid phosphoprotein and ORF10 protein.
TABLE 1 SARS-CoV-2 coronavirus encoded protein
| SEQ ID No. | Protein name | Protein abbreviation | Genbank number |
| 969 | orf1ab polyprotein | QHD43415.1 | |
| 970 | surface glycoprotein | S | QHD43416.1 |
| 971 | ORF3a protein | QHD43417.1 | |
| 972 | envelope protein | E | QHD43418.1 |
| 973 | membrane glycoprotein | M | QHD43419.1 |
| 974 | ORF6 protein | QHD43420.1 | |
| 975 | ORF7a protein | QHD43421.1 | |
| 976 | ORF8 protein | QHD43422.1 | |
| 977 | nucleocapsid phosphoprotein | N | QHD43423.2 |
| 978 | ORF10 protein | QHI42199.1 |
Preferably, the term "cover" refers to the complete identity of the SARS-CoV-2 coronavirus protein sequence after removal of the overlapping sequence portions of the group consisting of the sequences of the first polypeptide through the nth polypeptide.
Preferably, the adjacent polypeptides have 5 amino acid overlap.
Preferably, n is 900-1000, i.e. 900-1000 polypeptides are distributed on the polypeptide chip.
More preferably, n in the present invention is 950-1000, i.e. 950-1000 polypeptides are distributed on the polypeptide chip.
Particularly preferably, n is 968, i.e. 968 polypeptides are distributed on the polypeptide chip.
Preferably, the polypeptide of the present invention is a polypeptide of table 2:
TABLE 2 polypeptide sequences for use in the chips of the invention
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
Preferably, the substrate includes, but is not limited to, glass, silicon, ceramic, mica, metal, plastic, polymer film, etc., preferably glass, silicon and ceramic.
Preferably, the substrate is a three-dimensional D-modified slide.
Preferably, the substrate array of the present invention is a 48×42 array.
Preferably, the substrate according to the invention further comprises a positive control and/or a negative control.
Those skilled in the art will appreciate that the polypeptide chips of the present invention may be prepared by conventional methods of preparing polypeptide chips. In one embodiment of the present invention, the method for preparing a polypeptide chip according to the present invention comprises: synthesizing the 1 st to nth polypeptides, taking a three-dimensional D modified glass slide as a substrate, and spotting the synthesized n polypeptides and/or positive control points on the surface of the glass slide by a microarray spotter. Preferably, the spotting array of the substrate is a 48×42 array.
The invention also provides a detection method of 2019 novel coronavirus SARS-CoV-2, which comprises the following steps:
sealing the surface of the chip; adding a sample to be tested into a chip, and incubating; washing; adding a secondary anti-fluorescent dye; washing again; scanning is performed with a chip scanner.
Preferably, the chip surface sealing step includes: 400 μl/well of 5% milk was added to the chip and blocked for 0.5-2h at room temperature. More preferably, the milk is 0.5g of skimmed milk powder added to 10ml of PBST (phosphate Tween buffer). More preferably, the closing time is 1h.
Preferably, the sample to be tested is serum, and 4 μl of the sample to be tested is added into 400 μl of 5% milk to be mixed uniformly. More preferably, the milk is 0.5g of skimmed milk powder added to 10ml of PBST (phosphate Tween buffer).
Preferably, the sample to be tested is incubated for 2 hours at room temperature after being added to the chip.
Preferably, the washing is 0.05% PBST washing 1-4 times, each time for 5-15min; then ddH2O is washed 1-4 times for 1-5min each time. More preferably, the wash is 3 times at 10min each with 0.05% PBST, followed by 3 washes at 2min each with ddH 2O.
Preferably, the secondary antibody in the secondary-fluorescent dye is an IgM, igG or IgA antibody, more preferably, the secondary antibody is a goat-anti-hIgG or goat-anti-hIgA.
Preferably, the fluorescent dye in the secondary anti-fluorescent dye is Cy3 or dye 647.
Preferably, the re-washing includes 0.05% PBST washing 1-4 times, each time 1-10min; more preferably, the re-wash includes 0.05% PBST wash 3 times, each for 5min.
In a further aspect, the invention provides the use of the polypeptide chip of the invention in the detection 2019 of novel coronavirus SARS-CoV-2.
In a further aspect, the invention provides application of the polypeptide chip in preparing a product for detecting 2019 novel coronavirus SARS-CoV-2.
In yet another aspect, the invention provides the use of a polypeptide as described in Table 2 for the preparation of a product for detecting 2019 a novel coronavirus SARS-CoV-2.
In a further aspect, the invention provides the use of the polypeptide chip of the invention in screening and/or identifying antibody marker molecules for the detection of 2019 novel coronavirus SARS-CoV-2.
Drawings
Fig. 1A: monoclonal antibodies against the N protein of SARS-CoV-2 react specifically with the polypeptide chips of the present invention.
Fig. 1B: polyclonal antibodies against the N protein of SARS-CoV-2 react specifically with the polypeptide chip of the invention.
Fig. 2: an antibody against the N protein of SARS-CoV-2 reacts specifically with the polypeptide chip of the invention.
Fig. 3: the polypeptide chip of the invention detects the anti-SARS-CoV-2 protein antibody in the serum of the infected patient.
Fig. 4: SARS-CoV-2 coronavirus encoded protein sequence.
Detailed Description
EXAMPLE 1 preparation of polypeptide chips
(1) Solid phase synthesis was performed according to 968 polypeptides shown in table 2;
(2) Selecting a three-dimensional D modified glass slide as a substrate of a protein chip, and designing a sample application array of the substrate into a 48X 42 array;
(3) All synthesized polypeptide fragments and positive controls were spotted on the surface of the slide by a microarray spotter.
EXAMPLE 2 effectiveness test of polypeptide chip for detecting SARS-CoV-2 protein antibody
(1) Closing: the chip prepared in example 1 was then enclosed for 0.5h at room temperature by adding a rail, 400. Mu.l/well 5% milk (0.5 g nonfat milk powder+10 ml PBST);
(2) Sample preparation: monoclonal antibodies of N protein of anti SARS-CoV-2 and polyclonal antibodies of N protein of anti SARS-CoV-2 (all are commercially available with original concentration of 1 mg/ml) are added into 400 mul of 5% milk (1:2000 dilution) and mixed uniformly;
(3) Sample adding: adding the sample into a chip, 400 mu l/well, and incubating for 20min at room temperature;
(4) Washing: washing with 0.05% PBST 3 times for 5min each;
(5) Adding fluorescent dye: the Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, alexa Fluor 555 (original concentration 2mg/ml, used after 1:1000 dilution in 400 μl 5% milk) was added to the chip array, 400 μl/well incubated at room temperature for 20min under light-shielding conditions;
(6) And (5) washing in dark: washing with 0.05% PBST 3 times for 5min each; then ddH2O is washed for 2 times, each time for 2min;
(7) And (3) chip detection, namely airing the chip and detecting the chip by using a chip scanner.
The results of monoclonal antibodies against N protein of SARS-CoV-2 and polyclonal antibodies against N protein of SARS-CoV-2 are shown in FIGS. 1A and 1B, wherein the amino acid sequences specifically reacting with the polypeptide chip are shown in FIG. 2.
EXAMPLE 3 polypeptide chip detection of serum from patients infected with SARS-CoV-2 Virus
(1) Closing: the chip prepared in example 1 was then enclosed for 1h at room temperature by adding a rail, 400. Mu.l/well 5% milk (0.5 g nonfat dry milk+10 ml PBST);
(2) Sample preparation: adding 4 μl of serum to be tested into 400 μl of 5% milk, and mixing;
(3) Sample adding: adding the detection sample into a chip, and incubating for 2 hours at room temperature, wherein the volume of the detection sample is 400 mu l/well;
(4) Washing: washing with 0.05% PBST 3 times for 10min each; then ddH2O is washed for 3 times, each time for 2min;
(5) Adding fluorescent dye: the coat-anti-hIgG (Fc) -Cy3 (original concentration 1.5mg/ml, 4. Mu.l+1200. Mu.l mill) was added to the chip array and incubated for 1h at room temperature in the absence of light;
(6) And (5) washing in dark: washing with 0.05% PBST 3 times for 5min each;
(7) And (3) chip detection, namely airing the chip and detecting the chip by using a chip scanner.
The serum detection result of the polypeptide chip is shown in figure 3.
The present invention has been described in connection with the specific embodiments hereinabove, but it should be understood that these descriptions and illustrations are only for the purpose of better understanding of the present invention and are not to be construed as limiting the invention in any way. Those skilled in the art, having benefit of this disclosure, may make any necessary alterations to the embodiments of the invention without departing from the spirit and scope of the invention. The scope of the invention is defined by the appended claims, and equivalents of the claims to be embraced therein.
Claims (17)
1. A polypeptide chip comprising a substrate and n polypeptides distributed on the substrate in an array, wherein the polypeptide sequence has 10-20 amino acids, the group consisting of the sequences of the first polypeptide to the n-th polypeptide cover at least 95% of the SARS-CoV-2 coronavirus protein sequence, and there is a 5 amino acid overlap between adjacent polypeptides, wherein n is 810-1370;
the sequences of the polypeptides include those shown as SSRGTSPARMAGNGG (SEQ ID No. 806), LLLLDRLNQLESKMS (SEQ ID No. 808), TQAFGRRGPEQTQGN (SEQ ID No. 813) and TVTLLPAADLDDFSK (SEQ ID No. 825).
2. The polypeptide chip of claim 1, wherein the viral proteins are rf1ab polyprotein, S glycioprotein, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8protein, N nucleocapsid phosphoprotein, and ORF10 protein.
3. The polypeptide chip of claim 1, wherein the sequence of the first to nth polypeptides comprises the sequence of the SARS-CoV-2 coronavirus protein.
4. The polypeptide chip of claim 1, wherein said adjacent polypeptides have 5 amino acid overlaps.
5. The polypeptide chip of claim 1, wherein n is 900-1000, i.e., 900-1000 polypeptides are distributed on the polypeptide chip.
6. The polypeptide chip of claim 5, wherein n is 968, and 968 polypeptides are distributed on the polypeptide chip.
7. The polypeptide chip of claim 1, wherein the sequence of the polypeptide is SEQ ID nos. 1-968.
8. A method for detecting 2019 novel coronavirus SARS-CoV-2 for non-diagnostic purposes, said method comprising:
sealing the surface of the polypeptide chip of any one of claims 1-7; adding a sample to be tested into a chip, and incubating; washing; adding a secondary anti-fluorescent dye; washing again; scanning is performed with a chip scanner.
9. The method of claim 8, wherein the step of sealing the surface of the chip comprises: 400 μl/well of 5% milk was added to the chip and blocked for 0.5-2h at room temperature.
10. The detection method of claim 8, wherein the sample to be detected is serum, and 4 μl of the sample to be detected is added into 400 μl of 5% milk to be mixed uniformly;
and adding the sample to be tested into the chip, and then incubating for 2 hours at room temperature.
11. The assay of claim 8, wherein the wash is 0.05% PBST wash 1-4 times for 5-15min each time; then ddH 2 O is washed for 1-4 times, each time for 1-5min.
12. The detection method of claim 8, wherein the secondary antibody in the secondary antibody-fluorescent dye is an IgM, igG or IgA antibody.
13. The assay of claim 12 wherein said secondary antibody is a coat-anti-hIgG or coat-anti-hIgA.
14. The detection method of claim 8, wherein the fluorescent dye in the secondary antibody-fluorescent dye is Cy3 or dye 647.
15. The assay of claim 8, wherein said re-washing comprises 1-4 washes of 0.05% PBST for 1-10min each.
16. Use of the polypeptide chip of any one of claims 1-7 in the preparation of a product for detecting 2019 novel coronavirus SARS-CoV-2.
17. Use of the polypeptide chip of any one of claims 1-7 for screening and/or identifying antibody marker molecules for the detection of 2019 novel coronavirus SARS-CoV-2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020102030740 | 2020-03-20 | ||
| CN202010203074 | 2020-03-20 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112034174A CN112034174A (en) | 2020-12-04 |
| CN112034174B true CN112034174B (en) | 2024-03-29 |
Family
ID=73578822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010215184.9A Active CN112034174B (en) | 2020-03-20 | 2020-03-24 | Polypeptide chip and application thereof in virus detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112034174B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3922262A1 (en) * | 2020-06-08 | 2021-12-15 | Consejo Superior De Investigaciones Científicas (CSIC) | Assay for the detection of the cys-like protease (mpro) of sars-cov-2 |
| CN112194711A (en) * | 2020-10-15 | 2021-01-08 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | B cell linear epitope of novel coronavirus S protein, antibody, identification method and application |
| US20240067679A1 (en) * | 2021-01-05 | 2024-02-29 | Onco Therapy Science, Inc. | Sars-cov-2 protein-derived peptide and vaccine containing same |
| WO2022149295A1 (en) * | 2021-01-05 | 2022-07-14 | オンコセラピー・サイエンス株式会社 | SARS-CoV-2 PROTEIN-DERIVED PEPTIDE AND VACCINE CONTAINING SAME |
| CN112881685B (en) * | 2021-01-21 | 2023-02-17 | 北京市肝病研究所 | Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof |
| CN113980099A (en) * | 2021-03-29 | 2022-01-28 | 军事科学院军事医学研究院生命组学研究所 | N antigen specific epitope of new coronavirus and application thereof |
| EP4322995A1 (en) * | 2021-04-12 | 2024-02-21 | La Jolla Institute for Immunology | Coronavirus t cell epitopes and uses thereof |
| CN113624965B (en) * | 2021-08-05 | 2024-02-09 | 中国人民解放军军事科学院军事医学研究院 | Application of N-protein specific IgG4 in screening novel coronavirus infected person and vaccinated person |
| CN113735947A (en) * | 2021-09-22 | 2021-12-03 | 四川大学 | Specific T cell epitope peptide P48 screened by novel coronavirus S protein holoproteome and application thereof |
| CN113943349A (en) * | 2021-10-22 | 2022-01-18 | 华瑞同康生物技术（深圳）有限公司 | Core amino acid sequence group for targeted recognition of anti-new coronavirus neutralizing antibody N-IgY-pAbs and application thereof |
| WO2023104154A1 (en) * | 2021-12-08 | 2023-06-15 | 安达生物药物开发(深圳)有限公司 | Antigenic polypeptide and use thereof |
| IT202200005633A1 (en) * | 2022-03-22 | 2023-09-22 | Univ Degli Studi Di Palermo | PEPTIDES FOR THE PREPARATION OF VACCINES AGAINST SARS-COV-2 |
| CN114832099B (en) * | 2022-04-08 | 2023-11-28 | 国科宁波生命与健康产业研究院 | Polypeptide preparation for treating SARS-CoV-2 variant strain infection |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580073A (en) * | 2003-11-28 | 2005-02-16 | 深圳大学 | Human SARS virus surface film protein antigen determinant polypeptide, polynucleotide sequence and its use |
| FR2866025A1 (en) * | 2005-03-14 | 2005-08-12 | Pasteur Institut | Use of proteins and peptides coded by the genome of corona virus associated with severe acute respiratory syndrome (SARS) to detect infections by corona virus associated with SARS |
| CN2735340Y (en) * | 2003-12-25 | 2005-10-19 | 上海生物芯片有限公司 | Preparation of SARS antibody screening polypeptide chip and detecting kit |
| CN1826356A (en) * | 2003-07-22 | 2006-08-30 | 克鲁塞尔荷兰公司 | Binding molecules against sars-coronavirus and uses thereof |
| CN101575361A (en) * | 2009-03-20 | 2009-11-11 | 北京健诺威生物科技有限公司 | Novel specific polypeptide and application thereof in preparing medicament for diagnosing, preventing and treating severe acute respiratory syndrome caused by coronaviruses |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050100883A1 (en) * | 2003-11-12 | 2005-05-12 | Wang Chang Y. | Peptide-based diagnostic reagents for SARS |
-
2020
- 2020-03-24 CN CN202010215184.9A patent/CN112034174B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826356A (en) * | 2003-07-22 | 2006-08-30 | 克鲁塞尔荷兰公司 | Binding molecules against sars-coronavirus and uses thereof |
| CN1580073A (en) * | 2003-11-28 | 2005-02-16 | 深圳大学 | Human SARS virus surface film protein antigen determinant polypeptide, polynucleotide sequence and its use |
| CN2735340Y (en) * | 2003-12-25 | 2005-10-19 | 上海生物芯片有限公司 | Preparation of SARS antibody screening polypeptide chip and detecting kit |
| FR2866025A1 (en) * | 2005-03-14 | 2005-08-12 | Pasteur Institut | Use of proteins and peptides coded by the genome of corona virus associated with severe acute respiratory syndrome (SARS) to detect infections by corona virus associated with SARS |
| CN101575361A (en) * | 2009-03-20 | 2009-11-11 | 北京健诺威生物科技有限公司 | Novel specific polypeptide and application thereof in preparing medicament for diagnosing, preventing and treating severe acute respiratory syndrome caused by coronaviruses |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
Non-Patent Citations (2)
| Title |
|---|
| 吕世静 等.临床免疫学检验.中国医药科技出版社,2020,第35页. * |
| 新型冠状病毒基因组结构与蛋白功能;刘彬;秦照玲;戚中田;;微生物与感染(第01期);第56-61页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112034174A (en) | 2020-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112034174B (en) | Polypeptide chip and application thereof in virus detection | |
| CN111647053A (en) | Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening | |
| Majdoubi et al. | A majority of uninfected adults show preexisting antibody reactivity against SARS-CoV-2 | |
| Tappe et al. | Occupation-associated fatal limbic encephalitis caused by variegated squirrel bornavirus 1, Germany, 2013 | |
| EP2089712A2 (en) | Autoimmune disease biomarkers | |
| JP2004035534A (en) | Hcv anti-core monoclonal antibody | |
| CN107064518A (en) | Determine method, array and purposes that cancer of pancreas is present | |
| US20190359679A1 (en) | Diagnostic, prognostic, therapeutic and screening protocols | |
| CN111856027A (en) | New coronavirus antibody detection kit suitable for examination of patients without obvious symptoms | |
| CN109738653B (en) | Antigen-protein combination for detection, diagnosis or risk prediction of alzheimer's disease and kit comprising same | |
| CN111978377B (en) | COVID-19 antigen, preparation method and application | |
| Seong | Microimmunoassay using a protein chip: optimizing conditions for protein immobilization | |
| WO2011037827A2 (en) | Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual | |
| Arrieta et al. | Evaluation of serological test of Zika in an endemic area of flavivirus in the Colombian Caribbean | |
| Allartz et al. | Detection of bornavirus-reactive antibodies and BoDV-1 RNA only in encephalitis patients from virus endemic areas: a comparative serological and molecular sensitivity, specificity, predictive value, and disease duration correlation study | |
| US20210239713A1 (en) | Synergistic combination of biomarkers for detecting and assessing hepatic fibrosis | |
| Bonhomme et al. | Serological microarray for a paradoxical diagnostic of Whipple’s disease | |
| Germain et al. | Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France | |
| JP7199384B2 (en) | Novel peptides and their use in diagnostics | |
| CN113702636B (en) | Application of plasma autoantibody marker in early diagnosis of breast cancer and molecular subtype characterization thereof | |
| EP2669296A1 (en) | Anti-il28b antibody and method for assaying il28b using same | |
| Ma et al. | Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array | |
| Sompuram et al. | Accurate identification of paraprotein antigen targets by epitope reconstruction | |
| Ding et al. | A novel ELISA method to determine human MrgX2 in chronic urticaria | |
| RU2133472C1 (en) | Method for diagnosing active stage of human cytomegalovirus infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |