CN112034174A - Polypeptide chip and application thereof in virus detection - Google Patents
Polypeptide chip and application thereof in virus detection Download PDFInfo
- Publication number
- CN112034174A CN112034174A CN202010215184.9A CN202010215184A CN112034174A CN 112034174 A CN112034174 A CN 112034174A CN 202010215184 A CN202010215184 A CN 202010215184A CN 112034174 A CN112034174 A CN 112034174A
- 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.)
- Granted
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 81
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 81
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 81
- 241000700605 Viruses Species 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 title claims description 19
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
- 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 22
- 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 11
- 239000008267 milk Substances 0.000 claims description 11
- 210000004080 milk Anatomy 0.000 claims description 11
- 239000007850 fluorescent dye Substances 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 9
- 108700010904 coronavirus proteins Proteins 0.000 claims description 7
- 238000007789 sealing Methods 0.000 claims description 6
- 101710087110 ORF6 protein Proteins 0.000 claims description 5
- 108010089430 Phosphoproteins Proteins 0.000 claims description 5
- 102000007982 Phosphoproteins Human genes 0.000 claims description 5
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 101710193592 ORF3a protein Proteins 0.000 claims description 4
- 101710128341 ORF7a protein Proteins 0.000 claims description 4
- 101710096370 ORF8 protein Proteins 0.000 claims description 4
- 101710193546 Tegument protein VP16 homolog Proteins 0.000 claims description 4
- 101710135104 Uncharacterized protein p6 Proteins 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
- 101710091045 Envelope protein Proteins 0.000 claims description 3
- 108010076039 Polyproteins Proteins 0.000 claims description 3
- 101710188315 Protein X Proteins 0.000 claims description 3
- 101710172711 Structural protein Proteins 0.000 claims description 3
- 102100021696 Syncytin-1 Human genes 0.000 claims description 3
- 101710167605 Spike glycoprotein Proteins 0.000 claims description 2
- 230000000903 blocking effect Effects 0.000 claims description 2
- 239000000975 dye Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 208000025721 COVID-19 Diseases 0.000 abstract description 3
- 108010026552 Proteome Proteins 0.000 abstract description 3
- 206010035664 Pneumonia Diseases 0.000 abstract description 2
- 238000013461 design Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 13
- 101710141454 Nucleoprotein Proteins 0.000 description 7
- 239000011521 glass Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 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
- 238000002360 preparation method Methods 0.000 description 5
- 241000711902 Pneumovirus Species 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 238000003745 diagnosis Methods 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
- 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
- 238000000018 DNA microarray Methods 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
- -1 Sglycoprotein Proteins 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000002156 mixing Methods 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
- 229960005486 vaccine Drugs 0.000 description 2
- 208000009421 viral pneumonia Diseases 0.000 description 2
- 101000748061 Acholeplasma phage L2 Uncharacterized 16.1 kDa protein Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101000947615 Clostridium perfringens Uncharacterized 38.4 kDa protein Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 208000001528 Coronaviridae Infections Diseases 0.000 description 1
- 101000964391 Enterococcus faecalis UPF0145 protein Proteins 0.000 description 1
- 101800001632 Envelope protein E Proteins 0.000 description 1
- 101000748063 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 11.1 kDa protein in rep-hol intergenic region Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000790840 Klebsiella pneumoniae Uncharacterized 49.5 kDa protein in cps region Proteins 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 108091005774 SARS-CoV-2 proteins Proteins 0.000 description 1
- 101000779242 Severe acute respiratory syndrome coronavirus 2 ORF3a protein Proteins 0.000 description 1
- 101000596353 Severe acute respiratory syndrome coronavirus 2 ORF7a protein Proteins 0.000 description 1
- 101710198378 Uncharacterized 10.8 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101710095001 Uncharacterized protein in nifU 5'region Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012472 biological sample Substances 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
- 238000006243 chemical reaction Methods 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
- 238000003748 differential diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002032 lab-on-a-chip Methods 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
- 230000006916 protein interaction Effects 0.000 description 1
- 238000011160 research Methods 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
Images
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 manner, wherein each polypeptide sequence has 10-20 amino acids, the group consisting of the sequences of the first polypeptide to the nth polypeptide covers at least 95 percent of the viral protein sequence, and the adjacent polypeptides have 3-8 amino acid overlaps, wherein n is 810-1370. The invention adopts proteomics and system biology strategies to extract all coding protein sequences of 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 novel pneumonia virus infected patients.
Description
Technical Field
The invention belongs to the technical field of biomedicine, relates to a polypeptide chip, and particularly 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 polypeptide, protein, carbohydrate, etc.) which is fixed on a support medium at high density according to a preset sequence, and realizes accurate, rapid and high-flux detection of biological components according to the principle of specific binding between molecules. At present, the common biochips mainly include gene chips, polypeptide chips, lab-on-a-chip, etc., wherein the polypeptide chips are gaining more and more attention and application due to the advantages of direct analysis with crude biological samples (serum, urine, body fluid, etc.) and wide functions. Polypeptide chips, also known as protein chips or protein microarrays, were first proposed by Roger Ekin in the last 80 th century, are an accurate, rapid, high-throughput protein analysis technique, and can be used for studying the interaction between proteins, even the interaction between DNA-protein and RNA-protein, screening protein targets for drug action, protein expression profiling, and the like.
Since 12 months in 2019, many viral pneumonia cases are found in various parts of the world, and the diagnosis results are all viral pneumonia. The world health organization formally named the novel coronavirus 2019 (SARS-CoV-2) at 11/2/2020, and the similarity with the SARS virus reaches 82%. However, the novel virus still has no effective vaccine, strong infectivity, wide spreading range, long hiding time, few virus infected patients have no disease symptoms or light symptoms, and if people who contact the virus are infected rapidly, the virus can induce secondary or even tertiary spread. In addition, without the development of an effective vaccine, new pneumovirus may be present in humans for a long time.
The current clinical main detection means for SARS-CoV-2 is nucleic acid detection, and the sensitivity and specificity are very high. However, it has been found that some patients with clinical symptoms are nucleic acid negative, probably because RNA instability is prone to degradation during sample handling and the human immune system will eliminate 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 the severity of the disease course 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 diagnosis of suspected cases of novel coronavirus infection.
After the virus invades human body, the virus is rapidly recognized by the immune system of the human body, and specific IgM, IgG and IgA (mucosal infection) antibodies appear in the blood of the human body within 1-7 days and maintain relatively high level in a period of time. For some convalescent patients, neutralizing antibodies may be generated in vivo, and the successful identification of the neutralizing antibodies can block virus and target cell receptors and prevent invasion of cells, thereby achieving the purpose of treatment. The existing research shows that only a small part of proteins of the virus can generate antibodies, and most of the proteins have no immunogenicity, so that the detection of the antibodies in the blood of patients infected by the patients from the proteome range is proved to be very important for finding antibody marker molecules with high specificity and sensitivity. Therefore, the marker molecules for the COVID-19 novel pneumovirus early detection, differential diagnosis and treatment rehabilitation guidance can be identified by comprehensively detecting the antibody level change rule of the COVID-19 novel pneumovirus infected patients in different severity (asymptomatic, mild and severe) and before and after treatment, and further the development of a rapid diagnosis reagent and a neutralizing antibody drug which is expected to be used for clinical treatment have very important significance for the prevention and control and treatment of the long-term novel pneumovirus.
The invention adopts proteomics and system biology strategies to extract all the coding protein sequences of SARS-CoV-2 from NCBI data, design and prepare SARS-CoV-2 virus proteome polypeptide chip, and realize panoramic scanning of all SARS-CoV-2 virus antibodies in blood of novel pneumonia virus infected patients.
Disclosure of Invention
The invention provides a polypeptide chip, which comprises a substrate and n polypeptides distributed on the substrate in an array manner, wherein each polypeptide sequence has 10-20 amino acids, the group consisting of the sequences of the first polypeptide to the nth polypeptide covers at least 95% of the viral protein sequence, and the adjacent polypeptides have 3-8 amino acid overlap, 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 ORF1ab polyprotein, Sglycoprotein, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8 protein, N nucleocapsed phosphoprotein and ORF10 protein.
More preferably, the SARS-CoV-2 coronavirus protein is rf1ab polyprotein, S glycoprotein, ORF3a protin, E envelope protin, M structural protin, ORF6 protin, ORF7a protin, ORF8 protin, N nucleocapesid phosphoprotein and ORF10 protin protein.
Preferably, the group consisting of the sequences of the first to nth polypeptides covers at least 98% of the SARS-CoV-2 coronavirus protein sequence.
More preferably, the group consisting of the sequences of the first to nth polypeptides covers at least 99% of the SARS-CoV-2 coronavirus protein sequence.
Particularly preferably, the group consisting of the sequences of said first to nth polypeptides covers the entire sequence of the SARS-CoV-2 coronavirus protein.
In a specific embodiment of the present invention, the group consisting of the sequences of the first to nth polypeptides covers the entire sequences of SARS-CoV-2 coronavirus rf1ab polyprotein, S glycoprotamine, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8 protein, N nucleocapside phosphoprotein and ORF10 protein.
TABLE 1 SARS-CoV-2 coronavirus encoded proteins
| SEQ ID No. | Name of protein | Abbreviation of protein | 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 "overlap" means that the sequence of the first polypeptide to the nth polypeptide is completely identical to the protein sequence of SARS-CoV-2 coronavirus after the overlapping sequence portions are removed.
Preferably, there is a 5 amino acid overlap between adjacent polypeptides.
Preferably, n in the invention is 900-1000, that is, 900-1000 polypeptides are distributed on the polypeptide chip.
More preferably, n in the invention is 950-1000, that is, 950-1000 polypeptides are distributed on the polypeptide chip.
Particularly preferably, n is 968, that is, 968 polypeptides are distributed on the polypeptide chip.
Preferably, the polypeptide of the present invention is a polypeptide in 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 wafer, ceramic, mica, metal, plastic, polymer film, etc., preferably glass, silicon wafer and ceramic.
Preferably, the substrate of the present invention is a three-dimensional D-modified glass slide.
Preferably, the array of substrates of the present invention is a 48 x 42 array.
Preferably, the substrate of the present invention further comprises a positive control and/or a negative control.
As will be appreciated by those skilled in the art, the polypeptide chip of the present invention may be prepared by conventional methods. In one embodiment of the present invention, the method for preparing the polypeptide chip of the present invention comprises: synthesizing the 1 st to the nth polypeptides, taking a three-dimensional D-modified glass slide as a substrate, and preparing the synthesized n polypeptides and/or positive control spots on the surface of the glass slide by a microarray spotting instrument. Preferably, the array of spots of the substrate is a 48 x 42 array.
The invention also provides a 2019 novel coronavirus SARS-CoV-2 detection method, which comprises the following steps:
sealing the surface of the chip; adding a sample to be detected into the chip, and incubating; washing; adding a secondary antibody-fluorescent dye; washing again; scanning was performed with a chip scanner.
Preferably, the chip surface sealing step includes: add 400. mu.l/well 5% milk to the chip and block at room temperature for 0.5-2 h. More preferably, the milk is 0.5g skimmed milk powder added to 10ml PBST (phosphate Tween buffer). More preferably, the blocking time is 1 h.
Preferably, the sample to be detected is serum, and the sample to be detected is 4 mul of serum to be detected, added into 400 mul of 5% milk and mixed evenly. More preferably, the milk is 0.5g skimmed milk powder added to 10ml PBST (phosphate Tween buffer).
Preferably, the sample to be tested is added to the chip and then incubated for 2h at room temperature.
Preferably, the washing is 0.05% PBST washing 1-4 times, 5-15min each time; then ddH2O was washed 1-4 times for 1-5min each. More preferably, the wash is 0.05% PBST wash 3 times, each time for 10min, then ddH2O wash 3 times, each time for 2 min.
Preferably, the secondary antibody in the secondary antibody-fluorescent dye is IgM, IgG or IgA antibody, and more preferably, the secondary antibody is coat-anti-hIgG or coat-anti-hIgA.
Preferably, the fluorescent dye in the secondary antibody-fluorescent dye is Cy3 or dye 647.
Preferably, the second washing comprises 0.05% PBST washing 1-4 times, each time for 1-10 min; more preferably, the second washing includes 0.05% PBST washing 3 times, each time for 5 min.
The invention also provides the application of the polypeptide chip in detecting 2019 novel coronavirus SARS-CoV-2.
The invention also provides the application of the polypeptide chip in preparing 2019 novel coronavirus SARS-CoV-2 products.
In still another aspect, the present invention provides the use of the polypeptides described in table 2 in the preparation of a product for detecting 2019 novel coronavirus SARS-CoV-2.
In still another aspect, the invention provides an application of the polypeptide chip in screening and/or identifying antibody marker molecules for 2019 novel coronavirus SARS-CoV-2 detection.
Drawings
FIG. 1A: the monoclonal antibody of the N protein of anti SARS-CoV-2 reacts specifically with the polypeptide chip of the invention.
FIG. 1B: the polyclonal antibody of the N protein of anti SARS-CoV-2 reacts specifically with the polypeptide chip of the invention.
FIG. 2: the antibody of the N protein of SARS-CoV-2 and the polypeptide chip of the invention have specific reaction amino acid sequence.
FIG. 3: the polypeptide chip of the present invention is used in detecting SARS-CoV-2 resisting protein antibody in serum of infected patient.
FIG. 4: protein sequence coded by SARS-CoV-2 coronavirus.
Detailed Description
EXAMPLE 1 preparation of polypeptide chips
(1) Performing solid phase synthesis 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 48 x 42 array;
(3) all the synthesized polypeptide fragments and positive control spots were prepared on the slide surface by a microarray spotter.
EXAMPLE 2 polypeptide chip test of the effectiveness of SARS-CoV-2 protein antibody
(1) And (3) sealing: the chip prepared in example 1 was enclosed in a pen, and 400. mu.l/well of 5% milk (0.5g skimmed milk powder +10ml PBST) was added and sealed at room temperature for 0.5 h;
(2) sample preparation: adding monoclonal antibody of N protein of anti-SARS-CoV-2, polyclonal antibody of N protein of anti-SARS-CoV-2 (all commercially available, original concentration is 1mg/ml), adding 400 μ l 5% milk (1: 2000 dilution), and mixing;
(3) sample adding: adding the sample to a chip, and incubating at room temperature for 20min in a manner of 400 mu l/well;
(4) washing: washing with 0.05% PBST for 5min 3 times;
(5) adding a fluorescent dye: goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (original concentration 2mg/ml, used after 1:1000 dilution in 400. mu.l 5% milk) was added to the chip array, 400. mu.l/well, and incubated for 20min at room temperature in the dark;
(6) washing in dark: washing with 0.05% PBST for 5min 3 times; then ddH2O was washed 2 times for 2min each;
(7) and (3) chip detection, namely drying the chip and detecting by using a chip scanner.
The results of the monoclonal antibody against the N protein of SARS-CoV-2 and the polyclonal antibody against the N protein of SARS-CoV-2 are shown in FIGS. 1A and 1B, and the amino acid sequences specifically reacting with the polypeptide chip are shown in FIG. 2.
EXAMPLE 3 detection of serum from SARS-CoV-2 Virus-infected patient by polypeptide chip
(1) And (3) sealing: the chip prepared in example 1 was enclosed in a pen, and 400. mu.l/well of 5% milk (0.5g skimmed milk powder +10ml PBST) was added and sealed at room temperature for 1 hour;
(2) sample preparation: adding 4 mul of serum to be detected into 400 mul of 5% milk, and mixing uniformly;
(3) sample adding: adding a detection sample to the chip, incubating at room temperature for 2h, wherein the volume of the detection sample is 400 mu l/well;
(4) washing: washing with 0.05% PBST for 3 times, each for 10 min; then ddH2O was washed 3 times for 2min each;
(5) adding a fluorescent dye: adding the goat-anti-hIgG (Fc) -Cy3 (original concentration is 1.5mg/ml, 4 mu l +1200 mu l mil) into the chip array, and incubating for 1h at room temperature in a dark place;
(6) washing in dark: washing with 0.05% PBST for 5min 3 times;
(7) and (3) chip detection, namely drying the chip and detecting by using a chip scanner.
The result of the polypeptide chip of the invention for detecting the serum of a patient is shown in figure 3.
While the invention has been described in conjunction with specific embodiments thereof, it is to be understood that both the foregoing description and the following description are intended to provide a better understanding of the invention, and are not intended to limit the invention in any way. Those skilled in the art, having read the present specification, may make necessary alterations to the particular 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 all equivalents to the claims are intended to be embraced therein.
Claims (10)
1. A polypeptide chip comprises a substrate and n polypeptides distributed on the substrate in an array manner, wherein each polypeptide sequence has 10-20 amino acids, the group consisting of sequences of first polypeptide to nth polypeptide covers at least 95% of a virus protein sequence, and adjacent polypeptides have 3-8 amino acid overlaps, wherein n is 810-1370.
2. The polypeptide chip of claim 1, wherein the virus is SARS-CoV-2 coronavirus, and the viral proteins are rf1ab polyprotein, S glycoprotein, ORF3a protein, E envelope protein, M structural protein, ORF6 protein, ORF7a protein, ORF8 protein, N nucleocapesid phosphoprotein and ORF10 protein.
3. The polypeptide chip of claim 2, wherein the sequences of the first to nth polypeptides cover the entire sequence of SARS-CoV-2 coronavirus protein.
4. The polypeptide chip of claim 3, wherein said adjacent polypeptides have an overlap of 5 amino acids.
5. The polypeptide chip of claim 3, wherein n is 900-1000, that is, 900-1000 polypeptides are distributed on the polypeptide chip.
6. The polypeptide chip of claim 5, wherein n is 968, i.e., 968 polypeptides are distributed on the polypeptide chip.
7. The polypeptide chip of claim 6, wherein the polypeptide sequence is SEQ ID No. 1-968.
8. A2019 method for detecting novel coronavirus SARS-CoV-2, the method comprises:
blocking the surface of the polypeptide chip of any one of claims 1-8; adding a sample to be detected into the chip, and incubating; washing; adding a secondary antibody-fluorescent dye; washing again; scanning with a chip scanner;
preferably, the chip surface sealing step includes: adding 400 mul/well 5% milk into the chip, sealing at room temperature for 0.5-2 h;
preferably, the sample to be detected is serum, and the sample to be detected is 4 mul of serum to be detected, added into 400 mul of 5% milk and mixed evenly;
adding a sample to be detected into the chip, and then incubating for 2h at room temperature;
preferably, the washing is 0.05% PBST washing 1-4 times, 5-15min each time; then ddH2O is washed for 1-4 times, each time for 1-5 min;
preferably, the secondary antibody in the secondary antibody-fluorescent dye is IgM, IgG or IgA antibody, more preferably, the secondary antibody is coat-anti-hIgG or coat-anti-hIgA;
preferably, the fluorescent dye in the secondary antibody-fluorescent dye is Cy3 or dye 647;
preferably, the second washing includes 0.05% PBST washing 1-4 times, each time 1-10 min.
9. Use of the polypeptide chip of any one of claims 1-8 for detecting 2019 novel coronavirus SARS-CoV-2.
10. Use of the polypeptide chip of any one of claims 1-8 for screening and/or identifying antibody marker molecules for 2019 novel coronavirus SARS-CoV-2 detection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010203074 | 2020-03-20 | ||
| CN2020102030740 | 2020-03-20 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112034174A true CN112034174A (en) | 2020-12-04 |
| CN112034174B 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) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112881685A (en) * | 2021-01-21 | 2021-06-01 | 北京市肝病研究所 | Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof |
| CN113624965A (en) * | 2021-08-05 | 2021-11-09 | 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 infectors and vaccinators |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| WO2022149549A1 (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 |
| CN114832099A (en) * | 2022-04-08 | 2022-08-02 | 国科宁波生命与健康产业研究院 | Polypeptide preparation for treating SARS-CoV-2 variant strain infection |
| WO2022221189A1 (en) * | 2021-04-12 | 2022-10-20 | La Jolla Institute For Immunology | Coronavirus t cell epitopes and uses 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 |
Citations (7)
| 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 |
| US20050100883A1 (en) * | 2003-11-12 | 2005-05-12 | Wang Chang Y. | Peptide-based diagnostic reagents for SARS |
| 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 |
-
2020
- 2020-03-24 CN CN202010215184.9A patent/CN112034174B/en active Active
Patent Citations (7)
| 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 |
| US20050100883A1 (en) * | 2003-11-12 | 2005-05-12 | Wang Chang Y. | Peptide-based diagnostic reagents for SARS |
| 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 |
|---|
| NCBI: "Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome，GenBank: MN908947.2", Retrieved from the Internet <URL:NCBI> * |
| 刘彬;秦照玲;戚中田;: "新型冠状病毒基因组结构与蛋白功能", 微生物与感染, no. 01, pages 56 - 61 * |
Cited By (17)
| 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 |
| WO2022149549A1 (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 |
| 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 |
| CN112881685A (en) * | 2021-01-21 | 2021-06-01 | 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 |
| WO2022221189A1 (en) * | 2021-04-12 | 2022-10-20 | La Jolla Institute For Immunology | Coronavirus t cell epitopes and uses thereof |
| CN113624965A (en) * | 2021-08-05 | 2021-11-09 | 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 infectors and vaccinators |
| 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 |
| WO2023066396A1 (en) * | 2021-10-22 | 2023-04-27 | 华瑞同康生物技术（深圳）有限公司 | Core amino acid sequence group for targeted recognition of anti-sars-cov-2 neutralizing antibodies n-igy-pabs, and use 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 |
| WO2023180966A1 (en) * | 2022-03-22 | 2023-09-28 | Universita' 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 |
| CN114832099A (en) * | 2022-04-08 | 2022-08-02 | 国科宁波生命与健康产业研究院 | Polypeptide preparation for treating SARS-CoV-2 variant strain infection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112034174B (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112034174A (en) | Polypeptide chip and application thereof in virus detection | |
| CN111647053A (en) | Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening | |
| JP6161542B2 (en) | Methods, arrays and uses thereof | |
| Fritzen et al. | Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach | |
| JP2004035534A (en) | Hcv anti-core monoclonal antibody | |
| RU2746815C1 (en) | Method for detecting antibodies - class g immunoglobulins in blood serum to pathogens of severe acute respiratory viral infections, including sars-cov-2, with simultaneous prognosis of covid-19 severity, based on hydrogel biochip | |
| JPH0767700A (en) | Htlv test using synthetic peptide | |
| CN111978377B (en) | COVID-19 antigen, preparation method and application | |
| Du et al. | Development and application of human coronavirus protein microarray for specificity analysis | |
| Seong | Microimmunoassay using a protein chip: optimizing conditions for protein immobilization | |
| Smith et al. | Age, disease severity and ethnicity influence humoral responses in a multi-ethnic COVID-19 cohort | |
| CN105378479A (en) | Method for predicting clinical effect of immunotherapy | |
| WO2011037827A2 (en) | Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual | |
| JP2023049050A (en) | Novel peptides and their use in diagnosis | |
| CN110818800A (en) | Detection method for indirectly detecting target analyte by constructing bridging compound | |
| CN1643378A (en) | Method for discrimination of metaplasias from neoplastic or preneoplastic lesions | |
| Manta et al. | Formulation rationale for the development of SARS-COV-2 immunochromatography rapid test kits in India | |
| 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 | |
| Aira et al. | Improving African swine fever surveillance using fluorescent rapid tests | |
| CN113444154A (en) | Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening | |
| Bennett et al. | Antibody epitope profiling of the KSHV LANA protein using VirScan | |
| Wang et al. | Antigenic characteristics of glycosylated protein 3 of highly pathogenic porcine reproductive and respiratory syndrome virus | |
| Igiri et al. | Diversity in typhoid diagnostic protocols and recommendation for composite reference standard | |
| Acharjee et al. | Protein microarrays for COVID-19 research: Biomarker discovery, humoral response, and vaccine targets | |
| 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 |