CN114773465B - Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application - Google Patents
Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application Download PDFInfo
- Publication number
- CN114773465B CN114773465B CN202210694199.7A CN202210694199A CN114773465B CN 114773465 B CN114773465 B CN 114773465B CN 202210694199 A CN202210694199 A CN 202210694199A CN 114773465 B CN114773465 B CN 114773465B
- Authority
- CN
- China
- Prior art keywords
- protein
- domain antibody
- omicron
- strain
- vhh
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- 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
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
-
- 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
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- 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
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
Abstract
The invention provides a specific single domain antibody aiming at the S protein of the new coronavirus omicron strain, a gene sequence and a coding sequence of VHH-3, and also discloses that the omicron strain single domain antibody can be efficiently expressed in eukaryotic cells, and can be applied to the research and development of a new coronavirus omicron strain detection reagent and targeted therapy.
Description
Technical Field
The invention relates to the field of biomedical or biotechnological application, in particular to a single domain antibody aiming at the S protein of a new coronavirus omicron strain, a coding sequence and application.
Background
In the early 2020, the global spread of new crown diseases brings serious challenges to human health and economic development, and the continuous variation of new crown viruses causes the new crown viruses to have stronger transmission capability, faster diffusion speed and wider infection range, thus becoming the key and hot areas of global public health attention. The prevention and control of the new crown can not be detected and identified in a laboratory, and is more dependent on the effective treatment of the new crown case. At present, the mainstream detection technology of the new coronavirus is a real-time fluorescence PCR method, the specificity and the sensitivity of the real-time fluorescence PCR method are closely related to the design of a primer probe, system optimization, an amplification instrument and the like, and detection personnel also need to be trained strictly. The operation and cost of the antigen detection method are relatively low, but the antibody used for the antigen detection at present mainly aims at the conserved N protein, and the specificity and the sensitivity of the detection cannot meet the market requirement. At present, the omicron strain is a main popular strain of new coronavirus which is abused worldwide, so that the development of a rapid detection kit by utilizing a single-domain antibody aiming at the S protein of the new coronavirus is of great significance, and simultaneously, the single-domain antibody of broad-spectrum targeted therapy can be developed.
The new corona virion is spherical (some is polymorphic), has surface protrusions, and is observed under electron microscope to form virus similar to crown, and its diameter is 75-160 nm. The viral gene is continuous linear single-stranded RNA, the whole genome sequence is about 29000 bp in length, and the viral gene comprises 14 main Open Reading Frames (ORF) in total and can encode 27 proteins. The S protein is rich in the new coronavirus, relatively conservative and highly immunogenic, is a target protein of a neutralizing antibody after infection, and is also a target of detection and a key point of treatment and vaccine design.
Single domain antibodies contain only the variable region of the heavy chain (VHH), which is of small molecular weight and can be isolated and screened from camelids by molecular cloning. The single domain antibody not only has the specificity and reactivity of the traditional antibody, but also has higher stability, solubility, permeability and the like.
However, there is currently no satisfactory single domain antibody against the S protein of the novel coronavirus in the art. Therefore, there is an urgent need in the art to develop a new specific single domain antibody against the S protein, which is effective for establishing a highly efficient detection method, and provides experimental basis and candidate materials for developing anti-new coronavirus single domain antibody drugs.
Disclosure of Invention
The invention aims to provide a single domain antibody aiming at the S protein of the novel coronavirus omicron strain, and also provides a coding sequence of the single domain antibody and application of the single domain antibody in detection and treatment.
The technical scheme adopted by the invention is as follows:
the first aspect of the invention provides 1 VHH-3 of single domain antibody of the S protein of the novel coronavirus omicron strain, which is characterized in that the VHH comprises a CDR1 shown in SEQ ID NO. 13, a CDR2 shown in SEQ ID NO. 14 and a CDR3 shown in SEQ ID NO. 15. In a specific embodiment, the amino acid sequence of the VHH is a VHH chain comprising the above 3 CDRs and an amino acid sequence with a length identical to SEQ ID No. 6 and with more than 95% homology; in another specific embodiment, the amino acid sequence of the VHH is as shown in SEQ ID NO 6.
The second aspect of the present invention provides a nucleic acid encoding the above-described single domain antibody VHH-3; in a specific embodiment, the sequence of said nucleic acid has more than 95% homology with the nucleic acid molecule as shown in SEQ ID NO. 3 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 6; in another specific embodiment, the sequence of said nucleic acid has more than 99% homology with the nucleic acid molecule as shown in SEQ ID NO. 3 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 6; in another specific embodiment, the nucleic acid has the sequence shown in SEQ ID NO 3.
The third aspect of the invention provides a method for preparing the single domain antibody VHH-3 of the S protein of the novel coronavirus omicron strain, which comprises the steps of inserting nucleic acid for coding the VHH-3 into an expression vector, transferring the expression vector into host cells, culturing the host cells, and purifying to obtain the single domain antibody. In one specific embodiment, the expression vector is a baculovirus expression vector pFastBac and the host cell is sf9 insect cell.
The fourth aspect of the invention provides the application of the S protein single domain antibody of the new coronavirus omicron strain in the preparation of products for diagnosing the new coronavirus omicron strain. The product includes but is not limited to a kit, a test strip and the like.
The fifth aspect of the invention provides the application of the S protein single domain antibody VHH-3 of the new coronavirus omicron strain of the invention in the preparation of products for treating the new coronavirus omicron strain; in a specific embodiment, the therapeutic product includes, but is not limited to, oral, injectable, or nasal spray.
The sixth aspect of the present invention provides a pharmaceutical composition comprising the novel coronavirus omicron strain S protein single domain antibody VHH-3 of the present invention described in the present invention and a pharmaceutically acceptable carrier.
The invention has the beneficial effects that:
the method comprises the steps of utilizing a natural single domain antibody phage display library established by established alpaca peripheral blood lymphocytes, using biotinylated S protein as an antigen, carrying out 3 rounds of immune panning, coating an ELISA plate with the S protein, using an antibody expressed by a panned monoclonal colony as a primary antibody, screening positive monoclonal colonies, amplifying corresponding bacterial liquid, sequencing, and finally obtaining a gene sequence of a single domain antibody VHH chain specific to the S protein. Specific single-domain antibodies against the S protein, which were efficiently expressed in insect sf9 cells, were obtained by amplification cloning of the specific single-domain antibodies into baculovirus expression vectors. Through WB test identification, the antibody has a good immunodetection effect, and a key foundation is laid for establishing a rapid and accurate antigen method for detecting the new coronavirus. And the single-domain antibody is identified to have strong specificity by using western blot. By virus neutralization assay, serum neutralizing titers for immunization against the omitron strain reached 1:32, the sensitivity is high, and the research direction is provided for the targeted therapy of the new coronavirus.
Drawings
FIG. 1 shows the expression and purification of S protein single domain antibody of new coronavirus omicron strain in Sf9 cells and the identification of Western blot specificity; wherein 1A is an expression and purification result of the S protein single domain antibody in Sf9 cells, and 1B is a specificity identification result of the S protein single domain antibody;
FIG. 2 shows the identification of the neutralizing activity of the S protein single domain antibody of the novel coronavirus omicron strain (neutralizing titer of 1: 32).
Detailed Description
The invention is further described with reference to specific examples and the accompanying drawings, but it should be understood that the scope of the invention is not limited to the following examples.
The invention will be further illustrated with reference to specific examples:
example 1: immunopanning process for natural single domain antibodies to S protein
(1) Amplifying the established natural single-domain antibody phage library: adding a 2 XYT culture medium into a 100 mu L glycerol bacterial library, adding 20 MOI auxiliary phage when OD600=0.5, standing for 30min, resuspending the precipitate with the 2 XYT culture medium after centrifugation, culturing for 1h, adding antibiotics, culturing for 16h, centrifuging, precipitating the supernatant with precooled PEG-NaCl (1/4 volume), and resuspending with 1mL PBS to obtain the amplified single domain antibody library;
(2) immune tube panning: coating an immune tube with 50 micrograms/tube of S protein (omicron strain S gene ORF full-length expression, biotin labeling and purification) overnight, removing coating liquid, washing for 3 times, sealing for 2h with 2mL BSA (1%), washing for 3 times with PBST, adding 100 muL of the single-domain antibody library amplified in the step 1 as a primary antibody, acting for 2h at 37 ℃, washing for 3 times with PBST, eluting with Glycine-HCI (PH2.2), adjusting the eluent with Tris-HCI to PH 7.4, and obtaining a first round of natural single-domain antibody library after elutriation;
(3) amplifying the 1 st round of natural single-domain antibody library obtained in the step (2) according to the step (1), obtaining a 1 st round of natural single-domain antibody heavy suspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 1 st round of natural single-domain antibody heavy suspension library as primary antibody, and finally obtaining a panned 2 nd round of natural single-domain antibody library;
(4) amplifying the 2 nd round natural single-domain antibody library obtained in the step (3) according to the step (1), obtaining a 2 nd round natural single-domain antibody resuspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 2 nd round natural single-domain antibody resuspension library as a primary antibody, and finally obtaining a panned 3 rd round natural single-domain antibody library.
Example 2: ELISA identification of Individual clones
(1) Panning for single positive clonal expansion of the shake bacteria for antibody expression: the 3 rd round natural single domain antibody library panned in example 1 was inoculated into 2 XYT medium, OD 600nm If =0.5, adding 20 MOI helper phage, standing for 30min, centrifuging, then resuspending the precipitate with 2 XYT medium, culturing for 1h, then coating on 2 XYT plates containing antibiotics, culturing overnight, selecting 40 single colonies to inoculate into 2 XYT medium the next day, if OD600=0.5, then adding 20 MOI helper phage, standing for 30min, centrifuging, then resuspending the precipitate with 2 XYT medium, culturing again, and adding IPTG to induce expression for 8 h.
(2) And (3) ELISA identification: coating an ELISA plate with an S protein (an omicron strain S gene ORF full-length expression, biotin labeling and purification, concentration of 1 ng/muL) 100 muL/hole overnight, washing 3 times after removing a coating solution, sealing for 2h by using 200 muL/hole BSA (3%), washing for 3 times by using PBST, adding a single domain antibody library which is respectively amplified in the step of 1 muL/hole as a primary antibody (a library construction carrier with M13), acting at 37 ℃ for 2h, washing for 3 times by using PBST, adding M13-HRP into a secondary antibody, detecting an OD450nm value after termination, and judging the result: the test piece was determined to be positive by 3 times higher than the control group OD450nm value (see Table 1 for the results).
Table 1: screening ELISA experiment to obtain positive clone
Example 3: sequencing identification of Positive clones
The clones which were detected as positive in example 2 by ELISA were extracted with plasmids from the corresponding bacterial solutions and sequenced using the universal primers for the plasmid vectors. The gene sequences of the individual clones were analyzed according to the sequence alignment software MEGA 6.0, and the strains with the same CDR1, CDR2 and CDR3 sequences were identified as the same clones, while the strains with different sequences were identified as different clones, and finally 3 specific single domain antibody sequences against the S protein were obtained. The nucleotide sequence of the antibody is SEQ ID NO: 1-3, the amino acid sequence is SEQ ID NO: 4-6 comprising the CDR1-CDR2-CDR3 regions as shown, constituting the VHH chain of the subject.
Example 4: expression, purification and identification of S protein single-domain antibody in Sf9 insect cell
(1) Construction of a pFastBac-VHH recombinant plasmid for an S protein single domain antibody: the peptide represented by SEQ ID NO:3, extracting plasmid as a template, and adopting a primer VHH-F of a VHH chain: ggatccgatg tgcagctgca ggagtctgg (SEQ ID NO: 16) and VHH-R: aagcttgctg gagacggtga cctgggtcc (SEQ ID NO:18), the upstream and downstream primers have BamH I and Hind III sites, and the amplified fragment is about 400 bp. In addition, after the vector of the pFastBac is subjected to double enzyme digestion by BamH I and Hind III, the fragment is recovered, and then the VHH fragment is inserted into the vector of the pFastBac to form the pFastBac-VHH recombinant plasmid.
(2) Bacmid-VHH Bacmid screening for S protein single domain antibodies: and after the pFastBac-VHH recombinant plasmid is extracted aseptically, the recombinant plasmid is transformed into DH10Bac competent cells, the competent cells are coated on a Luria Agar selective plate, white spots are selected after 48 hours and inoculated into selective KTG/LB culture solution, the plasmid is extracted after culture, M13 primer is used for amplification and sequencing identification, and the positive clone is Bacmid-VHH Bacmid.
(3) Recombinant viral packaging of S protein single domain antibodies: sf9 cells are cultured to 80% in a 6-well plate by using a complete culture medium of 2 mL/well, the obtained recombinant Bacmid-VHH plasmid is used for transfecting Sf9 cells by using a liposome Cellffectine reagent, the cells are swollen and rounded after 96 h, the refractivity is enhanced, the intercellular tight connection disappears, the cell nucleus is enlarged, vacuoles appear in the nucleus, the cells in the late stage of infection gradually fall off and other typical CPE, and the normal control Sf9 cells are uniform in size, compact in cell nucleus and free of cell lesions, so that the successful packaging of the recombinant baculovirus is indicated.
(4) Secretory expression and identification of S protein single domain antibody: after successful packaging of the recombinant baculovirus, the pFastBac vector (vector with 6 × His tag) was used, so that the expression of the gene could be identified with anti-His antibody. Western blot detection is carried out on the supernatant and cells after the recombinant baculovirus infects Sf9 cells, and the result shows that 15kD target bands appear in the supernatant and cells of the Sf9 cells infected with the recombinant virus (see 1A: Marker: protein Marker; Lane 1: protein electrophoresis control; Lane 2: S protein single domain antibody SDS electrophoresis in figure 1), and the molecular weight of the recombinant baculovirus are consistent with that of the single domain antibody.
(5) Purification of single domain antibody of protein S: transferring the High Five cells into a T75 cell bottle, infecting the High Five cells with recombinant baculovirus Bacmid-VHH by MOI = 8 after the cells grow to 70% -80% abundance in an adherent manner, collecting culture supernatant of the infected cells after the High Five cells grow to be large, round and shed and other lesions after infecting for 96 h, centrifuging for 10 min at 4 ℃ and 2000 rpm, collecting the culture supernatant, firstly precipitating with 35% ammonium sulfate, and then passing through Ni + NTA affinity column purification and quantification of each recovery of purified protein using the Qubit 2.0 quantification kit.
Example 5: specificity identification of S protein single domain antibody
Identifying specificity of Western blot of S protein single domain antibody:
protein A electrophoresis: respectively diluting the purified S protein, influenza A HA protein, influenza B HA protein, parainfluenza virus HN protein and G protein of respiratory syncytial virus to 60 mu G by using 6 xSDS protein electrophoresis sample adding buffer solution, boiling for 5min at 100 ℃, pre-staining Marker 2 mu L, carrying out protein electrophoresis to concentrate glue 80 v and separate glue 120 v.
B. Film transfer: placing the gel on a nitrocellulose membrane (NC membrane), placing 3 Whatman 3 mm filter papers on the upper and lower sides respectively, and soaking the above materials in a membrane-transfer electrophoresis buffer for 15min to remove the air bubbles remained on the filter membrane. The electrotransfer device is installed in sequence, 3 pieces of filter paper, gel, NC membrane and 3 pieces of filter paper are sequentially placed on the negative electrode plate, each layer is ensured to be accurately aligned (from bottom to top), bubbles between each layer are removed, the position is marked, and the anode plate is closed. 2 mA/cm 2 And (5) rotating the membrane under the constant current condition of 2 h.
C. Western blot identification: sealing with 5% skimmed milk powder at 4 deg.C overnight; adding purified His-tagged S protein single-domain antibody (5% skimmed milk diluted at 1: 3000), and incubating the membrane at room temperature for 3 h; washing membrane with PBST (Tween 1 ‰) for 10 min for three times; adding Dy800 antibody (diluted with 5% skimmed milk at a ratio of 1: 20000) of goat anti-His, and incubating at room temperature in dark for 40 min; and finally, scanning and identifying by using 700 and 800 fluorescence channels of Odyssey (see figure 1B of figure 1, wherein Lane 3 is used for carrying out WB identification on protein loading, Lane 4-Lane 7 is used for carrying out WB identification on influenza A HA protein, influenza B HA protein, parainfluenza virus HN protein and respiratory syncytial virus G protein), and the specific binding of the S protein single-domain antibody and the S protein is shown, and the cross reaction with the proteins of other 4 respiratory pathogens is not generated.
Example 6: neutralization activity and stability identification of S protein single-domain antibody
Validation was performed in the P3 laboratory using a micro cell neutralization assay. The S protein single domain antibody is diluted by 1: 2-1: 128 in a multiple ratio by using normal saline, 1 part of immune serum is used as a positive control, and the dilution is also carried out in a multiple ratio of 1: 2-1: 128, and each dilution is provided with 5 times. Adding virus solution containing 100TCID50 into each well of the culture plate with diluted sample to be detected, setting a multiple ratio dilution hole of negative serum control, and adding equal amount of dilution solution. All plates were placed in a 37 ℃ cell incubator and neutralized for 2 h. And adding the Vero cell suspension into each well after neutralization, and placing the mixture in an incubator at 37 ℃ for 5 days. Meanwhile, a normal cell control is set. After 5 days, each culture well was observed and counted for the presence of lesions, i.e., the highest dilution of serum capable of protecting 50% of the cells from infection by 100TCID50 virus was the antibody titer of the serum, and the whole experiment was repeated 3 times. FIG. 2 is the identification of the neutralizing activity of the S protein single domain antibody of the new coronavirus omicron strain. The neutralization titer of the positive control serum was 1:32 (≧ 1: 4, positive). The result of the neutralization activity test shows that the S protein single-domain antibody has higher neutralization titer (1: 32), and the coefficient of variation of 3 repeated tests is less than 3%, which indicates that the single-domain antibody has excellent stability.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> prevention and control center of diseases in Beijing
<120> single domain antibody VHH-3 aiming at new coronavirus omicron strain S protein, coding sequence and application
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gatgtgcagc tgcaggagtc tgggggaggc ttggtgcagc ctgggggatc tctgagactc 60
tcctgtgcag cctctggatt ccgtttggat ggttatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcaggt attagccggg gtggcactgc cccagactgt 180
ttagactccg tgaagggccg attcaccatc tccagagaca acgccgcgaa cacggtgtat 240
ttacaaatga acagcctgaa acctgaggac acagccattt attactgtgc agctttcgct 300
tcgccgctcc ggtatgcctc atcctgtcgc agccaggcct atgagtactg gggccaggga 360
acccaggtca ccgtctccag c 381
<210> 2
<211> 378
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatgtgcagc tgcaggagtc tgggggaggc tcggtggaac ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacgttgaat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attacaaaag gcggtaccat aatctataca 180
gactccgtga agggccgatt caccgcctcc agagacaacg ccaagaacac ggtgtatctg 240
caaatgaaca gcctgatgcc tgaggacacg gccgtttatt actgtgcagc ggatcgcgga 300
gcaccgtatt acggctgttc aaaccacccc aacaggtatg acgcctgggg gcaggggacc 360
caggtcaccg tctccagc 378
<210> 3
<211> 372
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gatgtgcagc tgcaggagtc tgggggagga ttggtgcaac ctggggactc tctgagtctc 60
tcctgtgcag cctctggacg caccttcagt agctatacca tggcctggtt tcgccaggct 120
ccaggaaagg agcgtgagtt tgtagcagct attagtaagc ctggtaaaag tacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctacaaatgg tcagcctgaa acctgaggac acggccgtgt attactgtgc ggcgcgactg 300
ccagtactgc ttataactac aacccgaggg tatgactact ggggccaggg gacccaggtc 360
accgtctcca gc 372
<210> 4
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Leu Asp Gly Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Gly Ile Ser Arg Gly Gly Thr Ala Pro Asp Cys Leu Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Phe Ala Ser Pro Leu Arg Tyr Ala Ser Ser Cys Arg Ser Gln
100 105 110
Ala Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Glu Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asn Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Thr Lys Gly Gly Thr Ile Ile Tyr Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Met Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Arg Gly Ala Pro Tyr Tyr Gly Cys Ser Asn His Pro Asn Arg
100 105 110
Tyr Asp Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 124
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Lys Pro Gly Lys Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Val Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Leu Pro Val Leu Leu Ile Thr Thr Thr Arg Gly Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 7
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Phe Arg Leu Asp Gly Tyr Ala Ile Gly
1 5 10
<210> 8
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Ile Ser Arg Gly Gly Thr Ala Pro Asp
1 5 10
<210> 9
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Ala Phe Ala Ser Pro Leu Arg Tyr Ala Ser Ser Cys Arg Ser Gln Ala
1 5 10 15
Tyr Glu Tyr
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Phe Thr Leu Asn Tyr Tyr Ala Ile Gly
1 5 10
<210> 11
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Cys Ile Thr Lys Gly Gly Thr Ile Ile Tyr
1 5 10
<210> 12
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Ala Asp Arg Gly Ala Pro Tyr Tyr Gly Cys Ser Asn His Pro Asn Arg
1 5 10 15
Tyr Asp Ala
<210> 13
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Arg Thr Phe Ser Ser Tyr Thr Met Ala
1 5 10
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ala Ile Ser Lys Pro Gly Lys Ser Thr Tyr
1 5 10
<210> 15
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Ala Arg Leu Pro Val Leu Leu Ile Thr Thr Thr Arg Gly Tyr Asp Tyr
1 5 10 15
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggatccgatg tgcagctgca ggagtctgg 29
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aagcttgctg gagacggtga cctgggttcc 30
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aagcttgctg gagacggtga cctgggtcc 29
Claims (9)
1. A single domain antibody VHH-3 specifically binding to the S protein of the novel coronavirus omicron strain, characterized in that the VHH-3 comprises a CDR1 shown in SEQ ID NO. 13, a CDR2 shown in SEQ ID NO. 14 and a CDR3 shown in SEQ ID NO. 15.
2. The single domain antibody VHH-3 that specifically binds to the S protein of the novel coronavirus omicron strain of claim 1, wherein the amino acid sequence of VHH-3 is as shown in SEQ ID NO. 6.
3. A nucleic acid molecule encoding the single domain antibody VHH-3 of claim 1 or 2.
4. The nucleic acid molecule of claim 3, wherein the sequence of said nucleic acid molecule is set forth in SEQ ID NO. 3.
5. A method for preparing the single domain antibody VHH-3 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2, which comprises inserting a nucleic acid encoding the VHH-3 into an expression vector, transferring the expression vector into a host cell, culturing the host cell, and purifying to obtain the single domain antibody.
6. The method of claim 5, wherein the expression vector is a baculovirus expression vector pFastBac and the host cell is an sf9 insect cell.
7. Use of the single domain antibody VHH-3 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2 for the preparation of a product for the diagnosis of the novel coronavirus omicron strain.
8. Use of the single domain antibody VHH-3 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2 for the preparation of a product for the treatment of the novel coronavirus omicron strain.
9. A pharmaceutical composition comprising the single domain antibody VHH-3 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2 and a pharmaceutically acceptable carrier thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210694199.7A CN114773465B (en) | 2022-06-20 | 2022-06-20 | Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210694199.7A CN114773465B (en) | 2022-06-20 | 2022-06-20 | Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114773465A CN114773465A (en) | 2022-07-22 |
CN114773465B true CN114773465B (en) | 2022-08-26 |
Family
ID=82421978
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210694199.7A Active CN114773465B (en) | 2022-06-20 | 2022-06-20 | Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114773465B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022020668A1 (en) * | 2020-07-23 | 2022-01-27 | The United State Of America, As Represented By The Secretary, Department Of Health And Human Services | Nanobodies directed to coronavirus spike protein receptor binding domain and uses thereof |
WO2022060906A1 (en) * | 2020-09-15 | 2022-03-24 | Duke University | Coronavirus antibodies and uses thereof |
CN114231497A (en) * | 2022-02-24 | 2022-03-25 | 广州伯尼兹生物科技有限公司 | Monoclonal antibody hybridoma cell line for expressing novel coronavirus S1 protein and neutralizing active antibody |
CN114634556A (en) * | 2022-04-01 | 2022-06-17 | 中国科学院微生物研究所 | New coronavirus Delta and Omicron variant chimeric antigen, and preparation method and application thereof |
-
2022
- 2022-06-20 CN CN202210694199.7A patent/CN114773465B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022020668A1 (en) * | 2020-07-23 | 2022-01-27 | The United State Of America, As Represented By The Secretary, Department Of Health And Human Services | Nanobodies directed to coronavirus spike protein receptor binding domain and uses thereof |
WO2022060906A1 (en) * | 2020-09-15 | 2022-03-24 | Duke University | Coronavirus antibodies and uses thereof |
CN114231497A (en) * | 2022-02-24 | 2022-03-25 | 广州伯尼兹生物科技有限公司 | Monoclonal antibody hybridoma cell line for expressing novel coronavirus S1 protein and neutralizing active antibody |
CN114634556A (en) * | 2022-04-01 | 2022-06-17 | 中国科学院微生物研究所 | New coronavirus Delta and Omicron variant chimeric antigen, and preparation method and application thereof |
Non-Patent Citations (3)
Title |
---|
Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern;Irina A. Favorskaya等;《Frontiers in Immunology》;20220224;第13卷;第1-9页 * |
新型冠状病毒变异株以及疫苗研究现况;新宇等;《安徽预防医学杂志》;20220220;第28卷(第01期);第1-8、12页 * |
新型冠状病毒疫苗的研发策略及进展;李佳乐,王汀;《中国药剂学杂志》;20211113;第19卷(第06期);第193-202页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114773465A (en) | 2022-07-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112010965B (en) | Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof | |
CN111778218B (en) | Phage display antibody library and monoclonal antibody aiming at novel coronavirus SARS-CoV-2 obtained based on panning of phage display antibody library | |
CN111116752B (en) | Immunoglobulin-binding single domain antibody, anti-avian influenza single domain antibody, bifunctional antibody and application thereof | |
CN114773464B (en) | Single-domain antibody VHH-2 aiming at new coronavirus omicron strain S protein, coding sequence and application | |
CN114957454B (en) | anti-CSFV E2 protein nano antibody, fusion protein, preparation method and application thereof | |
CN111138533A (en) | Single domain antibody against hepatitis A virus and derived protein thereof | |
CN113698475B (en) | Monoclonal antibody of anti-porcine delta coronavirus N protein and porcine delta coronavirus colloidal gold rapid detection test strip | |
CN111138532B (en) | Use of single domain antibodies against hepatitis a virus | |
CN114773465B (en) | Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application | |
CN114773463B (en) | Single-domain antibody VHH-1 aiming at new coronavirus omicron strain S protein, coding sequence and application | |
CN109503711B (en) | Difunctional nanobody for detecting PCV2 virus by hemagglutination method, coding gene and application thereof | |
CN106928355B (en) | CD105 nano antibody Nb184 | |
CN106928358B (en) | CD105 nano antibody Nb168 | |
CN111393528B (en) | Single-chain antibody targeting folate receptor alpha and application thereof | |
CN111454171A (en) | Paraquat hapten PH-A, artificial antigen and antibody as well as preparation methods and applications thereof | |
CN106928360B (en) | CD105 nano antibody Nb68 | |
CN106928359B (en) | CD105 nano antibody Nb59 | |
CN111499734B (en) | Single-chain antibody for resisting duck circovirus and preparation method and application thereof | |
CN114773462B (en) | Recombinant single-chain antibody for detecting bovine CRP protein and application thereof | |
CN116496392B (en) | Anti-novel coronavirus N protein single domain antibody, fusion protein, encoding gene and application thereof | |
CN114751963B (en) | Protein for detecting foot-and-mouth disease virus antibody and application thereof | |
CN109096394B (en) | Nano antibody of B subunit of anti-staphylococcal protein A, nucleic acid molecule and application | |
CN116143913A (en) | Shark source single domain antibody combined with SARS-CoV-2RBD, preparation method and application thereof | |
CN117686722A (en) | S100A4 nano antibody and application thereof | |
CN117106075A (en) | Nanometer antibody for resisting H7 subtype influenza virus and application thereof |
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 |