CN114773464A - Single-domain antibody VHH-2 aiming at S protein of new coronavirus omicron strain, coding sequence and application - Google Patents
Single-domain antibody VHH-2 aiming at S protein of new coronavirus omicron strain, coding sequence and application Download PDFInfo
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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-2, 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 detection reagent of the new coronavirus omicron strain and targeted therapy.
Description
Technical Field
The invention relates to the field of application of biomedicine or biotechnology, in particular to a single-domain antibody aiming at the S protein of a new coronavirus omicron strain, a coding sequence and application thereof.
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 detecting the antigen at present mainly aims at conservative N protein, and the specificity and sensitivity of the detection cannot meet the market requirements. At present, the omicron strain is a main prevalent strain in which new coronavirus is abused worldwide, so that the development of a rapid detection kit by using a single-domain antibody against the S protein of the new coronavirus is of great significance, and a single-domain antibody for broad-spectrum targeted therapy can be developed.
The new corona virion is spherical (some are polymorphic), has protrusions on surface, and is similar to a virus of crown shape when observed under an electron microscope, and the 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 a lack of satisfactory single domain antibodies against the S protein of the novel coronavirus. 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 chain of single domain antibody of S protein of the new coronavirus omicron strain named VHH-2, characterized in that the VHH-2 comprises CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12. In a specific embodiment, the amino acid sequence of VHH-2 is a VHH chain comprising the above 3 CDRs and is the same length as SEQ ID No. 5 with more than 95% homology; in another specific embodiment, the amino acid sequence of the VHH is as shown in SEQ ID NO 5.
The second aspect of the present invention provides a nucleic acid encoding the above-described single domain antibody VHH-2; 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. 2 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 5; in another embodiment, the sequence of said nucleic acid has more than 99% homology with the nucleic acid molecule as shown in SEQ ID NO. 2 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 5; in another specific embodiment, the nucleic acid has the sequence shown in SEQ ID NO. 2.
The third aspect of the invention provides a method for preparing the single domain antibody VHH-2 of the S protein of the new coronavirus omicron strain, wherein the method comprises the steps of inserting nucleic acid for coding the VHH-2 chain into an expression vector, transferring the expression vector into a host cell, culturing the host cell, 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; in one embodiment, the method is carried out by inserting the sequence shown in SEQ ID NO. 2 into a vector, transfecting a host cell, and expressing and purifying.
The fourth aspect of the invention provides the application of the single domain antibody VHH-2 of the S protein of the novel coronavirus omicron strain in the preparation of products for diagnosing the novel 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-2 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-2 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, performing 3 rounds of immune panning, coating the S protein on an ELISA plate, using an antibody expressed by a panned monoclonal colony as a primary antibody, screening positive monoclonal colonies, amplifying corresponding bacterial liquid, and sequencing to finally obtain a gene sequence of a single-domain antibody VHH-2 chain specific to the S protein. The specific single-domain antibody is amplified and cloned into a baculovirus expression vector, and the specific single-domain antibody aiming at the S protein, which is efficiently expressed in insect sf9 cells, is obtained. 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. Through virus neutralization test, the neutralizing titer of the antibody on the new crown omicron strain reaches 1: 64. the sensitivity is high, and a research direction is provided for the targeted therapy of the new coronavirus.
Drawings
FIG. 1 shows that S protein single domain antibody of new coronavirus omicron strain is expressed and purified in Sf9 cells and is identified by Western blot specificity;
FIG. 2 identification of neutralizing activity of S protein single domain antibody of the novel coronavirus omicron strain (neutralizing titer 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) to obtain 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 a 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, OD600nmAnd when the culture medium is =0.5, adding 20 MOI auxiliary phage, standing for 30min, centrifuging, then resuspending the precipitate with a 2 XYT culture medium, culturing for 1h, then coating the precipitate on a 2 XYT plate containing antibiotics, culturing overnight, selecting 40 single colonies the next day, inoculating the single colonies into the 2 XYT culture medium, when the OD600=0.5, adding 20 MOI auxiliary phage, standing for 30min, centrifuging, then resuspending the precipitate with the 2 XYT culture medium, culturing, and adding IPTG to induce expression for 8 h.
(2) And (3) ELISA identification: coating an ELISA plate with S protein (OMicron strain S gene ORF full length expression, biotin labeling and purification, concentration of 1 ng/muL) 100 muL/well overnight, washing 3 times after removing coating liquid, sealing 2h with BSA (3%) 200 muL/well, washing 3 times with PBST, adding a single-domain antibody library which is respectively amplified in the step of 100 muL/well (1) as a primary antibody (library construction vector belt M13), acting 2h at 37 ℃, washing 3 times with PBST, adding M13-HRP into secondary antibody, detecting OD450nm value after termination, and judging: 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 by the sequence alignment software MEGA 6.0, and 3 strains specific single domain antibody sequences against the S protein were finally obtained, with strains having the same CDR1, CDR2, and CDR3 sequences being identified as the same clones, and strains having different sequences being identified as different clones. 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 shown, constituting the VHH chain of the host.
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:2, 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 aseptic extraction, the pFastBac-VHH recombinant plasmid is transformed into DH10Bac competent cells, coated on a Luria Agar selective plate, and after 48 hours, white spots are selected and inoculated in a selective KTG/LB culture solution, after culture, the plasmid is extracted, 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: the 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 Cellfectin reagent, the cells are swelled 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 cytopathic effect, 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 a 15kD target band (shown in figure 1A: Marker: protein Marker; Lane 1: protein electrophoresis control; Lane 2: S protein single domain antibody SDS electrophoresis) appears in the supernatant and cells of the Sf9 cells infected with the recombinant virus, and the molecular weight of the target band is 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 infected cells after the High Five cells are infected for 96 h and have pathological changes such as enlargement, rounding and shedding, centrifuging the infected cells at 2000 rpm at 4 ℃ for 10 min, collecting the culture supernatant, precipitating by using 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 transferring: 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 sequentially arranged, 3 pieces of filter paper, gel, NC membrane and 3 pieces of filter paper are sequentially placed on the negative electrode plate to ensure that each layer is accurately aligned (from bottom to top), bubbles between layers are removed, the position is marked, and the anode plate is closed. 2 mA/cm2And (5) rotating the membrane under the constant current condition of 2 h.
C. Identifying Western blot: 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 three times, each for 10 min; 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 a figure 1B, wherein Lane 3 is used for carrying out WB identification on S protein, 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 G protein of respiratory syncytial virus), and the result shows that the S protein single-domain antibody is specifically combined with the S protein and does not have cross reaction with other 4 respiratory pathogen proteins.
Example 6: neutralization activity and stability identification of S protein single-domain antibody
Validation was performed in the P3 laboratory using a microcytoneutralization 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 diluted by 1: 2-1: 128 in a multiple ratio, and each dilution is provided with 5 times of dilution. In that100TCID is added into each hole of the culture plate with diluted sample to be detected50And simultaneously setting a multiple ratio dilution hole for negative serum control, and supplementing the same amount of dilution liquid. All plates were placed in a 37 ℃ cell incubator and neutralized for 2 h. After neutralization, Vero cell suspension is added into each well, and the mixture is placed 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 lesions, i.e., to protect 50% of the cells from 100TCID50The highest dilution of the virus-infected serum 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 novel coronavirus omicron strain. The final detection titer of the S protein single-domain antibody is 1:64, and the neutralization titer of the positive control serum is 1:32 (the determination result is positive if ≧ 1: 4). The neutralizing activity test result shows that the S protein single domain antibody has higher neutralizing titer (1: 32) and the coefficient of variation of 3 repeated tests<3%, indicating that the single domain antibody has excellent stability.
The foregoing shows and describes the general principles and features of the present invention, together with the 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 given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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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 (10)
1. A single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain, wherein the VHH-2 comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 shown in SEQ ID NO. 11 and a CDR3 shown in SEQ ID NO. 12.
2. The single domain antibody VHH-2 that specifically binds to the S protein of the novel coronavirus omicron strain of claim 1, wherein the amino acid sequence of VHH-2 is a VHH chain comprising the above 3 CDRs, and has an amino acid sequence that is identical in length to SEQ ID NO. 5 and has homology of 95% or more.
3. The single domain antibody VHH-2 that specifically binds to the S protein of the novel coronavirus omicron strain of claim 1 or 2, wherein the amino acid sequence of VHH is represented by SEQ ID NO. 5.
4. A nucleic acid molecule encoding the single domain antibody of any one of claims 1 to 3.
5. The nucleic acid molecule of claim 4, wherein the sequence of said nucleic acid molecule is set forth in SEQ ID NO. 2.
6. A method for preparing the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of claims 1 to 3, which comprises inserting a nucleic acid encoding the VHH-2 into an expression vector, transferring the expression vector into a host cell, culturing the host cell, and purifying to obtain the single domain antibody.
7. The method of claim 6, wherein the expression vector is a baculovirus expression vector pFastBac and the host cell is an sf9 insect cell.
8. Use of the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of any one of claims 1 to 3 for the preparation of a product for the diagnosis of the novel coronavirus omicron strain.
9. Use of a single domain antibody VHH-2 according to any one of claims 1 to 3 which specifically binds the S protein of the new coronavirus omicron strain in the manufacture of a product for the treatment of the new coronavirus omicron strain.
10. A pharmaceutical composition comprising the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of claims 1 to 3 and a pharmaceutically acceptable carrier thereof.
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| CN115724963A (en) * | 2022-12-07 | 2023-03-03 | 复旦大学 | Nano single-domain antibody targeting new coronavirus S-NTD protein and application thereof |
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| CN114773464B (en) | 2022-08-26 |
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