CN114933651B - Alpaca source nano antibody and application thereof - Google Patents

Abstract

The invention relates to an alpaca source nano antibody and application thereof, in particular to an alpaca source nano antibody R14 combined with SARS-CoV-2RBD or an antigen combining fragment thereof and application thereof, comprising a heavy chain variable region, wherein the heavy chain variable region comprises the following CDRs: CDR1 with an amino acid sequence shown as SEQ ID NO. 1, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and CDR3 with an amino acid sequence shown as SEQ ID NO. 3. The binding constant of the R14 nano antibody and SARS-CoV-2 is less than 1E-10M, and the invention can effectively inhibit SARS-CoV-2 pseudovirus infection. The R14 nano antibody of the invention has clinical application value in preventing, treating and/or detecting SARS-CoV-2 infection.

Description

Alpaca source nano antibody and application thereof

Cross-referencing

The present application claims priority from an invention patent application entitled "a strain of alpaca-derived nanobody and its use" filed on 16/9/2021 under application number 202111086956.4, which is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the field of biomedicine, in particular to an alpaca source nano antibody and application thereof, and more particularly relates to an alpaca source nano antibody combined with SARS-CoV-2RBD or an antigen binding fragment thereof, a polynucleotide encoding the alpaca source nano antibody, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a preparation method of the alpaca source nano antibody, a transformed cell and a pharmaceutical composition containing the alpaca source nano antibody, and application of the alpaca source nano antibody and the polynucleotide and the nucleic acid construct in preparation of medicines for preventing, treating and/or detecting new coronavirus infection.

Background

Since 12 months 2019, the epidemic caused by a novel coronavirus (SARS-CoV-2) of the family Coronaviridae (family Coronaviridae) has continued to spread worldwide. In addition, severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and the like belonging to the family Coronaviridae are also main pathogens of human respiratory system, are mainly transmitted by means of droplets, aerosols, contact and the like, have strong infectivity, and are easy to cause panic of the masses, so that the viruses causing respiratory system diseases seriously jeopardize public health and safety, and especially frequently cause respiratory infectious diseases and continuous variation of the viruses in recent years pose great threats to the health, life safety, national economic development and social stability of the masses.

The medicine is mainly combined with antigens on the surface of pathogenic microorganisms to prevent specific molecules expressed by the pathogenic microorganisms from being combined with cell surface receptors, so that the effect of neutralizing is achieved. Both SARS-CoV and SARS-CoV-2 virus have glycosylated spike protein (S) on their surface, which is capable of interacting with host cell receptor protein ACE2 and triggering membrane fusion, and thus blocking the binding of the S protein to ACE2 is an effective way to treat new coronavirus infections.

However, antibody strategies aimed at blocking virus from host cell receptors still require further optimization and upgrading. On one hand, the RNA virus such as the new coronavirus has the characteristics of easy mutation, easy immune escape and the like, and a single specific antibody hardly meets the long-term treatment requirement. In addition, the conventional monoclonal antibody has a certain defect in practical application due to its excessively large molecular weight.

Disclosure of Invention

Object of the Invention

The invention aims to provide an alpaca source nano antibody or an antigen binding fragment thereof combined with SARS-CoV-2RBD, a polynucleotide for coding the antibody, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a preparation method of the expression vector, a transformed cell, a pharmaceutical composition containing the nucleic acid construct, and application of the antibody and the polynucleotide in preparation of medicaments for preventing or treating new coronavirus. The alpaca source nano antibody or the antigen binding fragment thereof is a nano antibody with high neutralization activity, has strong binding capacity with SARS-CoV-2RBD protein, can effectively inhibit SARS-CoV-2 infection, has the advantages of small molecular weight (15 kDa), small immunogenicity, better solubility and stability and longer CDR3 region, can be atomized and administered, can directly reach the lung, has quicker effect, and provides a potential treatment strategy for new crown or other coronavirus infections.

Solution scheme

In order to achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the present invention provides an alpaca-derived nanobody or an antigen-binding fragment thereof that binds to SARS-CoV-2RBD, comprising a heavy chain variable region comprising the CDRs of:

CDR1 with an amino acid sequence shown in SEQ ID NO:1 (namely GFTLDYYAIG),

CDR2 having an amino acid sequence shown in SEQ ID NO:2 (i.e., CISSSDGSTSYADSVKG), and

the amino acid sequence is shown as CDR3 of SEQ ID NO:3 (namely TPATYYSGRYYYQCPAGGMDY).

Preferably, the heavy chain variable region further comprises 4 framework regions FR1-4, wherein the FR1-4 is staggered with the CDR1, CDR2 and CDR3 in sequence.

In a preferred embodiment, the amino acid sequences of FR1-4 are shown in SEQ ID NO 4 (i.e., QVQLQESGGGLVQPGGSLRLSCAVS), SEQ ID NO 5 (i.e., WFRQAPGKEREGVS), SEQ ID NO 6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA) and SEQ ID NO 7 (i.e., WGQGTQVTVSS), respectively.

Further preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8:

QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSSwherein the underlined parts are the framework regions FR1-4, respectively, and the shaded parts are CDR1, CDR2 and CDR3, respectively, of the heavy chain variable region.

In a second aspect, the present invention provides a polynucleotide encoding the alpaca nanobody or the antigen-binding fragment thereof that binds to SARS-CoV-2 RBD.

Further, the polynucleotide is DNA or mRNA.

Further, the polynucleotide has a nucleotide sequence shown as SEQ ID NO. 9:

CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGTCTCTGGATTTACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATCGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCCTTTATTACTGTGCAGCAACCCCTGCTACATACTATAGTGGACGTTACTACTACCAATGTCCCGCGGGGGGCATGGACTACTGGGGCAAAGGGACCCAGGTGACCGTGAGCTCT。

in a third aspect, the invention provides a nucleic acid construct comprising said polynucleotide.

Further preferably, the polynucleotide further comprises at least one expression control element operably linked to the polynucleotide. Such as a histidine tag, stop codon, etc.

In a fourth aspect, the present invention provides an expression vector comprising said nucleic acid construct.

In a fifth aspect, the invention provides a transformed cell comprising a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above or an expression vector as described in the fourth aspect above.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising a nanobody of alpaca origin or an antigen-binding fragment thereof that binds to SARS-CoV-2RBD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above or a transformed cell as described in the fifth aspect above, and a pharmaceutically acceptable carrier and/or excipient.

Further preferably, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation.

Further preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray.

Further preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments.

Further preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus preparation.

In a seventh aspect, the present invention provides a use of the alpaca nanobody or the antigen-binding fragment thereof that binds to SARS-CoV-2RBD according to the first aspect, the polynucleotide according to the second aspect, the nucleic acid construct according to the third aspect, the expression vector according to the fourth aspect, or the transformed cell according to the fifth aspect, or the pharmaceutical composition according to the sixth aspect, in the preparation of a medicament for preventing, treating and/or detecting a neocoronaviral infection.

Further preferably, the new coronavirus is SARS-CoV-2 original strain and/or SARS-CoV-2 variant strain.

Further preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.

In an eighth aspect, the present invention provides a method for preventing or treating a new coronavirus infection, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of an alpaca nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2RBD as described in the above first aspect, a polynucleotide as described in the above second aspect, a nucleic acid construct as described in the above third aspect, an expression vector as described in the above fourth aspect or a transformed cell as described in the above fifth aspect or a pharmaceutical composition as described in the above sixth aspect.

Preferably, the new coronavirus is SARS-CoV-2 original strain and/or SARS-CoV-2 variant strain.

Further preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.

The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, the target organ, the symptom, the administration method, and the like, and can be determined by the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.

In a ninth aspect, the present invention provides a method for detecting a novel coronavirus, which comprises using the alpaca-derived nanobody or the antigen-binding fragment thereof that binds to SARS-CoV-2RBD as described in the above first aspect.

Preferably, the new coronavirus is SARS-CoV-2 original strain and/or SARS-CoV-2 variant strain.

Further preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.

Advantageous effects

The invention relates to a nano antibody drug development aiming at new coronavirus, which comprises using SARS-CoV-2RBD protein and SARS-CoV-2NTD protein to immunize alpaca simultaneously, constructing antibody library, screening specific nano antibody by using phage display technology, using SARS-CoV-2RBD protein as 'bait', screening nano antibody specifically combined with SARS-CoV-2RBD with high affinity, named as R14 nano antibody. The R14 nano antibody of the invention can be combined with SARS-CoV-2RBD with high affinity, the combination constant is less than 1E-10M, and in the pseudovirus neutralization experiment, the invention can neutralize SARS-CoV-2 pseudovirus with high neutralization activity, which shows that: the R14 nano antibody is a novel coronavirus (SARS-CoV-2) alpaca source nano antibody which can be combined with SARS-CoV-2RBD with high affinity and has high neutralization activity.

The invention provides a potential nano-antibody new drug for clinical prevention, treatment and detection of novel coronavirus (including original strains and a series of variant strains).

Drawings

One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a schematic diagram showing the results of molecular sieve chromatography and SDS-PAGE identification of SARS-CoV-2RBD protein in example 1 of the present invention;

FIG. 2 is a diagram showing the results of molecular sieve chromatography and SDS-PAGE identification of SARS-CoV-2NTD protein in example 1 of the present invention;

FIG. 3 is a diagram showing the results of molecular sieve chromatography and SDS-PAGE identification of the R14 nanobody of example 4 of the present invention;

FIG. 4 is a graphical representation of the kinetics of binding of R14 nanobodies to SARS-CoV-2RBD as determined in example 5 of the present invention; wherein, the dotted line refers to the original data, and the solid line refers to the fitted kinetic curve;

FIG. 5 is a graph showing the effect of R14 nanobody on neutralization of VSV-SARS-CoV-2 pseudovirus infection as determined in example 6 of the present invention, wherein A is a graph showing the effect of R14 nanobody on neutralization of pseudovirus infection of original strain SARS-CoV-2 WT; b is an effect graph of R14 nano antibody neutralizing the pseudovirus infection of SARS-CoV-2 variant strain Alpha (B.1.1.7); c is an effect graph of R14 nano antibody neutralizing the pseudovirus infection of SARS-CoV-2 variant strain Beta (B.1.351); d is a graph of the effect of R14 nano antibody in neutralizing the pseudovirus infection of SARS-CoV-2 variant strain Gamma (P.1); e is an effect graph of R14 nano antibody neutralizing the pseudovirus infection of SARS-CoV-2 variant strain Kappa (B.1.617.1); f is an effect chart of R14 nanometer antibody neutralizing the pseudo virus infection of SARS-CoV-2 variant strain Delta (B.1.617.2).

Fig. 6 shows the neutralizing activity of the R14 nanobody detected in example 9 of the present invention on the pseudovirus of the original strain of new coronavirus before and after nebulization.

FIG. 7 shows the efficacy of R14 nanobodies detected in example 10 of the present invention in preventing new coronavirus infection in mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some instances, materials, components, methods, procedures, and the like that are well known to those of skill in the art have not been described in detail so as not to obscure the present invention.

The present invention will be described in detail below.

Definition of

"Nanobodies", i.e., "heavy chain single domain antibodies", comprise only one heavy chain variable region (VHH), from which a light chain is naturally deleted compared to other antibodies.

Due to its biophysical advantages, nanobodies can be easily aerosolized and delivered directly to the lungs via an inhaler to treat respiratory virus-induced infections, and are considered to be very potential antibody-based drugs.

"specific" binding, when referring to ligand/receptor, antibody/antigen or other binding pairs, refers to determining the presence or absence of a binding reaction of a protein, such as a nanobody of the invention, to a SARS-CoV-2RBD protein in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The reagents, enzymes, media, antibiotics, and milk used in the following examples of the present invention are commercially available, and for example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

Some commonly used biomaterials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available products, e.g., pCAGGS vectors are available from MiaoLingPlasmid;293F cells, HEK293T cells, etc. were purchased from ATCC; coli TG1 cells purchased from Lucigen; VCSM13 helper phage was purchased from StrataGene; plasmid pMES4 was purchased from adddge; protein a chips were purchased from GE Healthcare; vero cells were purchased from ATCC CCL81.

Some synthetic biomaterials, such as primers, sequences, etc., requiring artificial synthesis are manufactured by synthetic companies, for example, the primers (SED ID NO: 13-18) of the present invention are manufactured by Beijing Ongchou Biotech, inc.

SARS-CoV-2RBD protein and SARS-CoV-2NTD protein of the present invention are obtained from the laboratory of the inventors (see example 1).

Example 1: expression and purification of SARS-CoV-2RBD and SARS-CoV-2NTD

The coding sequence of the SARS-CoV-2RBD protein (shown as SEQ ID NO: 10) is connected with the coding sequence of 6 histidine tags (hexa-His-tag) and a translation termination codon TGA at the 3' end, and the coding sequence is constructed into a pCAGGS vector through restriction enzyme sites EcoRI and XhoI and is transfected into 293F cells to express SARS-CoV-2RBD-His protein. Similarly, the coding sequence of SARS-CoV-2NTD protein (as shown in SEQ ID NO: 11) was ligated 3' to the coding sequence of the SARS-CoV-2NTD protein by the sequence of 6 histidine tags (hexa-His-tag) and the translation termination codon TGA, and these were constructed into pCAGGS vectors via EcoRI and XhoI restriction endonuclease sites, and transfected into 293F cells to express SARS-CoV-2NTD-His protein.

The cell culture fluid containing the target protein is subjected to nickel ion affinity chromatography (HisTrap) ^TM excel (GE Healthcare) and gel filtration chromatography (Superdex) ^TM After 200Increatase 10/300GL column (GE Healthcare)), purified target protein can be obtained. The size of the SARS-CoV-2RBD-his protein is identified by SDS-PAGE to be about 30KD, and the result is shown in figure 1; the size of SARS-CoV-2NTD-his protein was identified to be about 60KD by SDS-PAGE, and the results are shown in FIG. 2.

The 3' end of the SARS-CoV-2RBD protein coding sequence (shown as SEQ ID NO: 10) is connected with the coding sequence (shown as SEQ ID NO: 12) of the human Fc tag (hFc) and the translation stop codon TGA, and the coding sequence and the translation stop codon TGA are constructed into pCAGGS vectors by connecting EcoRI and XhoI, transfected into 293F cells for expression of SARS-CoV-2RBD-hFc protein and used for surface plasmon resonance analysis.

Example 2: alpaca immunization and antibody library construction

The SARS-CoV-2RBD protein and SARS-CoV-2NTD protein each having 6 histidine tags prepared in example 1 (200. Mu.g) were diluted with PBS to a final volume of 1mL, emulsified with 1mL of complete Freund's adjuvant for 5min, and simultaneously subjected to subcutaneous multi-point injection for immunization, followed by immunization every two weeks, and on day 12 after the fourth immunization, 50 to 60mL of blood was collected and PBMCs (peripheral blood mononuclear cells) were isolated. The isolated PBMCs were added to 1mL TRIzol (available from Invitrogen) and total RNA was extracted according to the procedure described in the specification. Using the extracted total RNA as a template, the Superscript II First-Strand Synthesis System for RT-PCR kit (purchased from Invitrogen) was used, and oligo-dT was used as a random primer _12-18 cDNA was synthesized using the primers. Using cDNA as a template, PCR was performed using specific primers CALL001 and CALL002 (primers shown in Table 1), and 700bp bands were cut and recovered. The purified DNA is used as a template, nested PCR is carried out by using nested primers VHH-BACK and PMCF to amplify a nano antibody (VHHs) sequence, and the VHHs sequence with the purified size of about 400bp is recovered.

The VHHs fragments were ligated into plasmid pMES4 by restriction of sites Pst I and BstEI using a two-enzyme cleavage method. The purified cloning vector and electrocompetent e.coli TG1 cells were mixed, the cloning vector was transformed into electrocompetent e.coli TG1 cells using an electrotransfer (BIO-RAD electrotransfer micropopulser), all of which were plated on selective medium containing ampicillin, after overnight culture at 37 ℃, all colonies were collected in LB medium, centrifuged and the supernatant was discarded, and the cells were resuspended with LB, which was an antibody library.

TABLE 1 reaction primers

Example 3: screening specific nano antibody by phage display technology

The E.coli TG1 transfected with the recombinant plasmid in example 2 was taken, VCSM13 helper phage was added at a ratio of multiplicity of infection (MOI) of about 20, after overnight culture, centrifugation was performed at 4000rpm, the supernatant was taken, membrane filtration was performed at 0.22 μm, PEG6000/NaCl was added at 1:4 in volume ratio, after mixing, the mixture was left at 4 ℃ for at least 1 hour, centrifugation was performed at 8000 Xg for 30min, the supernatant was discarded, the precipitate was resuspended in PBS, the collected phage particles were used, and phage titer was determined.

2 x10 to ¹¹ Each of the above-collected phages was mixed with an equal volume of 5% (w/v) skim milk, added to a 96-well plate coated with SARS-CoV-2RBD-His antigen, incubated at room temperature for 1h, and then specific phages were eluted with 0.2M glycine, and the eluted phages were neutralized with Tris-HCl (pH 9.1). Coli TG1 cells were then infected with the phage, and the phage were amplified. And preparing a 96-pore plate coated with SARS-CoV-2RBD-His antigen, performing 2 nd round of panning to enrich the bacteriophage expressing the specific nano antibody, and performing 3 rounds of panning. After each round of panning, randomly selecting different single colonies from agar plates with colonies, culturing in a shaker at 37 ℃, adding VCSM13 helper phage for overnight propagation, centrifuging the culture solution the next day, taking phage supernatant for ELISA experiment, and performing ELISA experiment when OD is obtained _450nM >At 0.2 timeAnd judging the reaction to be positive, taking the corresponding clone, sequencing the plasmid by using specific primers MP57 and GIII (the primers are shown in the table 2), and obtaining the sequence of the plasmid for coding VHHs. The core coding sequence for R14 was obtained by sequencing.

TABLE 2 reaction primers

Example 4: expression of R14 Nanobodies

In order to make the heavy chain variable region of R14 more complete, the 5 'end of the core coding sequence of R14 obtained in example 3 is connected with the coding sequence of QVQLQ (CAGGTGCAGCTGCAG), and the 3' end is connected with the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT), so as to obtain the nucleotide sequence shown as SEQ ID NO. 9, namely the coding sequence of the R14 nano antibody of the application; then, the coding sequence of 6 histidine tags (hexa-His-tag) and the translation termination codon TGA were ligated to the DNA fragment, and the DNA fragment was constructed into pCAGGS vectors by restriction endonuclease sites EcoRI and XhoI, transfected into 293F cells, cultured for 5 days, collected as a supernatant, centrifuged at 5000rpm for 30min, filtered through a 0.22 μm filter, and subjected to nickel ion affinity chromatography (HisTrap) ^TM excel ((GE Healthcare)) and gel filtration chromatography (Superdex) ^TM 75Increate 10/300GL column (GE Healthcare)), and then the purer target protein is obtained after purification. The peak of interest was determined by SDS-PAGE and the result is shown in FIG. 3, resulting in purified R14 nanobody.

Example 5: surface plasma resonance technology for detecting binding capacity of antibody and SARS-CoV-2RBD

Surface plasmon resonance analysis was performed using Biacore 8K (Biacore inc.). The method comprises the following specific steps:

the SARS-CoV-2RBD-hFc protein obtained in example 1 was immobilized on a protein A chip (purchased from GE Healthcare) at an amount of about 100RU by affinity of the protein A chip to hFc using a PBST buffer (2.7 mM KCl,137mM NaCl,4.3mM Na ₂ HPO ₄ ，1.4mM KH ₂ PO ₄ 0.05% tween) diluted the R14 protein from low concentration toThe samples are loaded one by one at high concentration. The kinetic profile of antibody binding to SARS-CoV-2RBD protein is shown in FIG. 4. Equilibrium dissociation constant (K) between R14 nanobody and SARS CoV-2RBD _D ) Less than 0.1nM. The calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, inc.). The R14 nano antibody is shown to be capable of binding with SARS-CoV-2RBD with higher affinity.

Example 6: SARS-CoV-2 original strain and variant strain pseudovirus package

1) The 18 th amino acid gene of S protein encoding SARS-CoV-2 original strain (WT) and variant strain (Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and Delta (B.1.617.2)) is removed, and the rest sequence of S protein is synthesized (providing synthesis service by Jin Weizhi of Suzhou) to obtain the nucleotide sequences of SARS-CoV-2-WT-S-del18, B.1.1.7-S-del18, B.1.351-S-del18, P.1-S-del18, B.1.617.1-S-del18 and B.1.617.2-S-del18 genes, which are respectively shown in SEQ ID NO: 19-24.

2) The protein gene obtained in 1) was cloned into pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.1.7-S-del18, pCAGGS-B.1.351-S-del18, pCAGGS-P.1-S-del18, pCAGGS-B.1.617.1-S-del18, and pCAGGS-B.1.617.2-S-del18.

The SARS-CoV-2 original strain and variant strain pseudovirus packaging steps are as follows:

a. cell preparation: HEK293T cells (ATCC CRL-3216 cells) were plated on 10cm cell culture dishes to reach a cell confluency of about 80% the next day. The culture solution was DMEM medium containing 10% FBS.

b. Transfection: taking the expression plasmid of each S protein in the step 2), transfecting 30 mu g of plasmid/10 cm of cell culture dish by using PEI, uniformly mixing the target plasmid and PEI according to the proportion of 1:3, transfecting, changing a culture solution (DMEM medium containing 10 percent FBS) for 4-6h, and culturing at 37 ℃ for 24h.

c. Adding poison: pseudovirus packaging frame virus G × VSV-delG (purchased from Wuhan Shunji Ministry of technology for brain science, inc.) was added to the above transfected HEK293T cells, incubated at 37 ℃ for 2h, the culture medium (DMEM medium containing 10% FBS) was replaced, VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added, and the culture was continued in the incubator for 30h.

d. And (3) toxin collection: collecting supernatant, centrifuging at 3000rpm for 10min, filtering with 0.45 μm sterile filter in ultra-clean bench, removing cell debris, packaging, and freezing at-80 deg.C.

Pseudoviruses of SARS-CoV-2 original strain (SARS-CoV-2 WT) and variant strain (Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and Delta (B.1.617.2)) were obtained, respectively.

Example 7: detection of R14 nano antibody neutralization SARS-CoV-2 pseudovirus infection

The purified R14 nanobody from example 4 was diluted 5-fold from 5. Mu.g/mL to the 9 th gradient (2.56 pg/mL) and 1.6x10 ⁴ TCID ₅₀ A series of pseudoviruses of the original strain and the variant strain of SARS-CoV-2 obtained in example 6 were mixed, incubated at 37 ℃ for 1 hour, and then added to a 96-well plate previously inoculated with Vero cells (purchased from ATCC CCL 81). After incubation for 18-20 hours, detection was performed by CQ1 structural Image Cytometer (Yokogawa). The neutralizing ability of the antibody against the pseudoviruses of the above-mentioned SARS-CoV-2 original strain and variant strain was calculated based on the number of cells having GFP fluorescence, and the results are shown in FIGS. 5A to 5F, respectively, and the statistics of the results are shown in Table 3.

TABLE 3 pseudovirus neutralization effect of R14 Nanobodies on New coronavirus

* Wherein, IC ₅₀ (μg/mL) ^a Is the half inhibitory concentration of the R14 nm antibody.

As can be seen from Table 3, the R14 nanobody can neutralize the pseudoviruses of the above-mentioned SARS-CoV-2 original strain and variant strain with high neutralizing activity.

In conclusion, the R14 nano antibody can be used as a novel coronavirus (SARS-CoV-2) alpaca source nano antibody with high neutralization activity.

Example 8: detection of R14 nano antibody neutralization SARS-CoV-2 live virus infection

In this example, the neutralizing effect of nanobody R14 on live viruses of new coronaviruses was determined by a live virus neutralization test based on cytopathic effect (CPE); the specific procedure is as follows:

the nanobody R14 was diluted 2-fold to the 11 th gradient, each gradient 4 replicate wells, 50. Mu.L per well, each dilution was mixed with an equal volume of 100TCID ₅₀ The original strain of SARS-CoV-2 or variant strains Alpha, beta and Delta thereof are incubated at 37 ℃; after 1 hour, the mixture was added to the suspended Vero cells and incubation continued for 3 days at 37 ℃; observing and recording cytopathic conditions; IC of the nanobody for inhibiting live virus infection of the new coronavirus was calculated using GraphPad Prism 7.0 ₅₀ 。

The above experiments were performed in the biosafety third-level laboratory (BSL 3) of the chinese centers for disease prevention and control.

The neutralizing effect of the nano antibody R14 on live viruses of the original strain and the variant strain of the new coronavirus is shown in the following table 4.

TABLE 4 neutralizing effect of R14 Nanobody on live coronavirus

Table 4 the results show that: the nano antibody R14 has good inhibition effect on live viruses of the original strain and the variant strain of the new coronavirus.

Example 9: detection of stability before and after atomization of R14 nano antibody

Nanobodies R14 were nebulized using an Aerogen sol (Aerogen inc., chicago, USA) nebulizer, and the nebulized antibodies were collected using full glass SKC (origin Four, PA, USA) containing 20mL of PBS and subjected to a pseudovirus neutralization assay as described in example 7.

The results are shown in figure 6 which shows, the results in FIG. 6 show that: the nanometer antibody R14 of the invention keeps stable neutralizing activity to pseudovirus of the new coronavirus original strain before and after atomization, which indicates that the antibody is suitable for administration by atomization.

Example 10: detection of effect of R14 nano antibody on prevention of new coronavirus infection in vivo

In the embodiment, the effect of the nano antibody R14 in preventing the infection of the new coronavirus is detected in 7-8 week-old female BALB/c mice (purchased from Beijing Wintolite laboratory animal technologies, inc.); the specific procedure is as follows:

1) Female BALB/c mice at 7-8 weeks were anesthetized and nasally infected with recombinant adenovirus Ad5-hACE2 (according to Jing Sun et al, "Generation of a broad Useful Model for covI-19pathenesis, vaccination, and treatment" cell.2020Aug 6;182 (3): 734-743);

2) On day 5, mice were dosed and challenged;

specifically, the administration was performed in the following groups:

nasal drop administration group (IN): after 5 mice are anesthetized, 50 mu L of 2mg/ml nano antibody R14 is instilled into the nostrils of the mice by using a pipette gun, and the administration dose is 5mg/kg;

nebulized administration group (IH): 8 mice are placed in an atomization bin, 20mg/mL of nano antibody R14 is continuously atomized, after 10 minutes, the mice are immediately taken out, 5 of the mice are placed in mouse cages, and a lung filling test is carried out after the rest 3 mice are anesthetized to determine the amount of the inhaled antibody, wherein the inhaled dose is 0.25mg/kg;

control group (Control): 5 mice were given 200 μ L PBS by nasal drip;

6 hours after administration, the mice were again anesthetized and nasally infected with live virus (5X 10) of the original strain of the new coronavirus ⁵ TCID50)；

3) On day 10, the mice were dissected, lung tissue was taken, RNA was extracted, and viral load was measured.

The above experiments were performed in the biosafety third-level laboratory (BSL 3) of the chinese centers for disease prevention and control.

The results are shown in fig. 7, and the results in fig. 7 show that the nanobody R14 of the present invention, whether administered by nasal drops or nebulization, can effectively reduce the pulmonary viral load and effectively prevent mice from being infected with new coronavirus.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> alpaca source nano antibody and application thereof

<130> 1087-210168F-1

<160> 24

<170> PatentIn version 3.3

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 amino acid sequence of heavy chain complementary region of camelid source nano antibody R14

<220>

<221> DOMAIN

<222> (1)..(10)

<400> 1

Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly

1 5 10

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 amino acid sequence of heavy chain complementary region of camelid source nano antibody R14

<220>

<221> DOMAIN

<222> (1)..(17)

<400> 2

Cys Ile Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 amino acid sequence of heavy chain complementary region of camelid source nano antibody R14

<220>

<221> DOMAIN

<222> (1)..(21)

<400> 3

Thr Pro Ala Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gln Cys Pro Ala

1 5 10 15

Gly Gly Met Asp Tyr

20

<210> 4

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1 framework region of camelid nanobody R14

<220>

<221> DOMAIN

<222> (1)..(25)

<400> 4

Gln 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 Val Ser

20 25

<210> 5

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2 framework region of camelid nanobody R14

<220>

<221> DOMAIN

<222> (1)..(14)

<400> 5

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser

1 5 10

<210> 6

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3 framework region of camelid nanobody R14

<220>

<221> DOMAIN

<222> (1)..(32)

<400> 6

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala

20 25 30

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4 framework region of camelid nanobody R14

<220>

<221> DOMAIN

<222> (1)..(11)

<400> 7

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 8

<211> 130

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain complementary region of camelid source nano antibody R14

<220>

<221> DOMAIN

<222> (1)..(130)

<400> 8

Gln 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 Val Ser Gly Phe Thr Leu Asp 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 Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Thr Pro Ala Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gln Cys

100 105 110

Pro Ala Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser

130

<210> 9

<211> 390

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of camel source nano antibody R14

<220>

<221> misc_feature

<222> (1)..(390)

<400> 9

caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag tctctggatt tactttggat tattatgcca taggctggtt ccgccaggcc 120

ccagggaagg agcgtgaggg ggtctcatgt attagtagta gtgatggtag cacatcgtat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaaaaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acagcccttt attactgtgc agcaacccct 300

gctacatact atagtggacg ttactactac caatgtcccg cggggggcat ggactactgg 360

ggcaaaggga cccaggtgac cgtgagctct 390

<210> 10

<211> 669

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of SARS-CoV-2RBD protein

<220>

<221> misc_feature

<222> (1)..(669)

<400> 10

agagtgcaac ctacagaatc aatcgtgaga tttcctaaca tcacaaacct ttgccctttc 60

ggcgaggtgt ttaacgcaac aagatttgca tcagtgtacg catggaacag aaagcgtata 120

tcaaactgcg tggcagatta ctcagtgctt tacaactcag catcattcag tacgtttaaa 180

tgctacggag tgtcacctac aaagctaaat gatctttgct ttacaaacgt gtacgcagat 240

tcatttgtga tcagaggaga tgaagtgaga caaatcgcac ctggacaaac aggaaagatt 300

gccgattaca actacaaact tcctgatgat ttcaccggct gcgtgatcgc atggaactca 360

aacaaccttg attcaaaggt aggtggtaat tataattatt tgtataggct ctttcgtaag 420

agcaacttaa agccatttga gcgagatatc tcaacagaaa tctaccaagc aggatcaaca 480

ccttgcaacg gagtggaagg atttaactgc tactttcctc ttcaatcata cggatttcaa 540

cctacaaacg gagtgggata ccaaccttac agagtggtgg tgctttcatt tgaacttctt 600

cacgcacctg caacagtgtg cggacctaag aagagcacga accttgtgaa gaataagtgc 660

gtgaacttt 669

<210> 11

<211> 858

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of SARS-CoV-2NTD protein

<220>

<221> misc_feature

<222> (1)..(858)

<400> 11

acacggaccc agctccctcc cgcctacaca aactctttca cccggggcgt gtactacccc 60

gacaaggtgt tccggtctag cgtgctccac tctacacagg acctgttcct ccctttcttc 120

agcaacgtga catggttcca cgccatccac gtgtctggca caaacggcac aaagcggttc 180

gacaaccccg tgctcccttt caacgacggc gtgtacttcg ccagcaccga gaagtctaac 240

attatccggg gctggatttt cggcaccaca ctcgactcta agacacagtc cctcctgatt 300

gtgaacaacg ccacaaacgt ggtgattaag gtgtgcgagt tccagttctg caacgaccct 360

ttcctgggcg tgtactacca caagaacaac aagtcttgga tggagtctga gttcagagtg 420

tactctagcg ccaacaactg caccttcgag tacgtgtccc agcctttcct catggacctg 480

gagggcaagc agggcaactt caagaacctg agagagttcg tgttcaagaa cattgacggc 540

tacttcaaga tttactctaa gcacacccca attaacctcg tgagggacct ccctcagggc 600

ttctccgcct tagaaccact ggtggacctc cctattggca ttaacatcac acgcttccag 660

acactgctcg ccctccaccg gtcttacctg accccaggcg actctagctc tggctggaca 720

gccggcgccg ccgcctacta cgtgggctac ctgcagccta ggaccttcct cctgaagtac 780

aacgagaacg gcacaattac cgacgccgtg gactgcgccc tggacccact gtccgagaca 840

aagtgcacac tgaagtcc 858

<210> 12

<211> 681

<212> DNA

<213> Artificial Sequence

<220>

<223> encoding nucleotide sequence of human Fc tag

<220>

<221> misc_feature

<222> (1)..(681)

<400> 12

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaactatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> CALL001 primer sequence

<220>

<221> misc_feature

<222> (1)..(23)

<400> 13

gtcctggctg ctcttctaca agg 23

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> CALL002 primer sequence

<220>

<221> misc_feature

<222> (1)..(23)

<400> 14

ggtacgtgct gttgaactgt tcc 23

<210> 15

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> VHH-BA primer sequences

<220>

<221> misc_feature

<222> (1)..(29)

<400> 15

gatgtgcagc tgcaggagtc tggrggagg 29

<210> 16

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> PMCF primer sequences

<220>

<221> misc_feature

<222> (1)..(34)

<400> 16

ctagtgcggc cgctgaggag acggtgacct gggt 34

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> MP57 primer sequence

<220>

<221> misc_feature

<222> (1)..(20)

<400> 17

ttatgcttcc ggctcgtatg 20

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> GIII primer sequences

<220>

<221> misc_feature

<222> (1)..(19)

<400> 18

ccacagacag ccctcatag 19

<210> 19

<211> 3786

<212> DNA

<213> Artificial Sequence

<220>

<223> coding nucleotide sequence of novel coronavirus SARS-CoV-2-WT-S-del18

<220>

<221> misc_feature

<222> (1)..(3786)

<400> 19

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60

agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120

aaggtgttca gaagcagcgt cctccacagc acccaagacc tgttcctgcc cttcttcagc 180

aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240

gtgctgccct tcaacgacgg cgtgtacttc gctagcaccg agaagagcaa catcatcaga 300

ggctggatct tcggcaccac cctggacagc aaaacacaga gcctgctgat cgtgaacaac 360

gccacaaacg tggtgatcaa ggtgtgcgag tttcagttct gcaacgaccc cttcctgggc 420

gtgtaccaca agaacaacaa gagctggatg gagagcgagt tccgggtgta cagcagcgcc 480

aacaactgca ccttcgagta cgtgagccaa cccttcctga tggacctgga gggcaagcaa 540

ggcaatttta agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600

tacagcaagc acacccccat caacctggtg agagacctgc cccaaggctt cagcgccctg 660

gagcccctgg tggacctgcc catcggcatc aacatcacaa gatttcagac cctgctggcc 720

ctgcacagaa gctatctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780

gcttactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840

acaatcaccg acgccgtcga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900

aagagcttca ccgtggagaa gggcatctat cagacaagca acttcagagt gcagcccacc 960

gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020

gccacaagat tcgctagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080

gactacagcg tgctgtacaa cagcgctagc ttcagcacct tcaagtgcta cggcgtcagc 1140

cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200

ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1260

aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggactcc 1320

aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380

ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggcgtg 1440

gagggcttca actgctactt ccccctgcag agctacggct ttcagcccac ctacggcgtg 1500

ggctatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgcacgc ccccgccacc 1560

gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca ccgggaccgg cgtgctgacc gagagcaaca agaagttcct gcctttccaa 1680

cagttcggca gagacatcga cgacaccacc gacgccgtca gagaccctca gaccctggag 1740

atcctggaca tcacaccctg cagcttcggc ggcgtgagcg tgatcacccc cggcaccaac 1800

acaagcaacc aagtggccgt gctgtaccaa ggcgtgaact gcaccgaggt gcccgtggcc 1860

atccacgccg atcagctgac ccccacctgg agagtgtaca gcaccggcag caacgtgttt 1920

cagacaagag ccggctgcct gatcggcgcc gagcacgtga acaacagcta cgagtgcgac 1980

atccccatcg gcgccggcat ctgcgctagc tatcagacac agaccaacag ccacagaaga 2040

gctagaagcg tggctagcca aagcatcatc gcctacacca tgagcctggg cgccgagaac 2100

agcgtggcct acagcaacaa cagcatcgcc atccccacca acttcaccat cagcgtgacc 2160

accgaaatcc tgcctgtgag catgaccaag acaagcgtgg actgcaccat gtacatctgc 2220

ggcgacagca ccgagtgcag caacctgctc ctgcagtacg gcagcttctg cattcagctg 2280

aacagagccc tgaccggcat cgccgtggag caagacaaga acacccaaga ggtgttcgcc 2340

caagtgaagc agatctacaa gacccccccc atcaaggact tcggcggctt caacttcagc 2400

caaatcctgc ctgaccctag caagcctagc aagagaagct tcatcgagga cctgctgttc 2460

aacaaggtga ccctggccga cgccggcttc atcaagcagt acggcgactg cctgggcgac 2520

atcgccgcta gagacctgat ctgcgctcag aagttcaacg gcctgaccgt gctgcccccc 2580

ctgctgaccg acgagatgat cgctcagtac acaagcgccc tgctcgctgg caccatcaca 2640

agcgggtgga ccttcggcgc cggggccgcc ctgcagatcc ccttcgccat gcagatggcc 2700

tacagattca acggcatcgg cgtgacacag aacgtgctgt acgagaatca gaagctgatc 2760

gccaatcagt tcaacagcgc catcggcaag atccaagaca gcctgagcag caccgctagc 2820

gccctgggca agctgcaaga cgtggtgaat cagaacgccc aagccctgaa caccctggtg 2880

aagcagctga gcagcaactt cggcgccatc agcagcgtgc tgaacgacat cctggctaga 2940

ctggacaagg tggaggccga ggtgcagatc gatagactga tcaccggcag actgcagagc 3000

ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctggggcaga gcaagagagt ggacttctgc 3120

ggcaagggct accacctgat gagcttccct cagagcgccc cccacggcgt ggtgttcctg 3180

cacgtgacct acgtgcccgc ccaagagaag aacttcacca ccgcccccgc catctgccac 3240

gacggcaagg cccacttccc tagagagggc gtgttcgtga gcaacggcac ccactggttc 3300

gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacaacac cttcgtgagc 3360

ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc tctgcagccc 3420

gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac aagccccgac 3480

gtggacctgg gcgacatcag cgggatcaac gctagcgtgg tgaacattca gaaggaaatc 3540

gacagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600

ggcaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt catcgccggc 3660

ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgctcctgt 3720

ctgaaggggt gctgcagctg cggcagctgc tgcaaggact acaaggacga tgacgacaag 3780

ggcccc 3786

<210> 20

<211> 3786

<212> DNA

<213> Artificial Sequence

<220>

<223> coding nucleotide sequence of novel coronavirus SARS-CoV-2-B.1.1.7-S-del18

<220>

<221> misc_feature

<222> (1)..(3786)

<400> 20

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60

agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120

aaggtgttca gaagcagcgt cctccacagc acccaagacc tgttcctgcc cttcttcagc 180

aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240

gtgctgccct tcaacgacgg cgtgtacttc gctagcaccg agaagagcaa catcatcaga 300

ggctggatct tcggcaccac cctggacagc aaaacacaga gcctgctgat cgtgaacaac 360

gccacaaacg tggtgatcaa ggtgtgcgag tttcagttct gcaacgaccc cttcctgggc 420

gtgtaccaca agaacaacaa gagctggatg gagagcgagt tccgggtgta cagcagcgcc 480

aacaactgca ccttcgagta cgtgagccaa cccttcctga tggacctgga gggcaagcaa 540

ggcaatttta agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600

tacagcaagc acacccccat caacctggtg agagacctgc cccaaggctt cagcgccctg 660

gagcccctgg tggacctgcc catcggcatc aacatcacaa gatttcagac cctgctggcc 720

ctgcacagaa gctatctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780

gcttactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840

acaatcaccg acgccgtcga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900

aagagcttca ccgtggagaa gggcatctat cagacaagca acttcagagt gcagcccacc 960

gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020

gccacaagat tcgctagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080

gactacagcg tgctgtacaa cagcgctagc ttcagcacct tcaagtgcta cggcgtcagc 1140

cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200

ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1260

aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggactcc 1320

aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380

ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggcgtg 1440

gagggcttca actgctactt ccccctgcag agctacggct ttcagcccac ctacggcgtg 1500

ggctatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgcacgc ccccgccacc 1560

gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca ccgggaccgg cgtgctgacc gagagcaaca agaagttcct gcctttccaa 1680

cagttcggca gagacatcga cgacaccacc gacgccgtca gagaccctca gaccctggag 1740

atcctggaca tcacaccctg cagcttcggc ggcgtgagcg tgatcacccc cggcaccaac 1800

acaagcaacc aagtggccgt gctgtaccaa ggcgtgaact gcaccgaggt gcccgtggcc 1860

atccacgccg atcagctgac ccccacctgg agagtgtaca gcaccggcag caacgtgttt 1920

cagacaagag ccggctgcct gatcggcgcc gagcacgtga acaacagcta cgagtgcgac 1980

atccccatcg gcgccggcat ctgcgctagc tatcagacac agaccaacag ccacagaaga 2040

gctagaagcg tggctagcca aagcatcatc gcctacacca tgagcctggg cgccgagaac 2100

agcgtggcct acagcaacaa cagcatcgcc atccccacca acttcaccat cagcgtgacc 2160

accgaaatcc tgcctgtgag catgaccaag acaagcgtgg actgcaccat gtacatctgc 2220

ggcgacagca ccgagtgcag caacctgctc ctgcagtacg gcagcttctg cattcagctg 2280

aacagagccc tgaccggcat cgccgtggag caagacaaga acacccaaga ggtgttcgcc 2340

caagtgaagc agatctacaa gacccccccc atcaaggact tcggcggctt caacttcagc 2400

caaatcctgc ctgaccctag caagcctagc aagagaagct tcatcgagga cctgctgttc 2460

aacaaggtga ccctggccga cgccggcttc atcaagcagt acggcgactg cctgggcgac 2520

atcgccgcta gagacctgat ctgcgctcag aagttcaacg gcctgaccgt gctgcccccc 2580

ctgctgaccg acgagatgat cgctcagtac acaagcgccc tgctcgctgg caccatcaca 2640

agcgggtgga ccttcggcgc cggggccgcc ctgcagatcc ccttcgccat gcagatggcc 2700

tacagattca acggcatcgg cgtgacacag aacgtgctgt acgagaatca gaagctgatc 2760

gccaatcagt tcaacagcgc catcggcaag atccaagaca gcctgagcag caccgctagc 2820

gccctgggca agctgcaaga cgtggtgaat cagaacgccc aagccctgaa caccctggtg 2880

aagcagctga gcagcaactt cggcgccatc agcagcgtgc tgaacgacat cctggctaga 2940

ctggacaagg tggaggccga ggtgcagatc gatagactga tcaccggcag actgcagagc 3000

ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctggggcaga gcaagagagt ggacttctgc 3120

ggcaagggct accacctgat gagcttccct cagagcgccc cccacggcgt ggtgttcctg 3180

cacgtgacct acgtgcccgc ccaagagaag aacttcacca ccgcccccgc catctgccac 3240

gacggcaagg cccacttccc tagagagggc gtgttcgtga gcaacggcac ccactggttc 3300

gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacaacac cttcgtgagc 3360

ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc tctgcagccc 3420

gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac aagccccgac 3480

gtggacctgg gcgacatcag cgggatcaac gctagcgtgg tgaacattca gaaggaaatc 3540

gacagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600

ggcaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt catcgccggc 3660

ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgctcctgt 3720

ctgaaggggt gctgcagctg cggcagctgc tgcaaggact acaaggacga tgacgacaag 3780

ggcccc 3786

<210> 21

<211> 3786

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding SARS-CoV-2-B.1.351-S-del18

<220>

<221> misc_feature

<222> (1)..(3786)

<400> 21

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cttcaccaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgcc 240

aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360

aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt cagagtgtac 480

tctagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctcat ggacctggag 540

ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat tgacggctac 600

ttcaagattt actctaagca caccccaatt aacctcgtga ggggcctccc tcagggcttc 660

tccgccttag aaccactggt ggacctccct attggcatta acatcacacg cttccagaca 720

ctgcacatct cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780

gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840

acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900

aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960

gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcggcga ggtgttcaac 1020

gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080

gactactccg tgctgtacaa ctccgcctct ttctctacat tcaagtgcta cggcgtgtcc 1140

cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200

ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260

aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa cctggactct 1320

aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380

ttcgagaggg acattagcac cgagatttac caggccggct ctaccccatg caacggcgtg 1440

aagggcttca actgctactt cccactgcag tcctacggct tccagcctac atacggcgtg 1500

ggctaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560

gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680

cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740

attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800

acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860

atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920

cagacccggg ccggctgcct cattggcgcc gagcacgtga acaactctta cgagtgcgac 1980

atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaactc tccacggaga 2040

gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgtggagaac 2100

tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160

accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220

ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280

aaccgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340

caggtgaagc agatttacaa gaccccccca attaaggact tcggcggctt caacttctct 2400

cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460

aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520

attgccgccc gggacctgat ttgcgcccag aagttcaacg gcctcacagt gctcccccca 2580

ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640

tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700

taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760

gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820

gccctgggca agctgcagga cgtggtgaac cagaacgccc aggccctgaa cacactggtg 2880

aagcagctgt cttctaactt cggcgccatt tctagcgtgc tgaacgacat tctgtcgcgg 2940

ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000

ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120

ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180

cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240

gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300

gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360

ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420

gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480

gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540

gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600

ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660

ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720

ctgaagggct gctgctcttg cggctcttgc tgcaaggact acaaggacga cgatgacaag 3780

ggacct 3786

<210> 22

<211> 3765

<212> DNA

<213> Artificial Sequence

<220>

<223> coding nucleotide sequence of novel coronavirus SARS-CoV-2-P.1-S-del18

<220>

<221> misc_feature

<222> (1)..(3765)

<400> 22

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgacaaac 60

agaactcaat taccctctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa ttatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagt gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaac gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgtta aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

tatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg gtgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aatatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctat taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcatttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaa 3765

<210> 23

<211> 3795

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence coding for SARS-CoV-2-B.1.617.1-S-del18

<220>

<221> misc_feature

<222> (1)..(3795)

<400> 23

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60

agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120

aaggtgttca gaagcagcgt gctgcactcc acccaagacc tgttcctgcc cttcttcagc 180

aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240

aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcta gcaccgagaa gagcaacatc 300

atcagaggct ggatcttcgg caccaccctc gatagcaaga cacagagcct gctgatcgtg 360

aataatgcta ccaacgtggt gatcaaggtg tgcgagtttc agttctgcaa cgaccccttc 420

ctggacgtgt actaccacaa gaacaacaag agctggatga agagcgagtt cagagtgtac 480

agcagcgcta acaactgcac cttcgagtac gtgagccaac ccttcctgat ggacctggag 540

ggcaagcaag gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600

ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc ccaaggcttc 660

agcgccctgg agcccctggt ggacctgccc atcggcatca acatcacaag atttcagacc 720

ctgctggccc tgcacagatc ctacctcacc cccggcgata gcagcagcgg gtggacagcc 780

ggcgctgccg cctactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840

gagaacggca ccattaccga cgctgtggac tgtgccctgg accccctgag cgagaccaag 900

tgcaccctga agagcttcac cgtggagaag ggcatctatc agacaagcaa cttcagagtg 960

cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020

gtgttcaacg ccacaagatt cgctagcgtg tacgcctgga atagaaagag aatcagcaac 1080

tgcgtggccg actacagcgt gctgtacaac agcgctagct tcagcacctt caaatgctat 1140

ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200

gtgatcagag gcgacgaggt gagacagatc gcccccgggc agaccggcaa gatcgccgat 1260

tacaactaca aactgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320

ctggacagca aggtcggcgg caactacaac tacagataca gactgttcag aaagagcaac 1380

ctgaagccct tcgagagaga catcagcacc gagatctacc aagccggcag caccccctgc 1440

aacggcgtgc aaggcttcaa ctgctacttc cccctgcaga gctacggctt tcagcccacc 1500

aacggcgtgg gctatcagcc ctacagagtg gtcgtgctga gcttcgagct gctgcacgct 1560

cccgccaccg tctgcggccc caagaagtcc accaacctcg tgaagaacaa gtgcgtgaac 1620

ttcaacttta acggcctgac cgggaccggc gtgctgaccg agagcaacaa gaagtttctg 1680

cccttccaac agttcggcag agacatcgcc gacaccaccg acgccgtgag agaccctcag 1740

accctggaga tcctggacat caccccttgt agcttcggcg gcgtgagcgt gatcaccccc 1800

ggcaccaaca caagcaacca agtggccgtg ctgtaccaag gcgtgaactg caccgaggtg 1860

cccgtggcca tccacgccga tcagctgacc cccacctgga gagtctacag caccggcagc 1920

aacgtgtttc agacaagagc cggctgcctg atcggcgccg agcacgtgaa caacagctac 1980

gagtgcgaca tccccatcgg cgccggcatc tgcgctagct atcagacaca gaccaacagc 2040

agacggagag ctagaagcgt ggctagccaa agcatcatcg cctacaccat gagcctgggc 2100

gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160

agcgtgacca ccgagatcct gcccgtcagc atgaccaaga caagcgtgga ctgcaccatg 2220

tacatctgcg gcgacagcac cgagtgcagc aacctgctcc tgcagtacgg cagcttctgc 2280

acacagctga acagagccct gaccggcatc gccgtggagc aagacaagaa cacccaagag 2340

gtgttcgccc aagtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400

aacttcagcc aaatcctccc cgatcctagc aagcctagca agagaagctt catcgaggac 2460

ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520

ctgggcgaca tcgccgctag agacctgatc tgcgctcaga agtttaacgg cctgaccgtg 2580

ctcccccccc tgctgaccga cgagatgatc gctcagtaca caagcgccct gctggccggc 2640

accatcacaa gcggctggac atttggcgcc ggcgccgctc tgcagatccc cttcgccatg 2700

cagatggcct acagattcaa cggcatcggc gtgacacaga acgtgctgta cgagaatcag 2760

aagctgatcg ccaatcagtt caacagcgcc atcggcaaga tccaagacag cctgagcagc 2820

accgctagcg ccctgggcaa gctgcaagac gtggtgaatc agaacgccca agccctgaac 2880

accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgatatc 2940

ctgagcagac tggacaaggt ggaggccgag gtgcagatcg atagactgat taccggcaga 3000

ctgcagagcc tgcagaccta cgtgacacag cagctgatca gagccgccga gatcagagct 3060

agcgccaacc tggccgccac caagatgagc gagtgcgtgc tggggcagag caagagagtg 3120

gacttctgcg gcaagggcta ccacctgatg agcttccctc agagcgcccc ccacggcgtg 3180

gtgttcctgc acgtgaccta cgtgcccgcc cacgaaaaga acttcacaac cgcccctgcc 3240

atctgccacg acggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300

gactggttcg tgacacagag aaacttctac gagcctcaga tcatcaccac cgacaacacc 3360

ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgaccct 3420

ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaatcacaca 3480

agccccgacg tggacctggg ggacatcagc ggcatcaacg ctagcgtggt gaacattcag 3540

aaggagatcg acagactgaa cgaagtggcc aagaacctga acgagagcct gatcgacctg 3600

caagagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gacaagctgc 3720

tgcagctgcc tgaagggctg ctgctcctgc ggcagctgct gcaaggacta caaggacgat 3780

gacgacaagg gcccc 3795

<210> 24

<211> 3789

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding SARS-CoV-2-b.1.617.2-S-del18

<220>

<221> misc_feature

<222> (1)..(3789)

<400> 24

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240

aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360

aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc 480

gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540

cagggcaact tcaagaacct gagagagttc gtgttcaaga acattgacgg ctacttcaag 600

atttactcta agcacacccc aattaacctc gtgagggacc tccctcaggg cttctccgtg 660

ttagaaccac tggtggacct ccctattggc attaacatca cacgcttcca gacactgctc 720

gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780

gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840

ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca 900

ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960

acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020

aacgccaccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080

gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140

tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200

agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260

tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320

tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380

ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440

gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500

gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560

acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620

ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680

cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740

gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800

aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860

gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920

ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980

gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040

agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100

aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160

accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220

tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280

ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340

gcccaggtga agcagattta caagaccccc ccaattaagg acttcggcgg cttcaacttc 2400

tctcagattc tccccgaccc atccaagcct agcaagcggt ccttcattga ggacctcctg 2460

ttcaacaagg tgacactggc cgacgccggc ttcattaagc agtacggcga ctgcctgggc 2520

gacattgccg cccgggacct gatttgcgcc cagaagttca acggcctcac agtgctcccc 2580

ccactgctca ccgacgagat gattgcccag tacacatctg ccctcctggc cggcacaatt 2640

acatctggct ggaccttcgg cgccggcgcc gccctgcaga tccctttcgc catgcagatg 2700

gcctaccgct tcaacggcat cggcgtgaca cagaacgtgc tgtacgagaa ccagaagctg 2760

atcgccaacc agttcaacag cgccattggc aagattcagg actctctgag cagcacagcc 2820

agcgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacacactg 2880

gtgaagcagc tgtcttctaa cttcggcgcc atttctagcg tgctgaacga cattctgtcg 2940

cggctggaca aggtggaggc cgaggtgcag attgacaggc tcatcacagg cagactgcag 3000

tctctgcaga catacgtgac ccagcagctg attagagccg ccgagattag agcctccgcc 3060

aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120

tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180

ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240

cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300

ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360

agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420

cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480

gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540

attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600

ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660

ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720

tgcctgaagg gctgctgctc ttgcggctct tgctgcaagg actacaagga cgacgatgac 3780

aagggacct 3789

Claims (24)

1. An alpaca source nano antibody or antigen binding fragment thereof combined with SARS-CoV-2RBD, which comprises a heavy chain variable region,

the heavy chain variable region comprises the following CDRs: CDR1 with an amino acid sequence shown as SEQ ID NO. 1, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and CDR3 with an amino acid sequence shown as SEQ ID NO. 3.

2. The nanobody of alpaca source or antigen-binding fragment thereof binding to SARS-CoV-2RBD of claim 1, wherein the variable region of the heavy chain further comprises 4 framework regions FR1-4, and the FR1-4 is staggered with the CDR1, CDR2 and CDR3 in this order.

3. The nano-antibody or the antigen-binding fragment thereof derived from alpaca that binds to SARS-CoV-2RBD according to claim 2, wherein the amino acid sequences of FR1-4 are shown in SEQ ID NOs 4, 5, 6 and 7, respectively.

4. The nano-antibody or the antigen-binding fragment thereof derived from alpaca combined with SARS-CoV-2RBD as claimed in claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 8.

5. A polynucleotide encoding the alpaca-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2RBD of any one of claims 1 to 4.

6. The polynucleotide of claim 5, wherein the polynucleotide is DNA or mRNA.

7. The polynucleotide of claim 5, wherein the polynucleotide has the nucleotide sequence set forth in SEQ ID No. 9.

8. A nucleic acid construct comprising the polynucleotide of any one of claims 5-7.

9. The nucleic acid construct of claim 8, further comprising at least one expression control element operably linked to the polynucleotide.

10. An expression vector comprising the nucleic acid construct of claim 8 or 9.

11. A transformed cell comprising the polynucleotide of any one of claims 5-7, the nucleic acid construct of claim 8 or 9, or the expression vector of claim 10.

12. A pharmaceutical composition comprising the alpaca nanobody or antigen-binding fragment thereof of any one of claims 1 to 4 that binds to SARS-CoV-2RBD, the polynucleotide of any one of claims 5 to 7, the nucleic acid construct of claim 8 or 9, the expression vector of claim 10 or the transformed cell of claim 11, and a pharmaceutically acceptable carrier and/or excipient.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation.

14. The pharmaceutical composition of claim 13, wherein the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

15. The pharmaceutical composition of claim 13, wherein the oral formulation is selected from the group consisting of tablets, powders, pills, granules, soft/hard capsules, film coatings and ointments.

16. The pharmaceutical composition of claim 15, wherein the tablet is a sublingual tablet.

17. The pharmaceutical composition of claim 15, wherein the granule is a fine granule.

18. The pharmaceutical composition of claim 15, wherein the powder is a powder.

19. The pharmaceutical composition of claim 15, wherein the pill is a pellet.

20. The pharmaceutical composition according to claim 13, wherein the parenteral formulation is a transdermal formulation, an ointment, a plaster, a topical liquid formulation or an injectable formulation.

21. The pharmaceutical composition of claim 20, wherein the injectable formulation is a bolus formulation.

22. Use of the nano-antibody derived from alpaca or the antigen-binding fragment thereof binding to SARS-CoV-2RBD according to any one of claims 1 to 4, the polynucleotide according to any one of claims 5 to 7, the nucleic acid construct according to claim 8 or 9, the expression vector according to claim 10, the transformed cell according to claim 11, or the pharmaceutical composition according to any one of claims 12 to 21 for the preparation of a medicament for the prevention, treatment and/or detection of a neocoronaviral infection.

23. The use of claim 22, wherein the new coronavirus is a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain.

24. The use of claim 23, wherein the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.

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