CN118307665A

CN118307665A - Anti-novel coronavirus broad-spectrum neutralization single-domain antibody, bivalent humanized single-domain antibody and application thereof

Info

Publication number: CN118307665A
Application number: CN202310024053.6A
Authority: CN
Inventors: 许莎莎; 李胜华; 李莹莹; 徐剑波
Original assignee: Beijing Newanbo Biotechnology Co ltd
Current assignee: Beijing Newanbo Biotechnology Co ltd
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2024-07-09

Abstract

The invention discloses a broad-spectrum neutralization single-domain antibody for resisting novel coronavirus, a bivalent humanized single-domain antibody and application thereof. The invention discloses a single domain antibody for resisting 2019-novel coronavirus broad spectrum neutralization obtained by constructing antibody gene library screening of immune alpaca, which has strong neutralizing or binding capacity to SARS, SARS-Cov2 and mainly popular variant strains of SARS-Cov2, broad spectrum neutralizing activity, strong specificity and high affinity. The invention further carries out humanization transformation on the single-domain antibody to obtain the bivalent humanized single-domain antibody through linker connection, and the bivalent humanized single-domain antibody also has broad-spectrum neutralization activity, strong specificity and high affinity. The broad-spectrum neutralizing single-domain antibody and the bivalent humanized single-domain antibody for resisting the novel coronavirus provided by the invention can be applied to preparation of reagents for detecting the novel coronavirus or medicines for treating diseases caused by the novel coronavirus.

Description

Anti-novel coronavirus broad-spectrum neutralization single-domain antibody, bivalent humanized single-domain antibody and application thereof

Technical Field

The invention relates to a single domain antibody, in particular to a broad-spectrum neutralization single domain antibody for resisting novel coronaviruses and a bivalent humanized single domain antibody constructed by the humanized single domain antibody, and further relates to application of the antibody in detecting novel coronavirus infection or preparing a medicament for treating diseases caused by the novel coronaviruses, belonging to the field of the single domain antibody for resisting the novel coronaviruses and application thereof.

Background

Transmission of 2019novel coronavirus (2019 Novel coronavirus, simply 2019-nCoV or 2019novel coronavirus or SARS-CoV 2) is predominantly through the respiratory system.

SARS-CoV2 is infected with human body through the receptor binding region (receptor binding domain, RBD) of its Spike glycoprotein (S, spike Protein, the most important surface membrane Protein) to bind with receptor-angiotensin converting enzyme II (angeotenin-converting enzyme, AEC 2) on the surface of cell membrane, the S Protein becomes allosteric, so that virus enters into cell, replicates in cell, assembles new virus, and releases virus out of cell, so that infection spreads and aggravates. The mechanism is also the action principle of vaccination epidemic prevention and therapeutic antibody, namely, the antibody for neutralizing virus is combined with RBD of virus S protein to block the combination of virus and ACE2, so that the virus can not enter into cell infection and replication, and the effects of preventing infection and treating diseases caused by the virus are achieved. However, SARS-CoV2 is continuously mutated in the world's outbreak infection over more than two years, and the most common of the new mutant strains which have been outbreak and epidemic are the ALPHA, BETA, DELTA, LAMBDA mutant strains and OMICRON which are currently in large-scale outbreak. Vaccines such as dead virus vaccines, adenovirus vaccines, mRNA vaccines, protein subunit vaccines, etc. that have been successfully marketed have been studied to date with poor protection against DELTA, LAMBDA and OMICRON. Human monoclonal antibody drugs such as Casirivimab/Imdevimab, bamlanivimab/Etesevimab and domestic An Bawei monoclonal antibody/romidepsin cocktail have been approved for the reduction of therapeutic effects on mutant DELTA, LAMBDA, and particularly for OMICRON. Small molecule chemical oral drugs such as REMDESIVIR (radevir), molnupiravir (Mo Lapi) and Proxalutamide (prochloraz) which are viral RNA replication inhibitors on the market have a certain therapeutic effect on diseases caused by new coronavirus infection, but have limited therapeutic effects on DELTA, LAMBDA and OMICRON, and the FDA has a conditional approval for the urgent use of PAXLOVID produced by new small molecule chemical oral drugs of the new coronavirus, american-dujie, and still have therapeutic effects on OMICRON mutant strains. The national drug administration (NMPA) promulgates emergency compliance at 2021, 2, 12, approving the drug to be marketed in China.

In summary, in view of the fact that SARS-CoV2 is continuously mutated in outbreak infections around the world, outbreak of new mutant strains is prevalent in large-scale infections, the effects of prevention and treatment are poor by researching on commercially available vaccines, macromolecular antibody drugs and most small molecular chemical drugs, and a new molecular biology method is utilized to research on broad-spectrum small molecular single domain antibody drugs against new coronaviruses, so that a new effective treatment means is provided for emergency prevention and treatment of diseases caused by the new coronavirus mutant strains.

The single domain antibody is an antibody research group led by Raymond Hamers professor of the university of free immunoresearch institute of belgium in the early nineties of the last century, and in the practical experimental study of students of the tutoring medical college, an antibody which naturally exists in camel blood and has complete functions and only heavy chains is found. After subsequent intensive research, only one variable region of the heavy chain antibody is called a single domain antibody (single domain antibody), which is called a nanobody (nanobody) due to its size of only 2-5 nm. The molecular weight (15 Kd) of the nano antibody (VhH) from the camel heavy chain antibody variable region is only one tenth of that of a common antibody, and the nano antibody has unique properties incomparable with common antibodies and other monovalent small molecular antibodies, such as good heat stability, high specificity, high affinity, good tissue penetrability, capability of adjusting in vivo half-life time according to the requirement of medicaments, low mass production cost and the like. The broad-spectrum neutralizing antibody for the new coronavirus is researched by utilizing a single-domain antibody technology platform, so that a new reliable medicine or product can be provided for emergently preventing and treating diseases caused by the new coronavirus mutant strain.

Disclosure of Invention

It is an object of the present invention to provide anti-2019-novel coronavirus single domain antibodies and genes encoding the same;

The second purpose of the invention is to carry out humanized transformation on the anti 2019-novel coronavirus single-domain antibody, and then connect the antibody through a linker to obtain a bivalent humanized single-domain antibody;

The third object of the present invention is to apply the single domain antibody or the bivalent humanized single domain antibody to the preparation of a reagent for detecting 2019-novel coronavirus or the preparation of a medicament for treating diseases caused by 2019-novel coronavirus.

The above object of the present invention is achieved by the following technical solutions:

The present invention first provides an anti-2019-novel coronavirus single domain antibody selected from any one of NAbkG-01 or NAbkG-03.

Wherein, the single domain antibody NAbkG-01 comprises a framework region and 3 complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the 3 complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively.

Further preferably, the amino acid sequence of the single domain antibody NAbkG-01 is selected from any one of (1) to (3):

(1) An amino acid sequence shown in SEQ ID No. 7;

(2) A protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.7, wherein the protein mutant has the same function as a protein before mutation;

(3) An amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID No.7, preferably an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No.7, more preferably an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID No. 7.

The invention further provides a coding gene of the single domain antibody NAbkG-01, wherein the nucleotide sequence of the single domain antibody NAbkG-01 coding gene is selected from any one of (1) - (3):

(1) A polynucleotide sequence shown in SEQ ID NO. 9; or (2) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions with the complement of the polynucleotide sequence shown in SEQ ID NO. 9; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 9; preferably, a polynucleotide sequence having at least 80% identity to the polynucleotide sequence set forth in SEQ ID NO. 9; further preferred is a polynucleotide sequence having at least 85% identity to the polynucleotide sequence shown in SEQ ID NO. 9; more preferably, a polynucleotide sequence having at least 95% identity to the polynucleotide sequence shown in SEQ ID NO. 9; most preferably, the polynucleotide sequence having more than 99% identity to the polynucleotide sequence shown in SEQ ID NO. 9.

The invention further provides a recombinant expression vector comprising the coding gene of the single domain antibody NAbkG-01; preferably, the recombinant expression vector is a prokaryotic cell expression vector or a eukaryotic cell expression vector.

The invention also provides a recombinant host cell comprising the recombinant expression vector described above. Preferably, the recombinant host cell is a recombinant prokaryotic expression cell, a recombinant eukaryotic expression cell, a recombinant fungal cell or a yeast recombinant parent cell, and the recombinant prokaryotic expression cell is preferably escherichia coli.

The single domain antibody NAbkG-03 comprises a framework region and 3 complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the 3 complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively.

Further preferably, the amino acid sequence of the single domain antibody NAbkG-03 is selected from any one of (1) to (3):

(1) An amino acid sequence shown in SEQ ID No. 8;

(2) A protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.8, wherein the protein mutant has the same function as a protein before mutation;

(3) An amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID No.8, preferably an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No.8, more preferably an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID No. 8.

The invention further provides a coding gene of the single domain antibody NAbkG-03, wherein the nucleotide sequence of the coding gene of the single domain antibody NAbkG-03 is selected from any one of (1) to (3):

(1) A polynucleotide sequence shown in SEQ ID NO. 10; or (2) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions with the complement of the polynucleotide sequence shown in SEQ ID NO. 10; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 10; preferably, a polynucleotide sequence having at least 80% identity to the polynucleotide sequence shown in SEQ ID NO. 10; further preferred is a polynucleotide sequence having at least 85% identity to the polynucleotide sequence shown in SEQ ID NO. 10; more preferably, a polynucleotide sequence having at least 95% identity to the polynucleotide sequence shown in SEQ ID NO. 10; most preferably, the polynucleotide sequence having more than 99% identity to the polynucleotide sequence shown in SEQ ID No. 10.

The invention further provides a recombinant expression vector comprising the coding gene of the single domain antibody NAbkG-03; preferably, the recombinant expression vector is a prokaryotic cell expression vector or a eukaryotic cell expression vector.

The invention further carries out humanized amino acid mutation on the single domain antibody NAbkG-01, NAbkG-02, NAbkG-03 or NAbkG-04 and then carries out linker connection to obtain the bivalent humanized single domain antibody, wherein the bivalent humanized single domain antibody is selected from any one of NAbkG-01-linker-NAbkG-02, NAbkG-02-linker-NAbkG-01, NAbkG-01-linker-NAbkG-03 or NAbkG-03-linker-NAbkG-01,

Wherein the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-02 is shown as SEQ ID No.11, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 15; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-02-linker-NAbkG-01 is shown as SEQ ID No.12, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 16; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-03 is shown as SEQ ID No.13, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 17; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-03-linker-NAbkG-01 is shown as SEQ ID No.14, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 18.

The anti-2019-novel coronavirus single-domain antibody obtained by screening has high activity, can be specifically combined with Delta-RBD and Omicron-RBD proteins, has broad-spectrum neutralization activity, has strong specificity and high affinity, and can be applied to preparation of reagents for detecting 2019-novel coronaviruses or medicines for preventing diseases caused by 2019-novel coronaviruses.

The bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-02, NAbkG-02-linker-NAbkG-01, NAbkG-01-linker-NAbkG-03 and NAbkG-03-linker-NAbkG-01 constructed by the invention have broad spectrum neutralization activity, strong specificity and high affinity, and can be applied to preparation of reagents for detecting 2019-novel coronaviruses or medicines for preventing diseases caused by 2019-novel coronaviruses.

As a preferred embodiment of the present invention, the present invention provides an ELISA kit for detecting 2019-novel coronavirus, comprising: primary antibody, enzyme-labeled secondary antibody, antibody diluent, washing solution, sealing solution and color development solution; wherein the primary antibody or the secondary antibody is an anti 2019-novel coronavirus broad-spectrum neutralization single-domain antibody or a bivalent humanized single-domain antibody.

As a preferred embodiment of the present invention, the present invention provides a neutralizing spray for preventing or treating 2019-novel coronavirus, comprising: the invention provides an anti 2019-novel coronavirus broad-spectrum neutralization single-domain antibody or a bivalent humanized single-domain antibody and an antibody buffer solution, wherein the antibody buffer solution comprises the following components: phosphate buffer or citric acid buffer, surfactant and protective agent, wherein the protective agent is preferably mannose or sucrose. Most preferably, the composition of the antibody buffer is: 0.01mM phosphate buffer+0.005% Tween-20 or Tween-80+3% mannose.

The single domain antibody with broad spectrum neutralization function against 2019-novel coronavirus obtained by screening has strong neutralization or binding capacity to SARS, SARS-Cov2 and mainly popular variant strains of SARS-Cov2, has broad spectrum neutralization activity, strong specificity and high affinity; the invention further carries out humanization transformation on the single-domain antibody to obtain the bivalent humanized single-domain antibody through linker connection, and the bivalent humanized single-domain antibody also has broad-spectrum neutralization activity, strong specificity and high affinity. The anti-novel coronavirus provided by the invention has a broad-spectrum neutralization single-domain antibody and a bivalent humanized single-domain antibody, and can be applied to preparation of reagents for detecting novel coronaviruses or preparation of medicines for treating diseases caused by the novel coronaviruses.

Definition of terms in connection with the present invention

The term "single domain antibody (sdAb)" as used herein refers to a fragment comprising a single variable domain in an antibody, also known as a Nanobody (Nanobody). As with intact antibodies, it can bind selectively to specific antigens. Single domain antibodies appear much smaller than the 150-160kDa mass of intact antibodies, approximately only 12-15kDa. The first single domain antibody was engineered from a heavy chain antibody of a camel and is referred to as the "VHH segment".

The term "Framework region", i.e., the Framework region, varies widely about 110 amino acid sequences near the N-terminus of the H and L chains of an immunoglobulin, and the amino acid sequences of the other parts are relatively constant, thereby distinguishing the light and heavy chains into variable (V) and constant (C) regions. The variable region comprises the hypervariable region HVR (hypervariable region) or complementarity determining region CDR (Complementarity-DETERMINING REGION) and the FR framework region.

The term "identity" of sequences as used herein is used interchangeably with "identity" and refers to the degree of similarity between sequences as determined by sequence alignment software such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. The engineered nucleotide sequence may be obtained by substitution, deletion and/or addition of one or several amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitutions, etc.), the sequences of the invention SEQ ID NO:1-198, and having substantially the same properties as those having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and which are within the scope of the present invention. Preferably, the present invention achieves sequence identity through conservative substitutions, but is not limited to conservative substitutions.

The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other after hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are complementary to each other in reverse when the sequences are all seen in the 5 'to 3' direction. It is also known in the art that two sequences which hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.

The term "amino acid sequence" refers to the order in which amino acids are linked to one another to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances on the amino acid chain, such as sugar, lipid, etc., and the present invention is not limited to the commonly used amino acids in 20.

The term "nucleotide sequence" or "polynucleotide sequence" refers to the arrangement of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, including rRNA, tRNA, mRNA. It should be understood that the presently claimed antibody genes encompass RNA (rRNA, tRNA, mRNA) and their complements in addition to DNA sequences.

The substitutions described in the present invention may be conservative substitutions, i.e. the substitution of a particular amino acid residue for one having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include those between aliphatic-containing amino acid residues (e.g., ile, val, leu or inter-substitutions between Ala), those between polar residues (e.g., between Lys and Arg, glu and Asp, gln and Asn), and the like. Mutants resulting from the deletion, substitution, insertion and/or addition of amino acids can be made by performing, for example, site-directed mutagenesis as known in the art on DNA encoding the wild-type protein (see, for example, nucleic ACID RESEARCH, vol.10, no.20, p.6487-6500, 1982, the entire disclosure of which is incorporated herein by reference).

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, the probe hybridizes to its target sequence to a greater degree of detectability (e.g., at least 2-fold over background) under stringent conditions than to other sequences. Stringent hybridization conditions are sequence dependent and will be different under different environmental conditions, longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For a detailed guidance on nucleic acid hybridization reference is made more particularly to the literature (Tijssen,Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acid assays.1993)., the stringent conditions are generally selected to be about 5-10℃below the thermal melting point (Tm) of the specific sequence at the defined ionic strength pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (50% of the probes at equilibrium are occupied at Tm because the target sequence is present in excess). Stringent conditions may be the following conditions: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30℃for short probes, including but not limited to 10 to 50 nucleotides, and at least about 60℃for long probes, including but not limited to greater than 50 nucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃; or 5 XSSC, 1% SDS, at 65℃in 0.2 XSSC and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.

In the present specification, "one or more amino acids" refers to amino acids that can be deleted, substituted, inserted, and/or added by a site-directed mutagenesis method, and is not limited, but is preferably 20 or less, 15 or less, 10 or less, or 7 or less, more preferably 5 or less. In the case of the site-directed mutagenesis method, for example, in addition to the desired mutation, i.e., the specific inconsistency, the synthesis of oligonucleotide primers complementary to the single-stranded phage DNA to be mutated can be performed as follows. That is, a strand complementary to phage is synthesized using the above synthetic oligonucleotide as a primer, and the obtained double-stranded DNA is used to transform a host cell. Cultures of transformed bacteria were plated on agar to form plaques from phage-containing single cells. Then, plaques hybridized with the probe were collected, cultured, and DNA was recovered. Furthermore, methods of deleting, substituting, inserting and/or adding one or more amino acids to the amino acid sequence of a biologically active peptide such as an enzyme while maintaining the activity thereof include methods of treating a gene with a mutagenesis source in addition to the above-mentioned site-directed mutagenesis, and methods of selectively cleaving a gene, deleting, substituting, inserting or adding a selected nucleotide, and then ligating.

The term "expression vector (Expression vectors)" refers to a vector in which expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic skeleton of a cloning vector so that a desired gene can be expressed. Expression vector four parts: a target gene, a promoter, a terminator and a marker gene. The invention includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, or other cellular expression vectors.

The terms "mutation" and "mutant" have their usual meaning herein, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, which are in the same sense as commonly known to those skilled in the art.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

Drawings

FIG. 1 shows the result of SDS-PAGE electrophoresis of the expression and purification of the screened single domain antibody of the present invention.

FIG. 2 is a graph showing ELISA assay results for specific binding activity of 7 purified single domain antibodies against Delta-RBD and Omicron-RBD proteins.

FIG. 3 is a schematic diagram of the construction of the bivalent humanized single domain antibody of the present invention.

FIG. 4 shows the results of expressing and purifying SSDS-PAGE of a bivalent humanized single domain antibody constructed according to the present invention in E.coli.

FIG. 5 is a schematic diagram of affinity determination of monovalent single domain antibodies and bivalent single domain antibodies.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

Example 1 design and construction of phage display immune alpaca Single domain antibody Gene library

(1) 2 Normal healthy alpacas (3 years old) were immunized with Spike protein extracellular domain (ECD-Spike) protein (Beijing Yi Qiaozhen biotechnology Co., ltd. And Beijing Baixin biotechnology Co., ltd.) at intervals of 1 time every 10-15 days, 4-6 times, 30-50 ml of venous whole blood was collected 1 week after 3,4,5,6 times before immunization, respectively, peripheral blood mononuclear cells were isolated, and Trizol or cell lysate (QIAGEN) was added for preservation at-80 ℃.

(2) Separating alpaca peripheral blood lymphocytes and extracting and purifying RNA: 2 alpaca whole blood was collected, alpaca peripheral blood lymphocytes were isolated, and total RNA was extracted from the obtained lymphocytes using an RNA extraction kit (QIAGEN).

(3) Obtaining alpaca heavy chain antibody variable region-VHH by a nested PCR method: in order to improve the amplification specificity, the reverse transcription primer adopts a specific primer of a heavy chain antibody to synthesize a cDNA first chain, and two sets of primers are respectively used for PCR amplification of a heavy chain antibody VHH gene fragment by using the template. The nested PCR method is adopted, the heavy chain gene fragment with the length of more than 800bp is common heavy chain gene fragment in the first PCR amplification, the heavy chain antibody gene fragment with the length of between 800 and 500bp is the missing light chain, the missing light chain heavy chain antibody gene fragment is recovered by cutting glue, and the VHH target gene (500 bp) is obtained by using VHH specific primers as templates through PCR amplification (figure 1).

Synthesis of diverse primers:

Heavy Chain Fd 5’primers:

YTL-1:GGTGGTCCTNGCTGCNCTN；

YTCh-2:GGG GTA CCT GTC ATC CAC GGA CCA GCT GA；

Heavy Chain Fd 3’primers:

YTVHH-F:GATCGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG；

YTVHH-B:

CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG；

(4) The VHH fragment and phage display vector were digested with sfII (NEB) and ligation was performed with T ₄ ligase (NEB) at appropriate ratios, followed by electrotransformation of TG1 competence.

(5) Identification and preservation of library capacity and diversity of VHH antibody gene library: the reservoir capacity was calculated from the titer measured after transformation multiplied by the total amount of transformation. Multiple electrotransformations were performed with a reservoir capacity of 1.2X10 ⁹; randomly selecting 50 clones growing on a plate for titer measurement after electrotransformation, carrying out PCR and sequencing identification, carrying out PCR on 50 colonies, and carrying out all positive PCR on the 50 colonies, wherein the size of a wide-amplified fragment is the same as that of the inserted VHH; the sequencing result of 50 clones shows that the VHH sequence is not repeated, and the library capacity and the diversity of the antibody gene library meet the design requirements.

Example 2 preparation of Receptor Binding Domain (RBD) Single Domain antibodies against novel coronavirus S proteins

(1) Screening specific single domain antibodies with broad spectrum neutralization against novel coronavirus mutant strains was performed by coating immune tubes with 2019-ConV S protein (Beijing Yi Qiaoshen Biotechnology Co., ltd.), SARS-CoV-RBD2 (wild type, alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lamda, omicron, etc. RBD proteins, proc. Natl.Acad. Microorganism Proc. Yan Jinghua professor, blocking with 4% skimmed milk PBST, adding the phage pool described above, binding for a certain period of time, washing to remove non-specifically bound phage, eluting specifically bound phage with TEA, and amplifying, and performing 2-3 rounds of screening.

As can be seen from the test results in Table 1, the titer of eluted phage for the next round of screening was increased, indicating that the single domain antibodies specific to the screening antigen were enriched, demonstrating the success of the screening test.

Table i enrichment effect of affinity screening on phage antibodies

Screening round	Titer of input phage	Titer of output phage
			1	1.0х10¹²	1.27х10⁷
2	1.2х10¹²	5.05х10⁷
			3	5.5x10¹¹	2.03х10⁸

(2) ELISA method for determining single clone culture supernatant and screening positive clone

Screening procedure one: single colonies were randomly picked from agar plates with better single colony growth and separation, inoculated in 96-well culture plates containing Amp 2YT liquid medium, cultured overnight, centrifuged, and the supernatants were separated, phage ELISA assays were performed simultaneously with each of the SARS-CoV1, SARS-CoV2 and SARS-CoV mutant RBD proteins of (1) above as antigens, clones were selected for all of these protein antigen positive wells, and DNA sequencing was performed to identify the gene sequences for their specific single domain antibody clones. The results of the monoclonal screening are shown in Table 2.

As can be seen from the results in Table 2, the clones listed showed binding positives for a variety of antigens.

TABLE 2SARS-CoV, SARS-CoV2 and important mutant RBD antigen Positive clones thereof

OD value is equal to or more than 1.0 and is +; more than or equal to 1.5 is++; more than or equal to 2.0 is++; is more than or equal to 3.0 as++ +.

Screening procedure II: screening the clone supernatant positive for various antigens simultaneously in the screening procedure I, coating a 96-well ELISA plate (Therme company) by taking human ACE2 protein (Beijing Yiqiao Shenzhou biotechnology limited company) as an antigen, and simultaneously adding culture supernatant of each clone and RBD-Delta, lambda, omicron protein of 2019-ConV for competitive binding screening, wherein the competitive binding screening is carried out according to a calculation formula: (x=od 450 value of clone well culture supernatant-blank well OD ₄₅₀ value/(RBD protein double well average OD ₄₅₀ value-blank well OD ₄₅₀ value), inhibition rate = (1-X) ×100%, clone with inhibition rate greater than 60% was selected, antibody clone was performed, expression purification was performed, and further activity function measurement was performed.

EXAMPLE 3 construction of specific Single-domain antibody expression plasmids

PCR amplification of the specific single domain antibody Gene obtained in example 2 to obtain a PCR product with restriction enzymes BbsI and BamHI sites, treatment of the PCR product and vector (pSJF vector) with restriction enzymes BbsI and BamHI, respectively (kim is. Biosic biochem.2002,66 (5): 1148-51), ligation recombination by T ₄ ligase to obtain plasmid NAbx-pSJF2 which can be efficiently expressed in E.coli, and gene sequencing to determine the sequence accuracy.

Through sequence determination, the amino acid sequences of CDR1, CDR2 and CDR3 of NAbkG-01 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively, the amino acid sequence of NAbkG-01 is shown as SEQ ID No.7, and the nucleotide sequence of the coding gene of NAbkG-01 is shown as SEQ ID No. 9.

The amino acid sequences of CDR1, CDR2 and CDR3 of NAbkG-03 are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively, the amino acid sequence of NAbkG-03 is shown as SEQ ID No.8, and the nucleotide sequence of the coding gene of NAbkG-03 is shown as SEQ ID No. 10.

EXAMPLE 4 specific Single Domain antibody expression, purification and neutralization Activity assay

1. Expression and purification of single domain antibodies

(1) The strain described in example 3, containing plasmid NAbkG-n-pSJF 2, was inoculated on LB plates containing ampicillin, at 37℃overnight. (2) Individual colonies were selected and inoculated into 4ml of LB medium containing ampicillin, and shake cultured overnight at 37 ℃. (3) Transferring the strain into 100ml 2YT culture solution containing ampicillin, performing shaking culture at 37 ℃ for 220 revolutions per minute, adding 0.1-0.5M IPTG when the OD value reaches 0.6-1.0, and continuously culturing overnight. Centrifuging at 5000 rpm for 20 min, and collecting bacteria. (4) Adding 0.05M Tris buffer solution to wash 2 times, extracting the soluble single domain antibody expressed in the periplasm of the bacterial cell by using hypertonic sucrose, and centrifuging to collect the soluble single domain antibody protein in the supernatant. (5) The single domain antibody protein with the purity of more than 90% is obtained by separating Ni ⁺ ion affinity chromatography magnetic beads (BeaverBeadsTM His-tag Protein Purification, the company of biomedical engineering, style, suzhou). The result of SDS-PAGE electrophoresis of single domain antibody proteins is shown in FIG. 1.

2. Activity assay

The activity of each purified single domain antibody was determined to specifically bind to the Delta-RBD and Omacron-RBD proteins.

Activity measurement: 96-well ELISA plates were coated with 1ug/ml Delta-RBD and Omicron-RBD protein, blocked with 2% skim milk PBST at 37℃for 1-1.5 hours, purified single domain antibodies at different dilution concentrations were added, the plates were washed 3 times at 37℃for 1 hour, 1:3000-fold diluted murine anti-myc-HRP secondary antibody (Beijing Yinqiao Shenzhou Biotechnology Co., ltd.) was added, the plates were washed 3 times at 37℃for 1 hour, TMB substrate was added, a terminator was added, OD ₄₅₀ values were determined, and the results were shown in FIG. 2.

According to the test results, the single domain antibodies NAbkG-01, NAbkG-02, NAbkG-03, NAbkG-04, NAbkG-05, NAbkG-06 and NAbkG-07 screened by the invention can be specifically combined with Delta-RBD and Omicron-RBD proteins, and have broad-spectrum neutralization activity.

EXAMPLE 5 construction of humanized amino acid mutations in Single-domain antibodies NAbkG-01, NAbkG-02, NAbkG-03, NAbkG-04

Mutations were carried out according to the method disclosed in patent document CN110964110a (invention name: anti-EGFR humanized single domain antibody, fc fusion protein, heavy chain Fab protein and uses thereof), with general mutation positions at the following key positions: 1-to Q or E, 14-to P,49, 50-to GL,87, 88-to LY, 95-to R, 123-to L; whether these positions require mutation or not and whether an amino acid at a position can be mutated or not depending on whether or not the affinity thereof for binding to an antigen is affected after mutation, expression purification, affinity assay are performed on the mutants to determine whether or not humanization is appropriate and successful.

EXAMPLE 6 construction of bivalent humanized Single-domain antibody

The bivalent single domain antibody is constructed according to the requirement of the application, and the construction strategy is shown in figure 3.

The bivalent single domain antibody constructed was as follows:

1.NAbkG-01-linker-NAbkG-02,2.NAbkG-02-linker-NAbkG-01,3.NAbkG-01-linker-NAbkG-03,4.NAbkG-03-linker-NAbkG-01,5.NabkG-02-linker-NAbkG-04,6.NAbkG-04-linker-NAbkG-02; The linker connecting the two single domain antibody genes had nucleotide sequences GGGGSGGGGS, GGSGGSGGGGSGGGGS or GGGGSGGGGSGGGGGGGGS, and the result of expressing and purifying SSDS-PAGE in E.coli is shown in FIG. 4.

Wherein the amino acid sequence of NAbkG-01-linker-NAbkG-02 is shown as SEQ ID No.11, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 15; the amino acid sequence of NAbkG-02-linker-NAbkG-01 is shown as SEQ ID No.12, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 16; the amino acid sequence of NAbkG-01-linker-NAbkG-03 is shown as SEQ ID No.13, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 17; the amino acid sequence of NAbkG-03-linker-NAbkG-01 is shown as SEQ ID No.14, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 18.

Test example 1 Single-Domain antibody Capacity to neutralize Virus infected cells test

1. Test method

VeroE6 cells, well grown, were seeded into 96 well cell plates, 2 x 10 ⁴ cells/well, incubated overnight at 37 ℃ with DMEM (Gibco) +10% heat-inactivated Fetal Bovine Serum (FBS) +1% l-glutamine and 1% penicillin/streptomycin medium. Serial dilution of single domain antibody to be tested and serial dilution with SARS-CoV-2, SARS-CoV-delta, SARS-CoV-OmicronBA2/BA2.75 virus of 100TCID ₅₀ (50% of tissue cell culture virus infection amount) are mixed homogeneously, and the mixture is added into the cell culture well at 37 deg.c for 2 hr, and the supernatant is removed, 200 ul/well culture solution is added or the supernatant is not removed, part of culture solution is added, and the mixture is cultured at 37 deg.c and 5% CO2 for 3-5 days, the cell is stained with crystal violet, OD value of 570nm/630nm is measured, and the cell hole without virus and antibody is used as blank and the cell hole without antibody with virus is used as virus control. Neutralization% = (sample well signal-virus control well signal)/(blank well control signal-virus control well signal) ×100%.

2. Test results

The virus neutralization efficiency of the single domain antibody ranges from 0.0005 to 0.65ug/ml (IC 50).

Test example 2 affinity assay for Single-Domain antibodies and bivalent humanized Single-Domain antibodies

1. Test method

1) Sample preparation

Single domain antibodies: sequentially diluted with 1 Xkinetic buffer to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM;

2) Sample testing

The antigen-human Fc fusion protein is loaded through an anti-human IgG-Fc antibody chip, appropriate antigen binding is carried out, and the affinity of fusion of all single domain antibodies is measured at 50nm, 20nm, 10nm, 1nm, 0.1nm and 0.01 nm. The principle of the test is shown in figure 5.

2. Measurement results

The measurement results are shown in Table 3.

Table 3 affinity assay results for single domain antibodies and bivalent humanized single domain antibodies

Single domain antibodies	Ka(1/Ms)	Kd(1/s)	KD(M)
				NAbkG-01	1.11E+06	1.46E-04	1.32E-10
NAbkG-03	2.87E+06	3.76E-04	1.31E-10
				Bivalent humanized single domain antibody
1.NAbkG-01-linker-NAbkG-02	1.18E+06	1.44E-05	1.22E-11
				2.NAbkG-02-linker-NAbkG-01	1.69E+06	1.46E-05	0.86E-11
3.NAbkG-01-linker-NAbkG-03	1.46E+06	1.33E-04	9.06E-11
				4.NAbkG-03-linker-NAbkG-01	1.48E+06	2.49E-05	1.68E-11

Test example 3 preparation of respiratory and skin surface Virus neutralization spray Using bivalent Single-domain antibody with broad-Spectrum neutralization

1. Test method

Preparing all buffer solutions (1) of spray, namely phosphate buffer solution, with the concentration of bivalent single-domain antibody of 2-100mg/ml, 0.001-0.1M and the pH of 5.5-8.0; (2) a citrate buffer, 0.01-0.1M, pH5.1-7.5; (3) a surfactant: 0.001-0.1% tween-20 or tween-80; (4) protective agent: 3.0-8.0% mannose or sucrose. Preferably 0.01mM phosphate buffer+0.005% Tween-20 or Tween-80+3% mannose as antibody buffer. The prepared antibody was dispensed into a spray bottle, the antibody solution was sprayed out within 5 minutes, collected with a 50ml sterile centrifuge tube, and the antibody activity was measured by the method described in experiment 4.

2. Test results

The activity of the antibody in the spray bottle is not reduced basically after the antibody is collected after being sprayed. See fig. 5 for a sample of the spray product. ELISA method Activity assay results are shown in Table 4.

Table 4 Activity detection results of neutralizing spray

Claims

1. A single domain antibody against 2019-novel coronavirus, wherein said single domain antibody is selected from any one of NAbkG-01 or NAbkG-03;

Wherein the single domain antibody NAbkG-01 comprises a framework region and 3 complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively;

The single domain antibody NAbkG-03 comprises a framework region and 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID No.4, SEQ ID No.4 and SEQ ID No.6 respectively.

2. The single domain antibody of claim 1, wherein the amino acid sequence of single domain antibody NAbkG-01 is selected from any one of (1) - (3):

(1) An amino acid sequence shown in SEQ ID No. 7;

(3) An amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID No.7, preferably an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No.7, more preferably an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID No. 7;

the amino acid sequence of the single domain antibody NAbkG-03 is selected from any one of (1) - (3):

(1) An amino acid sequence shown in SEQ ID No. 8;

3. The single domain antibody encoding gene of claim 2, wherein the single domain antibody NAbkG-01 encoding gene has a nucleotide sequence selected from any one of (1) - (3):

(1) A polynucleotide sequence shown in SEQ ID NO. 9; or (2) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions with the complement of the polynucleotide sequence shown in SEQ ID NO. 9; or (3) a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO. 9; preferably, a polynucleotide sequence having at least 80% identity to the polynucleotide sequence shown in SEQ ID NO. 9; further preferred is a polynucleotide sequence having at least 85% identity to the polynucleotide sequence shown in SEQ ID NO. 9; more preferably, a polynucleotide sequence having at least 95% identity to the polynucleotide sequence shown in SEQ ID NO. 9; most preferably, a polynucleotide sequence having more than 99% identity to the polynucleotide sequence shown in SEQ ID NO. 9;

The nucleotide sequence of the encoding gene of the single domain antibody NAbkG-03 is selected from any one of (1) to (3):

4. A recombinant expression vector comprising the coding gene of claim 3; preferably, the recombinant expression vector is a prokaryotic cell expression vector or a eukaryotic cell expression vector.

5. A recombinant host cell comprising the recombinant expression vector of claim 4.

6. The bivalent humanized single domain antibody is characterized in that the bivalent humanized single domain antibody is selected from any one of NAbkG-01-linker-NAbkG-02, NAbkG-02-linker-NAbkG-01, NAbkG-01-linker-NAbkG-03 or NAbkG-03-linker-NAbkG-01;

Wherein the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-02 is shown in SEQ ID No. 11; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-02-linker-NAbkG-01 is shown in SEQ ID No. 12; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-03 is shown as SEQ ID No. 13; the amino acid sequence of the bivalent humanized single-domain antibody NAbkG-03-linker-NAbkG-01 is shown in SEQ ID No. 14.

7. The coding gene of the bivalent humanized single domain antibody according to claim 6, wherein the nucleotide sequence of the coding gene of the bivalent humanized single domain antibody NAbkG-01-linker-NAbkG-02 is shown in SEQ ID No. 15; the nucleotide sequence of the coding gene of the bivalent humanized single-domain antibody NAbkG-02-linker-NAbkG-01 is shown as SEQ ID No. 16; the nucleotide sequence of the coding gene of the bivalent humanized single-domain antibody NAbkG-01-linker-NAbkG-03 is shown as SEQ ID No. 17; the nucleotide sequence of the coding gene of the bivalent humanized single-domain antibody NAbkG-03-linker-NAbkG-01 is shown as SEQ ID No. 18.

8. Use of the single domain antibody of claim 1 or 2, the bivalent humanized single domain antibody of claim 6 in the preparation of a reagent for detecting 2019-novel coronavirus or a medicament for treating a disease caused by 2019-novel coronavirus.

9. An ELISA detection kit to detect 2019-novel coronavirus comprising: primary antibody, enzyme-labeled secondary antibody, antibody diluent, washing solution, sealing solution and color development solution; the primary or secondary antibody is the single domain antibody of claim 1 or 2 or the bivalent humanized single domain antibody of claim 6.

10. A neutralizing spray for preventing or treating 2019-novel coronavirus comprising: the single domain antibody of claim 1 or 2 or the bivalent humanized single domain antibody of claim 6 and an antibody buffer, wherein the antibody buffer comprises: phosphate buffer solution or citric acid buffer solution, surfactant and protective agent, wherein the protective agent is mannose or sucrose; preferably, the composition of the antibody buffer is: 0.01mM phosphate buffer+0.005% Tween-20 or Tween-80+3% mannose.