Heavy chain antibodies against yersinia pestis lcrv antigen and related biological materials and uses thereof

Abstract

The application discloses a heavy chain antibody against Yersinia pestis LcrV antigen and related biological materials and application thereof, and belongs to the technical field of biotechnology. The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown in the 26th-33rd position, the 51st-58th position and the 97th-117th position of SEQ ID No. 1 in sequence. The antibody provided by the application can specifically bind to the protective antigen LcrV protein of Yersinia pestis, can neutralize Yersinia pestis, and can completely protect mice from 50 times LD 50 Pneumonic delivery challenge. The antibody provided by the application can be used for prevention and treatment of Yersinia pestis infection, and has important biological and medical significance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to heavy chain antibodies against Yersinia pestis LcrV antigen and related biomaterials and applications. Background Technology

[0002] Plague is a highly contagious and rapidly spreading infectious disease caused by Yersinia pestis (Yersinia pestis bacillus). Without timely treatment, it has an extremely high mortality rate. In my country, it is classified as a Class A infectious disease, subject to the strictest prevention and control measures. Plague is classified into three types: bubonic plague, pneumonic plague, and septicemic plague. Pneumonic plague, which can be transmitted through aerosols, is considered the most dangerous type. Although the current incidence of plague is low, its foci are widespread, making complete eradication difficult.

[0003] Currently, streptomycin is the first-line treatment for plague in clinical practice, followed by broad-spectrum antibiotics. Although antibiotics have significant therapeutic effects on plague, the course of pneumonic and septicemic plague is very rapid, and the mortality rate remains high even with timely antibiotic treatment. Furthermore, antibiotic use easily leads to drug-resistant strains, making the research of new treatment methods particularly important. Currently, there is no approved plague vaccine for human use. Current candidate subunit vaccines are formulated with two protective antigens: capsular protein F1 and low-calcium-response V (LcrV). Both antigens elicit a neutralizing antibody response, which can directly act on bacteria, thereby clearing the infection from the host. Therefore, the use of specific plague neutralizing antibodies for plague treatment is an important future direction of development. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a protective antibody against Yersinia pestis. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention claims to protect heavy chain antibodies against the Yersinia pestis LcrV antigen.

[0007] The present invention claims a heavy chain antibody against Yersinia pestis, wherein the heavy chain antibody or its antigen-binding fragment contains three complementarity-determining regions named HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are, respectively, positions 26-33 of SEQ ID No. 1, positions 51-58 of SEQ ID No. 1 and positions 97-117 of SEQ ID No. 1.

[0008] The sequence of the above complementarity determination regions is based on the Kabat definition.

[0009] Furthermore, the heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which is SEQ ID No. 1, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 1 (the inconsistency is preferably in the backbone region (FR)).

[0010] Within the scope of protection of this invention, the antibody may be in various forms such as full-length antibody, single-chain Fv fragment, Fab fragment, F(ab')2 fragment, etc.

[0011] Specifically, the heavy chain antibody is LcrVX19-R1-Fc, LcrVX19-R2-Fc, LcrVX19-R3-Fc, and LcrVX19-Fc. The amino acid sequence of LcrVX19-R1-Fc is SEQ ID NO.12, the amino acid sequence of LcrVX19-R2-Fc is SEQ ID NO.14, the amino acid sequence of LcrVX19-R3-Fc is SEQ ID NO.16, and the amino acid sequence of LcrVX19-Fc is SEQ ID No.4. Alternatively, it may have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more of identity with SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, or SEQ ID No.5 (the inconsistency is preferably in the backbone region (FR)).

[0012] Specifically, the antigen-binding fragment is a single-domain antibody. The amino acid sequence of the single-domain antibody may be SEQ ID No. 1.

[0013] The term "antigen-binding fragment" as used above refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0014] In a specific embodiment of the present invention, the heavy chain constant region of the full-length antibody is the IgG1 subtype.

[0015] Secondly, this invention claims protection for biological materials, said biological materials being any of the following:

[0016] 1) Monoclonal antibodies containing the aforementioned heavy chain antibodies;

[0017] 2) Small molecule antibodies containing the aforementioned heavy chain antibodies.

[0018] The small molecule antibody may be any of the following:

[0019] F1 and Fab antibodies;

[0020] F2 and Fv antibodies;

[0021] F3, single-chain antibody;

[0022] F4, Fab′ fragment.

[0023] The term "Fab' fragment" contains a portion of an antibody light chain and an antibody heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of two Fab' fragments to form F(ab'). 2 molecular.

[0024] The term "F(ab′)" 2 The "fragment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 fragment consists of two Fab′ fragments held together by disulfide bonds between the two heavy chains.

[0025] The term "nanobody (single-domain antibody)" (VHH) refers to a polypeptide composed of the variable region of the antibody heavy chain. Single-domain antibodies can be prepared by expressing the variable region (VH) of the antibody heavy chain using genetic engineering methods to obtain antibodies containing only the VH fragment. The ability of single-domain antibodies to bind to antigens and their stability are essentially the same as those of complete antibodies.

[0026] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0027] The term "Fab antibody" refers to a heterodimer formed by the heavy chain (Fd) and the intact light chain of an antibody linked by disulfide bonds, containing only one antigen-binding site. Fab antibodies can be prepared as follows: the coding genes for the heavy chain (Fd) and the intact light chain are linked, and a bacterial protein signal peptide gene is fused. Fab antibodies (Fab fragments) can then be secreted and expressed in *E. coli*, exhibiting complete stereofolding and intra- and inter-chain disulfide bonds. The heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0028] The term "Fv antibody" refers to a compound consisting solely of the heavy chain variable region and the light chain variable region of an antibody. The heavy chain and light chain variable regions are linked by non-covalent bonds. Fv antibodies can be prepared by: constructing vectors containing the VH and VL genes separately, co-transfecting cells to express them separately, and then assembling them into a functional Fv antibody; or by setting a stop codon between the VH and VL genes in the vector to express two small protein fragments, which are then bound together non-covalently to form an Fv antibody (Fv fragment).

[0029] The term "single-chain antibody" (ScFv) refers to a polypeptide formed by linking the heavy chain and light chain variable regions of an antibody with a short peptide. ScFv can be prepared by connecting the light and heavy chain variable region genes with an oligonucleotide linker, enabling the expression of a single polypeptide chain, known as a single-chain antibody (ScFv). The polypeptide chain spontaneously folds into its native conformation, maintaining the specificity and affinity of the Fv.

[0030] Thirdly, the present invention claims protection for a pharmaceutical composition.

[0031] The pharmaceutical compositions claimed in this invention comprise the antibodies described in the first aspect above and physiologically or pharmaceutically acceptable excipients, diluents or carriers.

[0032] In this article, the term "physiologically or pharmaceutically acceptable carriers or diluents" refers to those carriers and diluents that do not cause significant irritation to the organism and do not impair the biological activity and properties of the reagents in the pharmaceutical composition.

[0033] In this document, "physiologically or pharmaceutically acceptable excipients" refers to inert substances added to a pharmaceutical composition to further facilitate the administration of the agent. Carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred.

[0034] Fourthly, this invention claims protection for a nucleic acid molecule.

[0035] The nucleic acid molecule claimed in this invention is the nucleic acid molecule encoding the heavy chain antibody described in the first aspect above.

[0036] Furthermore, in the nucleic acid molecule, the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoding the antibody are as shown in SEQ ID No. 2, positions 76-99, 151-174 and 289-351 from the 5' end, respectively.

[0037] Furthermore, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No. 2 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 2 (the inconsistency is preferably in the backbone region (FR)).

[0038] More specifically, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain of the antibody is SEQ ID No. 3 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 3 (the inconsistency is preferably in the backbone region (FR)).

[0039] Fifthly, the present invention claims protection for expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria containing the nucleic acid molecules described in the fourth aspect above.

[0040] In a specific embodiment of the present invention, SEQ ID No. 3 (the gene encoding the antibody heavy chain) is cloned into the restriction enzyme sites Sal I and NheI of the pTSE-hFc vector to obtain a recombinant expression vector expressing the heavy chain of the antibody; the recombinant cells are recombinant cells obtained by transfecting HEK293-F cells with the above-mentioned recombinant expression vectors expressing the antibody heavy chain.

[0041] Sixthly, the present invention claims protection for any of the following applications:

[0042] (A1) The use of the nucleic acid molecules or expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria described in the fourth aspect above in the preparation of the antibodies described in the first aspect above or the pharmaceutical compositions described in the fourth aspect above;

[0043] (A2) The use of the antibody described in the first aspect above in the preparation of the pharmaceutical composition described in the fourth aspect above;

[0044] (A3) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of products for the prevention and / or treatment of diseases caused by Yersinia pestis infection;

[0045] (A4) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of a product for inhibiting Yersinia pestis infection;

[0046] (A5) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of a product for detecting Yersinia pestis;

[0047] (A6) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth or second aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of a product for neutralizing Yersinia pestis;

[0048] (A7) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of a product for detecting the LcrV antigen of Yersinia pestis;

[0049] (A8) The use of the antibody described in the first aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium described in the fifth aspect above, or the pharmaceutical composition described in the third aspect above, in the preparation of a product for binding to the LcrV antigen of Yersinia pestis.

[0050] In a specific embodiment of the present invention, the Yersinia pestis strain is Yersinia pestis strain 201.

[0051] This invention prepares a neutralizing antibody against Yersinia pestis. Experiments have shown that this antibody can specifically bind to the protective antigen LcrV protein of Yersinia pestis, neutralize the bacteria, and completely protect mice from 50 times the LD50. 50 Lung delivery attack with Yersinia pestis. The antibody provided by this invention can be used for the prevention and treatment of Yersinia pestis infection, and has important biological and medical significance. Attached Figure Description

[0052] Figure 1 The results of SDS-PAGE analysis of the purified LcrVX19-Fc antibody are shown.

[0053] Figure 2 The results of SDS-PAGE analysis of purified LcrV antigen are shown.

[0054] Figure 3 To detect the binding ability of antibody LcrVX19-Fc to LcrV.

[0055] Figure 4 To detect the neutralizing activity of antibody LcrVX19-Fc in mice.

[0056] Figure 5 To detect the binding ability of humanized heavy chain antibodies against plague to LcrV.

[0057] Figure 6 To detect the neutralizing activity of humanized heavy chain antibodies against plague in mice. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] Example 1: Discovery of Antibodies

[0061] I. Camel Immunity and Preparation of Antibody Library

[0062] One healthy camel, meeting immunization requirements and free from infectious diseases and experimental immunization history, was used. Blood was collected before immunization to obtain baseline serum. For the first immunization, 0.6 mg of recombinant Yersinia pestis LcrV protein (prepared in step one of Example 3) was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites on the camel. Two weeks later, booster immunizations were performed, with 0.6 mg of recombinant LcrV protein emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the camel. Blood was collected before each injection. A total of four booster immunizations were performed. Two weeks after the fifth immunization, 150 mL of peripheral blood was collected from the camel.

[0063] Peripheral blood lymphocytes were isolated using lymphocyte separation medium (Stemcell, 07851). RNA was extracted using an RNA extraction kit (Omega, R6834). Using the extracted total RNA as a template, cDNA was reverse transcribed using a reverse transcription kit (Invitrogen, 180180-051), with random primers provided in the kit. Then, using the reverse-transcribed cDNA as a template, primers were synthesized according to the reference (Journal of Immunological Methods, 201(1997), 35–55). The single-domain antibody heavy chain variable region gene was amplified by Susan PCR and cloned into the pADSCFV-S vector (see invention patent 201510097117.0) to construct a single-domain antibody library. The antibody library's capacity reached 2 × 10⁻⁶. 8 .

[0064] II. Screening of anti-Yersinia pestis LcrV protein single-domain antibody library

[0065] Using recombinant LcrV protein (prepared in step one of Example 3) as the antigen, the camel single-domain antibody library constructed above was screened using a solid-phase screening strategy (experimental protocol referred to "Phage Display: A Universal Laboratory Guide" edited by Clarkson, T. and Lowman, HB.; translated by Ma Lan et al., Chemical Industry Press, 2008.5) for three rounds of screening. Finally, a single-domain antibody that specifically binds to Yersinia pestis LcrV protein was obtained and named LcrVX19. The amino acid sequence of LcrVX19 is SEQ ID No. 1 (the nucleotide sequence of the corresponding coding gene is SEQ ID No. 2).

[0066] SEQ ID No. 1:

[0067] QLQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPKPEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTQVTVSS

[0068] SEQ ID No. 2:

[0069] cagctgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtcaagccactggatacatcatcgaagtcggcttcatgggctggttccgccagactccagggaaggagcgcgagggggtcgcggctattagcgctgctagtggtgcgacatactatatcgactccgtg aagggccgattcaccatctccagagacaaggccaagaacacaatgtatctgcagatggacagcccgaaacctgaggacactgccatgtactactgtgcggcagaaccaacccccctccccgaccgtaactggtggtttcttccacgggcttataatgtgtggggccaggggacccaggtcaccgtctcctca

[0070] Example 2: Preparation of Anti-Plague Heavy Chain Antibody

[0071] pTSE-hFc vector: described in the non-patent literature "Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against protective antigen V of plague bacteria [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03):266-271", this biomaterial is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0072] The selected camel single-domain antibody clone LcrVX19 was fused with human Fc and modified into a heavy chain antibody, named LcrVX19-Fc. The process is as follows:

[0073] I. Construction of Recombinant Plasmids

[0074] After adding four CGGT bases to the 5' end of the nucleotide sequence of LcrVX19 (SEQ ID No. 2), the sequence was inserted between the Sal I and Nhe I restriction sites of pTSE-hFc (which carries the human antibody Fc fragment) while keeping the other nucleotide sequences unchanged, resulting in the recombinant expression vector pTSE-hFc-LcrVX19-Fc, which expresses the heavy chain antibody LcrVX19-Fc fused with human Fc. The four CGGT bases are used to insert the sequence after adding four CGGT bases to the 5' end of the nucleotide sequence of LcrVX19 (SEQ ID No. 2) into the reading frame of the pTSE-hFc vector to complete the signal peptide.

[0075] The heavy chain antibody LcrVX19-Fc has the amino acid sequence shown in SEQ ID No. 4.

[0076] In SEQ ID No.4, amino acid residues from the N-terminus 1 to 128 form the variable region VH of the heavy chain (among which, amino acid residues from the 26th to 33rd form HCDR1, amino acid residues from the 51st to 58th form HCDR2, and amino acid residues from the 97th to 117th form HCDR3), and amino acid residues from the 122nd to 357th form the constant region and hinge region of the heavy chain.

[0077] The DNA molecule shown in SEQ ID No. 3 encodes the polypeptide (heavy chain) shown in SEQ ID No. 4. In SEQ ID No. 3, nucleotides 1-384 from the 5' end encode VH (where nucleotides 76-99 encode HCDR1, nucleotides 151-174 encode HCDR2, and nucleotides 289-351 encode HCDR3), nucleotides 364-1074 encode the heavy chain constant region and hinge region, and nucleotides 1071-1074 are stop codons.

[0078] The complementarity-determining region (CDR) sequence is defined according to Kabat. The hinge region, CH2, and CH3 of the heavy chain antibody LcrVX19-Fc are derived from IgG1.

[0079] SEQ ID No. 3

[0080]

[0081] SEQ ID No. 4

[0082] QLQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPKPEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0083] II. Antibody Preparation

[0084] 1. Antibody expression

[0085] The expression plasmid pTSE-hFc-LcrVX19-Fc constructed in step one above was transfected into FreeStyle using the transfection reagent FectoPRODNA Transfection Reagent (Polyplus, 116-001). TM HEK293-F cells (Invitrogen, R79007). Specifically, cells in good growth condition with a density of 2-3 × 10⁶ cells were selected one day before transfection. 6 FreeStyle at approximately / mL TM HEK293-F cells were centrifuged to remove the supernatant, resuspended in FreeStyle 293 medium (Gibco, 12338-018), and the cell density was adjusted to 1.0 × 10⁶ cells / year. 6 / mL, dispensed into 30mL cell suspensions / flask, and cultured at 37℃, 5% CO2, 125rpm in a cell shaker; on the day of transfection, the transfection complex was prepared: 24μL of FectoPRO transfection reagent was diluted in 3mL of FreeStyle 293 medium, gently mixed, and 24μg of heavy chain plasmid was added. After mixing, the mixture was incubated at room temperature for 10min; then the mixture was added to the prepared FreeStyle... TMIn HEK293-F cells, gently mix and return to the cell shaker for further culture. Monitor cell viability starting 48 hours after transfection. When cell viability drops to 80-85%, centrifuge at 8,000 rpm for 10 min to collect the culture supernatant for purification.

[0086] 2. Antibody purification

[0087] The collected antibody expression supernatant was filtered through a 0.45 μm filter membrane to remove impurities, and then processed using a HiTrap filter. TM Purification was performed using a MabSelectXtra pre-packed column (GE, 28-4082-58), eluted with 0.1M citrate buffer (pH 3.0), and the elution peak when UV280 > 100 was collected. Then, Hitrap was used for further purification. TM The replacement buffer for the Dedalting chromatography column was PBS (pH 7.4). A portion of the sample was used for SDS-PAGE analysis and concentration determination, and the remainder was aliquoted and frozen at -80°C for later use.

[0088] Figure 1 The image shows the SDS-PAGE results of the purified LcrVX19-Fc antibody. As can be seen from the figure, the purified LcrVX19-Fc antibody reducing sample shows a clear band with a molecular weight of approximately 40 kDa, consistent with expectations.

[0089] 3. Quantification of antibodies

[0090] The purified and replaced antibody solution was filtered through a 0.45 μm filter membrane for sterilization. The protein concentration was measured using a NanoDrop UV spectrophotometer (Thermo Scientific) and concentrated to 4 mg / mL.

[0091] Example 3: Detection of the specific binding ability of LcrVX19-Fc antibody

[0092] I. Preparation of LcrV recombinant protein

[0093] 1. Expression and purification of recombinant LcrV protein

[0094] 1.1 Synthesis of Yersinia pestis LcrV protein gene and construction of recombinant protein expression plasmid

[0095] Based on the LcrV gene sequence published by GENEBANK (ID: AE017043.1, nucleotide and amino acid sequences are as follows; the nucleotide sequence of the LcrV gene encodes the LcrV protein with the amino acid sequence shown below), Shanghai Sangon Biotech Co., Ltd. was commissioned to synthesize the whole gene. EcoRI and XhoI restriction enzyme sites were added to both ends, respectively. After the synthesized gene was digested, it was inserted into the prokaryotic expression vector pET32a (Novagen) that was digested with the same enzymes. After sequencing verification, it was named pET-LcrV.

[0096] The nucleotide sequence of the LcrV gene

[0097] atgattagagcctacgaacaaaacccacaacattttattgaggatctagaaaaagttagggtggaacaacttactggtcatggttcttcagttttagaagaattggttcagttagtcaaagataaaaatatagatatttccattaaatatgatcccagaaaagattcggaggtttttgccaatagagtaattactgatgatatcgaattgctcaagaaaatcctagcttattttctacccgaggatgccattcttaaaggcggtcattatgacaaccaactgcaaaatggcatcaagcgagtaaaagagttccttgaatcatcgccgaatacacaatgggaattgcgggcgttcatggcagtaatgcatttctctttaaccgccgatcgtatcgatgatgatattttgaaagtgattgttgattcaatgaatcatcatggtgatgcccgtagcaagttgcgtgaagaattagctgagcttaccgccg aattaaagatttattcagttattcaagccgaaattaataagcatctgtctagtagtggcaccataaatatccatgataaatccattaatctcatggataaaaatttatatggttatacagat gaagagatttttaaagccagcgcagagtacaaaattctcgagaaaatgcctcaaaccaccattcaggtggatgggagcgagaaaaaaatagtctcgataaaggactttcttggaagtgagaa taaaagaaccggggcgttgggtaatctgaaaaactcatactcttataataaagataataatgaattatctcactttgccaccacctgctcggataagtccaggccgctcaacgacttggtta gccaaaaaacaactcagctgtctgatattacatcacgttttaattcagctattgaagcactgaaccgtttcattcagaaatatgattcagtgatgcaacgtctgctagatgacacgtctggt

[0098] The amino acid sequence of LcrV protein

[0099] MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTA ELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSG

[0100] pET-LcrV is a recombinant expression vector that replaces the nucleotide sequence of the LcrV gene with the fragment between the EcoRI and XhoI restriction sites of pET32a while keeping other nucleotides unchanged. This vector expresses the LcrV protein.

[0101] 1.2 Expression and purification of recombinant LcrV protein

[0102] The recombinant plasmid pET-LcrV was transformed into E. coli BL21(DE3), and IPTG (0.4 mmol / L) was used to induce expression at 30°C. After 4 h, the cells were centrifuged at 8000 rpm for 30 min, the supernatant was discarded, and the cells were collected. The cells were resuspended in PBS buffer and mixed to an appropriate concentration (200 mL of bacterial culture was centrifuged and then resuspended in 100 mL of PB buffer). The cells were placed in an ice-water mixture and sonicated using an ultrasonic cell disruptor. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant, precipitate, uninduced bacterial culture, and induced whole cells were collected. The expression of the target protein was identified by SDS-PAGE.

[0103] Positive expression clones were cultured and induced to express by IPTG (0.4 mmol / L).

[0104] Purification of the target protein using a Ni-NTA column:

[0105] First, flush the tubing with deionized water until equilibration. During this process, connect the HisTrap™ HP (5 mL) purification column to the AKTA protein purification instrument. Then, replace the column with solution A1 (20 mM pH 7.5 phosphate buffer, 500 mM NaCl, 20 mM imidazole) to equilibrate the three column volumes. The flow rate for all three solutions is 3 mL / min. Load the supernatant containing LcrV from tubing A, wash away unbound protein with solution A1, and perform gradient elution with solution B1 (20 mM pH 7.5 phosphate buffer, 500 mM NaCl, 500 mM imidazole). Collect the elution peaks. Desalt the purified sample using HisTrap™ Desalting (5 mL): first equilibrate to pH 7.0 with deionized water, and finally equilibrate the three column volumes with PBS. The flow rate for all three solutions is 3 mL / min. Load the sample from the side tubing and collect 40 mL of sample. Analyze the elution peaks using SDS-PAGE. The results show that the purified LcrV protein has no impurities and a molecular weight of approximately 37 kDa. Figure 2 ( ), and store in portions frozen at -80℃.

[0106] II. Detection of the binding ability of LcrVX19-Fc antibody to LcrV

[0107] 1. Take LcrV recombinant protein (prepared in step 1), dilute it to 2 μg / mL with carbonate coating buffer (pH 9.6), add 100 μL to each well of the microplate (Corning, 9018), set 3 replicates for each sample, and coat overnight at 4°C;

[0108] 2. Take the above enzyme-labeled plate, wash it 6 times with PBST (PBS + 0.1% Tween 20), add PBS blocking solution containing 2% BSA, and incubate at 37°C for 2 hours;

[0109] 3. Take the above ELISA plate, discard the blocking solution, add 100 μL of LcrVX19-Fc antibody diluted 2 times in each well (maximum concentration is 10 μg / mL, 125 nmol / L), set a total of 23 gradients, and incubate at 37℃ for 90 min.

[0110] 4. Take the above enzyme-labeled plate, wash it 6 times with PBST (PBS + 0.1% Tween 20), add 100 μL of enzyme-labeled antibody-goat anti-human IgG-HRP (Zhongshan Jinqiao, ZB-2304) diluted 1:5000 to each well, and incubate at 37℃ for 45 min;

[0111] 5. Take the enzyme-labeled plate, wash it 6 times with PBST, add 100 μL of OPD substrate chromogenic solution to each well, and incubate at room temperature for 10 min;

[0112] 6. Add 100 μL of 2M sulfuric acid solution to each well to terminate the reaction;

[0113] 7. The optical density value was measured using a dual-wavelength microplate reader at 492nm / 630nm.

[0114] The results are as follows Figure 3 As shown in the figure. The horizontal axis represents the logarithm of protein molar concentration, and the vertical axis represents the optical density value. Analysis shows that the binding affinity between the LcrVX19-Fc antibody and the LcrV recombinant protein is EC50. 50 =0.01093nM.

[0115] Example 4: Detection of neutralizing activity of LcrVX19-Fc antibody in mice

[0116] The challenge dose was 1000 CFU (50 × LD50). 50 Strain 201 of Yersinia pestis (described in the article "Study on the sedimentation and decay of Yersinia pestis artificial aerosols" by Yang Wenhui et al., Military Medicine, September 2019) was obtained from the applicant in accordance with relevant national biosafety regulations. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes. Mice (female 6-8 week old Balb / C mice) were challenged by liquid aerosol lung delivery (using a lung liquid metering nebulizer, HRH-MAG4, a product of Beijing Huironghe Technology Co., Ltd.), six mice in each group. At 3, 6, 12 and 24 hours after challenge, the antibody group mice were injected via tail vein with the antibody LcrVX19-Fc obtained in Example 2, at a dose of 100 μg (i.e., 100 μg of LcrVX19-Fc obtained in Example 2). Figure 4 LcrVX19-Fc 100μg group), 200μg (i.e. Figure 4 (LcrVX19-Fc200μg group), 400μg (i.e. Figure 4 Mice in the LcrVX19-Fc 400μg group were injected with 100μL of physiological saline via the tail vein, while mice in the control group were injected with 100μL of physiological saline via the tail vein. The survival status of mice in each group was observed and recorded within 14 days after the challenge, and survival curves were plotted.

[0117] The results are as follows Figure 4 As shown, at 50×LD 50Under challenge with Yersinia pestis strain 201, all mice in the control group died by days 4-5. In the group receiving 400 μg of LcrVX19-Fc antibody at 3 and 6 hours post-challenge, mice were completely protected, with a survival rate of 100% (6 / 6). Injection of 400 μg of LcrVX19-Fc antibody at 24 hours post-challenge protected 50% of the mice, with a survival rate of 50% (3 / 6). Injection of 400 μg at 12 hours post-challenge... The LcrVX19-Fc antibody resulted in a mouse survival rate of 16.7% (1 / 6). In the 200μg LcrVX19-Fc antibody group, the survival rate was 83.3% (5 / 6) when administered 3 hours after challenge, 66.7% (4 / 6) when administered 6 and 12 hours after challenge, and 33.3% (2 / 6) when administered 24 hours after challenge. Similarly, in the 100μg LcrVX19-Fc antibody group, the survival rate was 66.7% (4 / 6) when administered 3 hours after challenge, 33.3% (2 / 6) when administered 6 and 24 hours after challenge, and 16.7% (1 / 6) when administered 12 hours after challenge. Overall, earlier administration and higher doses after challenge resulted in better protective efficacy.

[0118] Example 5: Humanization and Activity Evaluation of Anti-Plague Heavy Chain Antibody LcrVX19-Fc

[0119] I. Humanization of the anti-plague heavy chain antibody LcrVX19-Fc

[0120] The humanization of the anti-plague heavy chain antibody LcrVX19-Fc was completed by Sanyou Biopharmaceutical (Shanghai) Co., Ltd., resulting in 11 humanized sequences. Among them, LcrVX19-R1, LcrVX19-R2, and LcrVX19-R3 showed good neutralizing activity. The gene sequence of LcrVX19-R1 is SEQ ID NO.5, the amino acid sequence of LcrVX19-R1 is SEQ ID NO.6, the gene sequence of LcrVX19-R2 is SEQ ID NO.7, the amino acid sequence of LcrVX19-R2 is SEQ ID NO.8, the gene sequence of LcrVX19-R3 is SEQ ID NO.9, and the amino acid sequence of LcrVX19-R3 is SEQ ID NO.10.

[0121] SEQ ID NO.5:

[0122] Gagctgcagttggtggaatctggcggaggatctgttcaggctggcggctctctgagactgtcttgtcaggctaccggctacatcatcgaagtgggcttcatgggctggttcagacagacccctggcaaagagagagagggcgtcgccgctatctctgctgcttctggcgctacctactacatcgactccgtgaagggcagattcaccatctccagagacaaggccaagaacaccatgtacctgcagatggactcccctagagccgaggacaccgccatgtactactgtgccgctgagcctacacctctgcctgaccggaattggtggttcctgcctagagcctacaacgtgtggggccagggaacactggtcaccgtttcttct

[0123] SEQ ID NO.6:

[0124] QLQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSS

[0125] SEQ ID NO.7:

[0126] Gaggtgcagctggttgaatctggcggaggatctgttcaggctggcggctctctgagactgtcttgtcaggctaccggctacatcatcgaagtgggcttcatgggctggttcagacagacccctggcaaagagagagagggcgtcgccgctatctctgctgcttctggcgctacctactacatcgactccgtgaagggcagattcaccatctccagagacaaggccaagaacaccatgtacctgcagatggactcccctagagccgaggacaccgccatgtactactgtgccgctgagcctacacctctgcctgaccggaattggtggttcctgcctagagcctacaacgtgtggggccagggaacactggtcaccgtttcttct

[0127] SEQ ID NO.8:

[0128] EVQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSS

[0129] SEQ ID NO.9:

[0130] Gaggtgcagctggttgaatctggcggaggatctgttcaggctggcggctctctgagactgtcttgtcaggctaccggctacatcatcgaagtgggcttcatgggctggttcagacagaccgctggcaaagagagagagggcgtcgccgctatctctgctgcttctggcgctacctactacatcgactccgtgaagggcagattcaccatcagccgggacaacgccaagaacaccatgtacctgcagatggactccctgagagccgaggacaccgccatgtactactgtgccgctgagcctacacctctgcctgaccggaattggtggttcctgcctagagcctacaacgtgtggggccagggaacactggtcaccgtttcttct

[0131] SEQ ID NO.10:

[0132] EVQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDNAKNTMYLQMDSLRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSS

[0133] The nucleotide sequences of LcrVX19-R1 (SEQ ID No. 5), LcrVX19-R2 (SEQ ID No. 7), and LcrVX19-R3 (SEQ ID No. 9) were inserted between the Sal I and Nhe I restriction sites of pTSE-hFc, respectively, while keeping other nucleotide sequences unchanged. This yielded recombinant expression vectors that can express LcrVX19-R1, LcrVX19-R2, and LcrVX19-R3 humanized antibodies fused with human Fc heavy chain antibodies, namely pTSE-hFc-LcrVX19-R1-Fc, pTSE-hFc-LcrVX19-R2-Fc, and pTSE-hFc-LcrVX19-R3-Fc. After adding four CGGT bases to the 5' end of the nucleotide sequence of LcrVX19-R1 (SEQ ID No. 5), the nucleotide sequence of LcrVX19-R2 (SEQ ID No. 7), or the nucleotide sequence of LcrVX19-R3 (SEQ ID No. 9), the sequence is inserted into the pTSE-hFc vector. The four CGGT bases are used to complete the reading frame of the signal peptide on the pTSE-hFc vector.

[0134] Heavy chain antibody LcrVX19-R1-Fc, its nucleotide sequence is shown in SEQ ID No. 11, and its amino acid sequence is shown in SEQ ID No. 12. Heavy chain antibody LcrVX19-R2-Fc, its nucleotide sequence is shown in SEQ ID No. 13, and its amino acid sequence is shown in SEQ ID No. 14. Heavy chain antibody LcrVX19-R3-Fc, its nucleotide sequence is shown in SEQ ID No. 15, and its amino acid sequence is shown in SEQ ID No. 16.

[0135] In SEQ ID No. 12, SEQ ID No. 14 and SEQ ID No. 16, amino acid residues from the N-terminus 1 to 128 form the heavy chain variable region VH (among which amino acid residues from the 26th to 33rd form HCDR1, amino acid residues from the 51st to 58th form HCDR2, and amino acid residues from the 97th to 117th form HCDR3), and amino acid residues from the 122nd to 357th form the heavy chain constant region and hinge region.

[0136] The DNA molecules shown in SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15 encode the polypeptides (heavy chains) shown in SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 16, respectively. In SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15, nucleotides 1-384 from the 5' end encode VH (nucleotides 76-99 encode HCDR1, nucleotides 151-174 encode HCDR2, and nucleotides 289-351 encode HCDR3), nucleotides 364-1074 encode the constant region and hinge region of the heavy chain, and nucleotides 1071-1074 are stop codons.

[0137] The preparation of the antibody is described in Part 2 of Example 2.

[0138] SEQ ID NO.11:

[0139]

[0140] SEQ ID NO.12:

[0141] QLQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0142] SEQ ID NO.13:

[0143]

[0144] SEQ ID NO.14:

[0145] EVQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDKAKNTMYLQMDSPRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0146] SEQ ID NO.15:

[0147]

[0148] SEQ ID NO.16:

[0149] EVQLVESGGGSVQAGGSLRLSCQATGYIIEVGFMGWFRQTPGKEREGVAAISAASGATYYIDSVKGRFTISRDNAKNTMYLQMDSLRAEDTAMYYCAAEPTPLPDRNWWFLPRAYNVWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0150] II. Detection of the binding ability of humanized heavy chain antibodies against plague to LcrV

[0151] 1. Add Tween solution prepared with 1×PBS to each well of the probe fixation box to a final concentration of 0.02%. 20 and 0.2% BSA (IgG-Free, Protease-Free) Q Buffer solution, select an appropriate amount of ProA probe and put it into the fixation box for pre-soaking for 30 min.

[0152] 2. Preparation of the sample loading system: Dilute the test antibodies LcrVX19-R1-Fc, LcrVX19-R2-Fc, and LcrVX19-R3-Fc obtained in Part 1 of this example to 30 nM with Q Buffer solution, and dilute the antigen to 150 nM (i.e., Figure 5 The 150nM group), 75nM (i.e. Figure 5 The 75nM group), 37.5nM (i.e. Figure 5 The 37.5 nM group), 18.75 nM (i.e. Figure 5 The 18.75 nM group), 9.375 nM (i.e. Figure 5 (9.375 nM group).

[0153] 3. Set up the reaction program. After the Baseline, set up the program in the order of Loading-Association.

[0154] 4. Experimental Detection: Add the sample to be tested to the 96-well detection plate (200 μL / well). Place the probe holder and detection plate into the detector, and after confirming that they are properly secured, run the detection program.

[0155] 5. After the test is completed, import the data into DataAnalysis 7.0 software for processing to calculate the KD value. The experimental results are shown below. Figure 5 , Figure 5 In the figures, A, B, and C represent the humanized LcrVX19-R1-Fc, LcrVX19-R2-Fc, and LcrVX19-R3-Fc, respectively, and the binding curves show no significant differences. The KD values ​​of the antibody-antigen affinity were calculated: KD(M) = 5.61E-10 for LcrVX19-R1-Fc, 6.61E-10 for LcrVX19-R2-Fc, and 8.48E-10 for LcrVX19-R3-Fc. These values ​​are all on the same order of magnitude as the original parent antibody (6.12E-10), indicating that the humanized heavy chain antibody retains the antigen-binding properties of the parent antibody.

[0156] III. Detection of the neutralizing activity of humanized heavy chain antibodies against plague in mice

[0157] The challenge dose was 1000 CFU (50 × LD50). 50 Strain 201 of Yersinia pestis (described in the article "Yang Wenhui et al. Study on the sedimentation and decay of Yersinia pestis artificial aerosol. Military Medicine, September 2019"). According to relevant national biosafety regulations, this biological material can be obtained from the applicant and is only for repeating the relevant experiments of this invention; it cannot be used for other purposes. Mice (female 6-8 week old Balb / C mice) were challenged via liquid aerosol lung delivery (using a lung liquid metering nebulizer, HRH-MAG4, a product of Beijing Huironghe Technology Co., Ltd.), six mice per group. Three hours later, the mice in the antibody group were injected via tail vein with 400 μg of humanized heavy chain antibody LcrVX19-R1-Fc (i.e.,...). Figure 6 LcrVX19-R1-Fc and LcrVX19-R2-Fc 400μg (i.e. Figure 6 LcrVX19-R2-Fc or LcrVX19-R3-Fc 400μg (i.e. Figure 6 The mice were injected with LcrVX19-R3-Fc (100 μL) and the control group was injected with 100 μL of physiological saline via the tail vein. The survival of mice in each group was observed and recorded within 14 days after the challenge, and survival curves were plotted.

[0158] The results are as follows Figure 6 As shown, at 50×LD 50Under the challenge of Yersinia pestis strain 201, all mice in the control group died by day 5, with an average survival time of 3.83 days. All mice in the groups with 400 μg of LcrVX19-R1-Fc, LcrVX19-R2-Fc, and LcrVX19-R3-Fc antibodies survived until the end of the experiment, with a survival rate of 100% (6 / 6), which is comparable to the neutralizing activity of the parental LcrVX19-Fc before humanization.

[0159] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A heavy chain antibody against Yersinia pestis LcrV antigen, characterized in that, The heavy chain antibody contains three complementary determinant regions, HCDR1, HCDR2, and HCDR3, with the amino acid sequences of HCDR1, HCDR2, and HCDR3 being positions 26-33 of SEQ ID No. 1, positions 51-58 of SEQ ID No. 1, and positions 97-117 of SEQ ID No. 1, respectively.

2. The heavy chain antibody according to claim 1, characterized in that, The heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which has more than 75% identity with SEQ ID No.

1.

3. The heavy chain antibody according to claim 2, characterized in that, The heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which has more than 80% identity with SEQ ID No.

1.

4. The heavy chain antibody according to claim 3, characterized in that, The heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which has more than 85% identity with SEQ ID No.

1.

5. The heavy chain antibody according to claim 4, characterized in that, The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region has more than 90% identity with SEQ ID No.

1.

6. The heavy chain antibody according to claim 5, characterized in that, The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region has more than 95% identity with SEQ ID No.

1.

7. The heavy chain antibody according to claim 6, characterized in that, The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region has more than 99% identity with SEQ ID No.

1.

8. The heavy chain antibody according to claim 7, characterized in that, The heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which is SEQ ID No.

1.

9. The heavy chain antibody according to any one of claims 1-8, characterized in that, The heavy chain antibody is LcrVX19-R1-Fc, LcrVX19-R2-Fc, LcrVX19-R3-Fc, or LcrVX19-Fc, wherein the amino acid sequence of LcrVX19-R1-Fc is SEQ ID NO.12, the amino acid sequence of LcrVX19-R2-Fc is SEQ ID NO.14, the amino acid sequence of LcrVX19-R3-Fc is SEQ ID NO.16, and the amino acid sequence of LcrVX19-Fc is SEQ ID No.

4.

10. The heavy chain antibody according to any one of claims 1-8, characterized in that, The heavy chain antibody contains a heavy chain constant region, which is the IgG1 subtype.

11. An antigen-binding fragment of a heavy chain antibody against Yersinia pestis LcrV antigen, wherein the antigen-binding fragment contains HCDR1, HCDR2 and HCDR3 as described in claim 1; wherein the antigen-binding fragment is a single-domain antibody.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a heavy chain antibody as described in any one of claims 1-10 and a physiologically or pharmaceutically acceptable excipient, diluent, or carrier.

13. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain antibody as described in any one of claims 1-10.

14. An expression cassette, recombinant vector, or recombinant bacteria containing the nucleic acid molecule of claim 13.

15. Application, as shown in any of the following: (A1) The use of any of the heavy chain antibodies of claims 1-10, or the nucleic acid molecules of claim 13, or the expression cassettes, recombinant vectors, or recombinant bacteria of claim 14, or the pharmaceutical compositions of claim 12, in the preparation of a medicament for the prevention and / or treatment of diseases caused by Yersinia pestis infection; (A2) The use of any of the heavy chain antibodies of claims 1-10, or the nucleic acid molecule of claim 13, or the expression cassette, recombinant vector, or recombinant bacteria of claim 14, or the pharmaceutical composition of claim 12, in the preparation of a product for the detection of Yersinia pestis.

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