A monoclonal cell line 2D3 specifically secreting BP26 antibody, a single-chain antibody, and a preparation method thereof

Abstract

The present invention provides a monoclonal cell line 2D3 that specifically secretes BP26 antibodies, a single-chain antibody, and a preparation method, belonging to the field of veterinary biotechnology. The present invention expresses and purifies the Brucella canis BP26 protein. The spleen cells of mice immunized with the protein are fused with SP2 / 0 cells in vitro to obtain the monoclonal cell line 2D3 that specifically secretes BP26 antibodies. Subsequently, RNA of the monoclonal cell 2D3 is extracted, reverse transcribed into cDNA, and the V sequence expressing the monoclonal antibody is amplified. L and V H , and V L and V H Gene ligation was performed to obtain a single-chain antibody gene. This gene was ligated to the pCold-TF vector and transformed into BL21 competent cells for induced expression, yielding a single-chain antibody against the Brucella canis BP26 protein. The single-chain antibody against the Brucella canis BP26 protein of the present invention can bind to the BP26 protein and is of great significance for the development of new brucellosis detection reagents.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biotechnology, and in particular to a monoclonal cell line 2D3 that specifically secretes BP26 antibodies, a single-chain antibody, and a preparation method thereof. Background Art

[0002] Brucellosis, also known as brucellosis, is an important zoonotic bacterial infectious disease. Brucella canis is one of the main pathogens causing canine brucellosis and is also one of the causes of canine infertility. After sick dogs enter the kennel, they can spread the disease through the digestive tract, respiratory tract, skin and mucous membrane contact at a relatively fast rate. Most male dogs are latently infected and do not show clinical symptoms after infection. However, female dogs will experience miscarriage during pregnancy after infection. A very small number of male dogs with low immunity will show clinical symptoms such as orchitis, epididymitis, endocarditis, uveitis and intervertebral disc spondylitis. Brucella canis infection is not easy to detect, and the specific antibodies in the clinical serum will gradually decrease over time to undetectable levels, which makes clinical screening and treatment more difficult.

[0003] Canine brucellosis is primarily controlled through quarantine and culling. Compared to other Brucella species, such as bovine and melitensis, the surface lipopolysaccharide of Brucella canis lacks the O-antigen structure, resulting in a rough phenotype. O-antigen is the primary component of Brucella that induces host antibodies. The absence of this component makes conventional serological tests inapplicable to canine brucellosis infection. Therefore, immunogenic protein-based detection methods are crucial in the development of canine brucellosis detection methods.

[0004] BP26 protein is a major immunogenic protein of Brucella. Because BP26 is highly specific in the Brucella genus and not found in other bacterial genera, BP26 is recognized as a specific diagnostic marker for brucellosis. In addition, studies have shown that BP26 has strong reactivity with lipopolysaccharide, so BP26 has good application prospects in developing diagnostic reagents for brucellosis instead of lipopolysaccharide. However, according to literature reports, the use of BP26 protein as a diagnostic antigen has obvious non-specific reactions. Therefore, the key to solving this problem is to prepare monoclonal antibodies or single-chain antibodies against BP26 protein and then develop competitive detection reagents based on these specific antibodies. Among them, single-chain antibodies are small molecule engineered antibodies that are simple to prepare, easy to store, produce and transport. Single-chain antibodies developed against BP26 protein are of great value in the development of new brucellosis detection reagents. Summary of the Invention

[0005] The purpose of the present invention is to provide a monoclonal cell line 2D3 that specifically secretes BP26 antibodies, a single-chain antibody and a preparation method. The single-chain antibody prepared based on the monoclonal cell line 2D3 that specifically secretes BP26 antibodies can bind to the BP26 protein and has good reactivity with the BP26 protein.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a monoclonal cell line 2D3 that specifically secretes BP26 antibodies. The strain is deposited in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 4, 2024, and the deposit number is: CGMCC NO.46015.

[0008] Preferably, the method for constructing the monoclonal cell line that specifically secretes BP26 antibodies is:

[0009] After immunizing mice with Brucella canis BP26 protein, mouse spleen cells were taken and mixed with SP2 / 0 cells at a ratio of 10:1 for cell fusion, and the monoclonal cell line 2D3 that specifically secreted BP26 antibodies was screened.

[0010] The present invention also provides a single-chain antibody against Brucella canis BP26 protein, which is prepared based on the above-mentioned monoclonal cell line 2D3 that specifically secretes BP26 antibodies.

[0011] Preferably, the gene sequence of the single-chain antibody against Brucella canis BP26 protein is shown in SEQ ID NO.9.

[0012] Preferably, the amino acid sequence of the single-chain antibody against Brucella canis BP26 protein is shown in SEQ ID NO.10.

[0013] The present invention also provides a method for preparing a single-chain antibody against Brucella canis BP26 protein, characterized in that it comprises the following steps:

[0014] (1) RNA was extracted from the monoclonal cell line 2D3 and reverse transcribed into cDNA. The light chain variable region gene V that expressed the monoclonal antibody was amplified by PCR using degenerate primers. L and heavy chain variable region gene V H ; V L and V H The genes are connected via flexible peptide chains to obtain complete single-chain antibody genes;

[0015] (2) The single-chain antibody gene obtained in step (1) was connected to the pCold-TF vector and transformed into BL21 competent cells for induced expression to obtain a single-chain antibody against Brucella canis BP26 protein.

[0016] Preferably, in step (1), the V L The degenerate primer sequence is V shown in SEQ ID NO.1 L F and V shown in SEQ ID NO.2 L R; amplify the V H The degenerate primer sequence is V shown in SEQ ID NO.3 H F and V shown in SEQ ID NO.4 H R.

[0017] Preferably, the light chain variable region gene V L The gene sequence of the heavy chain variable region gene V H The gene sequence is shown in SEQ ID NO.6.

[0018] Preferably, the light chain variable region gene V L The amino acid sequence of the heavy chain variable region gene V H The amino acid sequence is shown in SEQ ID NO.8.

[0019] The present invention also provides an application of a single-chain antibody against Brucella canis BP26 protein in the preparation of a Brucella canis detection kit.

[0020] The single-chain antibody against Brucella canis BP26 protein of the present invention has the following advantages compared with the prior art:

[0021] (1) The present invention expresses and purifies Brucella canis BP26 protein using a prokaryotic expression system. Subsequently, mice are immunized with the protein, spleen cells are taken and fused with SP2 / 0 cells in vitro, and ELISA screening is performed to obtain a monoclonal cell line 2D3 that can specifically secrete BP26 antibodies. After the monoclonal cell line 2D3 is injected into mice, the ELISA titer of the monoclonal antibody prepared from the mouse ascites is 1:12800, and the monoclonal antibody secreted by the monoclonal cell line 2D3 has good reactivity with the BP26 protein.

[0022] (2) The present invention extracts RNA from the monoclonal cell line 2D3 that specifically secretes BP26 antibody, reverse transcribes it into cDNA, and uses degenerate primers to amplify the light chain variable region gene V of the monoclonal antibody by PCR. L and heavy chain variable region gene V H, and then V L and V H The genes were linked via flexible peptide chains to obtain a complete single-chain antibody gene. This gene was then linked to the pCold-TF vector and transformed into BL21(DE3) competent cells for induced expression, resulting in a single-chain antibody against the Brucella canis BP26 protein. This single-chain antibody against the Brucella canis BP26 protein can bind to the BP26 protein, exhibiting good reactivity with it and inhibiting the binding of monoclonal antibodies to the BP26 protein, making it of great significance for the development of novel brucellosis detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the PCR amplification of the Brucella bp26 gene and the construction of its expression vector in Example 1; wherein A is the PCR amplification of the bp26 gene; Figure B is the result of PCR identification using universal primers pCold-F and pCold-R after the bp26 gene was connected to the expression vector pCold-I; Lane M is the DL2000 Plus DNA Marker from Novozymes;

[0025] Figure 2 This is the prokaryotic expression of the BP26 protein in Example 1; wherein lane M is the DL2000 Plus DNA Marker produced by Novozymes; lane 1 is the whole bacteria after high-pressure disruption without IPTG induction; lane 2 is the whole bacteria after high-pressure disruption after IPTG induction; lane 3 is the supernatant after high-pressure disruption of IPTG-induced bacteria and centrifugation at 10,000 × g; lane 4 is the precipitate after high-pressure disruption of IPTG-induced bacteria and centrifugation at 10,000 × g;

[0026] Figure 3This is the purification of the BP26 protein in Example 1; wherein lane 1 is the supernatant after high-pressure disruption of IPTG-induced bacteria and centrifugation at 10,000×g; lanes 2-4 are the flow-through after washing the chromatography column with a wash buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH = 7.5); lanes 5-8 are the target protein eluted with eluents containing different imidazole concentrations (50 mM Tris-HCl, 500 mM NaCl, 100-300 mM imidazole, pH = 7.5), with the eluent in lane 5 containing 100 mM imidazole, the eluent in lane 6 containing 200 mM imidazole, the eluent in lane 7 containing 250 mM imidazole, and the eluent in lane 8 containing 300 mM imidazole;

[0027] Figure 4 This is the reactivity analysis of the anti-BP26 protein monoclonal antibody in Example 2; wherein lane M is the relative molecular mass of the protein; BP26 indicates that the monoclonal antibody ascites specifically binds to the BP26 protein;

[0028] Figure 5 The light chain variable region and heavy chain variable region genes of the monoclonal cell line were amplified by PCR in Example 3; wherein M represents DL2000 Plus DNA Marker from Novation; V H : Heavy chain variable region fragment; V L : light chain variable region fragment;

[0029] Figure 6 This is the T-cloning vector used in Example 3 for PCR identification of the light chain variable region and the heavy chain variable region. A represents the PCR identification of the light chain variable region after ligation to the T-cloning vector; B represents the PCR identification of the heavy chain variable region after ligation to the T-cloning vector; M represents the DL2000 Plus DNA Marker from Novozymes; lanes 1-8 represent monoclonal strains ligated with either the light chain variable region gene or the heavy chain variable region gene.

[0030] Figure 7 The single-chain antibody gene fragment is constructed by overlapping PCR fusion of the light chain variable region and the heavy chain variable region in Example 3; wherein M is the DL2000 Plus DNA Marker of Novozymes; V H V is the heavy chain variable region gene segment; L It is the light chain variable region gene fragment; scFv is the single-chain antibody gene amplification fragment.

[0031] Figure 8The following table shows the prokaryotic expression and purification of the single-chain antibody in Example 3; lane M shows the relative molecular weight of the protein; lane 1 shows the whole bacteria after high-pressure disruption induced by IPTG; lane 2 shows the whole bacteria after high-pressure disruption without IPTG induction; lane 3 shows the supernatant after high-pressure disruption of IPTG-induced bacteria and centrifugation at 10,000 × g; lane 4 shows the bacterial pellet after high-pressure disruption of IPTG-induced bacteria and centrifugation at 10,000 × g; lanes 5-8 show the target protein eluate (50 mM Tris-HCl, 500 mM NaCl, 100-300 mM imidazole, pH = 7.5), the eluate in lane 5 contains 100 mM imidazole, the eluate in lane 6 contains 200 mM imidazole, the eluate in lane 7 contains 250 mM imidazole, and the eluate in lane 8 contains 500 mM imidazole;

[0032] Figure 9 This is the immunoblot validation of the single-chain antibody binding activity to BP26 protein in Example 3; wherein M in A is the relative molecular weight of the protein, lanes 1 and 2 show the reaction between BP26 protein and the monoclonal antibody before incubation with the single-chain antibody; lanes 3 and 4 show the reaction between BP26 protein and the monoclonal antibody after incubation with the single-chain antibody; B is the grayscale value of the reaction band in Figure A scanned by Image J software, evaluating the inhibition of the reaction between the monoclonal antibody and BP26 protein by the single-chain antibody;

[0033] Figure 10 To evaluate the inhibitory effect of the single-chain antibody on the binding of the monoclonal antibody to the BP26 protein in Example 3, 30 μg of the single-chain antibody was pre-incubated and the monoclonal antibody ascites at different dilutions reacted with the BP26 protein to evaluate the inhibitory efficiency of the single-chain antibody. DETAILED DESCRIPTION

[0034] The following are detailed descriptions of the embodiments of the present invention. The embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature within the art or the product specifications are used. Reagents or instruments used without manufacturer's indication are commercially available conventional products.

[0035] The classification name of the monoclonal cell strain 2D3 that specifically secretes BP26 antibodies of the present invention is a cell strain that can secrete monoclonal antibodies against Brucella canis BP26 protein.

[0036] The sequences involved in the present invention are as follows:

[0037] V L F (SEQ ID NO.1): GAYATTSWRHTRACACARWS (Y:C / T; S:C / G; W:A / T; R:A / G; H:A / C / T; S:C / G);

[0038] V L R(SEQ ID NO.2):TCGTTTKRWYTYCMRSHTTG(K:G / T:A / G;W:A / TY:C / T:M:A / CH:A / C / TS:C / G);

[0039] V H F(SEQ ID NO.3):TAGTSMASCTSSAGTCWGG(S:C / G;M:A / C;W:A / T);

[0040] V H R(SEQ ID NO.4):TGAGGAGACKGTGACHGTGGTSCC(K:G / TH:A / C / TS:C / G);

[0041] V L Recombinant sequence (SEQ ID NO.5) of:

[0042] GACATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGACTA CACTCAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAAGATATTGCCACTTACTTTTGCCAACAGGGTAAAACGCATCCGTACACGTTCGGAGGGGGACCAAGCTGGAAATCAAACGA6

[0043] V H Recombinant sequence (SEQ ID NO.6) of:

[0044] TAGGTCAAGCTGCAGGAGTCAGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTACTAGCTACAA CCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAGGGCCTACTATGGTTACGAGGGCTATGGACTTCTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA;

[0045] V L The amino acid sequence (SEQ ID NO.7) is:

[0046] DIQMTQTTSSLSASLGDRVTISCRAS QDISN YLNWYQQKPDGT VKLL IYYTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GKTHPY TFG GGTKLEIKR;

[0047] V H The amino acid sequence (SEQ ID NO.8) is:

[0048] VKLQESGPELVKPGASMKISCKA SGYSFTGY TMNWVKQSHGKN LE WIGLINPYNGGTSYN QKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC AR RAYYGYERAMD FWGQGTTVTVSS;

[0049] The nucleotide sequence of the single-chain antibody gene (SEQ ID NO.9) is:

[0050] GACATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGACTACACTCAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAAAACGCATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGT GGTGGTGGATCC GAGGTCAAGCTGCAGGAGTCAGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAGGGCCTACTATGGTTACGAGAGGGCTATGGACTTCTGGGGTCAAGGAACCACAGTCACCGTCTCCTCATAA;

[0051] The amino acid sequence of the single-chain antibody (SEQ ID NO.10) is:

[0052] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTHPYTFGGGTKLEIKR GGGGSGGGGSGGGGSGGGGSEVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARRAYYGYERAMDFWGQGTTVTVSS;

[0053] Brucella canis (RM6 / 66) upstream primer BP26-F (SEQ ID NO.11):

[0054] 5'-CATATGGAGCTCGGTACCCTCGAGGGATCCCAGGAGAATC-3';

[0055] Brucella canis (RM6 / 66) downstream primer BP26-R (SEQ ID NO.12):

[0056] 5'-AGACTGCAGGTCGACAAGCTTTTACTTGATTTCAAAAACG-3';

[0057] pCold-I vector universal sequencing primer pCold-F (SEQ ID NO.13):

[0058] 5′-ACGCCATATCGCCGAAAGG-3′;

[0059] pCold-I vector universal sequencing primer pCold-R (SEQ ID NO.14):

[0060] 5'-GGCAGGGATCTTAGATTCTG-3';

[0061] The sequencing primer for the T cloning vector pLB-F (SEQ ID NO.15) is:

[0062] CGACTCACTATAGGGAGAGCGGC;

[0063] The sequencing primer for the T cloning vector pLB-R (SEQ ID NO.16) is:

[0064] AAGAACATCGATTTTCCATGGCAG;

[0065] Primer L2F (SEQ ID NO.17) is:

[0066] TCGGTACCCTCGAGGGATCCGACATTCAGATGACACAGAC;

[0067] L2R (SEQ ID NO. 18) is:

[0068] GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCAC CTCGTTTGATTTCCAGCTTGG;

[0069] Primer H1F (SEQ ID NO.19) is:

[0070] GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTCAAGCTGCA GGAGTC;

[0071] Primer H1R (SEQ ID NO. 20) is:

[0072] GACTGCAGGTCGACAAGCTTTTATGAGGAGACGGTGACTGTGG.

[0073] Example 1

[0074] In Example 1 of the present invention, BP26 protein was purified and extracted, and the specific method was as follows:

[0075] (1) Amplification of target fragment:

[0076] The bp26 gene sequence was amplified by PCR using the Brucella canis (RM6 / 66) genome as a template using the upstream primer BP26-F (SEQ ID NO. 11) and the downstream primer BP26-R (SEQ ID NO. 12). The PCR reaction system consisted of 50 μL of 2× PrimeSTARMax Premix (25 μL), 2 μL of each upstream and downstream primer, 2 μL of DNA template, and ddH2O to 50 μL. The PCR reaction conditions were 35 cycles of initial denaturation at 98°C for 3 min, denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. The PCR product was analyzed by electrophoresis on a 1% agarose gel, and the target fragment was recovered using an agarose gel extraction kit. The open reading frame of the Brucella canis bp26 gene is 753 bp long and encodes 250 amino acids.

[0077] The online bioinformatics software SignaIP 4.0 predicted that the 28 amino acids in the N segment of the BP26 protein were the signal peptide region. The PCR amplified the signal peptide coding region of the bp26 gene, totaling 669 bp. Figure 1 As shown in A, the amplified fragment is about 700 bp in size, and its position and size are consistent with the size of the PCR amplified fragment.

[0078] (2) Construction and identification of BP26 protein prokaryotic expression vector: The bp26 gene recovered from the gel was ligated with the linearized plasmid pCold-I double-digested with BamH I and Hind III using the homologous recombination kit of Novozymes, and the recombinant plasmid pCold I-bp26 was transformed into Escherichia coli DH5α competent cells. The cells were coated on LB solid plates containing ampicillin and cultured at 37°C for 10-16 hours. Monoclonal colonies on the LB plates were picked and inoculated into LB liquid medium containing ampicillin. The cells were shaken and cultured at 37°C for 10-16 hours. The bacterial liquid was identified by PCR using the universal sequencing primers pCold-F (SEQ ID NO.13) and pCold-R (SEQ ID NO.14) for the pCold-I vector.

[0079] The PCR reaction system (20 μL) consisted of 10 μL of 2× Taqmix, 1 μL each of upstream and downstream primers, 1 μL of bacterial culture, and ddH₂O to a total of 20 μL. The PCR reaction conditions were 35 cycles of initial denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. PCR products were analyzed by 1% agarose gel electrophoresis.

[0080] The results are as follows Figure 1 As shown in Figure B, the fragment length is approximately 800 bp, and the band size is consistent with the expected size. Positive clones were selected to extract the recombinant plasmid, which was sent to Qingke Biotechnology for sequencing. Correctly sequenced plasmids were stored at -20°C for future use.

[0081] (3) Prokaryotic expression of BP26 recombinant protein: The correctly sequenced recombinant plasmid pCold I-bp26 was transformed into Escherichia coli BL21 (DE3) competent cells, coated with LB solid plates containing ampicillin, and cultured at 37°C for 10-16 hours. A single colony was picked and inoculated into 5 mL of LB liquid medium containing ampicillin, and cultured with shaking for 6-8 hours. Subsequently, 5 mL of the bacterial solution was transferred to 1 L of LB liquid medium containing ampicillin, and cultured with shaking at 37°C and 180 rpm until the OD 600 When the value is 0.6-0.8, add IPTG to a final concentration of 1 mmol / L and induce expression at 16°C and 100 rpm for 10-15 h.

[0082] (4) SDS-PAGE electrophoresis detection of expression products: The bacterial suspension after IPTG induction was centrifuged at 8000×g for 5 min, the supernatant was discarded, and the bacterial pellet was resuspended in 40 mL PBS (pH = 7.6). The cells were disrupted by high-pressure crusher for 10 min, and centrifuged at 10000×g for 20 min. The pellet and supernatant were separated and the pellet was resuspended in 10 mL PBS (pH = 7.6). 40 μL of the bacterial suspension without IPTG induction, the bacterial suspension induced by IPTG, and the supernatant and pellet of the bacteria after high-pressure crusher were taken out respectively and placed in 1.5 mL EP tubes. 10 μL of 5× protein loading buffer and 10 μL of each were added and shaken to mix. The mixture was then incubated in a boiling water bath at 100°C for 5 min. The expression of BP26 protein was analyzed by SDS-PAGE electrophoresis.

[0083] The results are as follows Figure 2 As shown, E. coli not induced by IPTG did not express BP26 protein, while E. coli induced by IPTG successfully expressed BP26 protein; in the separated E. coli supernatant and precipitate fractions, it was found that BP26 protein was mainly expressed in the supernatant of E. coli, while only a trace amount was expressed in the precipitate.

[0084] (5) Purification of recombinant protein BP26: by Ni + The supernatant of E. coli lysis was purified by chromatography column, and the supernatant was added to the gravity Ni + In the chromatography column (filtered with a 0.22μM filter membrane before addition), control the flow rate to 5-10s / drop, pass the column in batches, then add 30 times the column volume of washing solution (50mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH7.5), control the flow rate to 5-10s / drop, and collect the flow-through liquid. Subsequently, add 5 times the volume of eluent containing different imidazole concentrations (50mM Tris-HCl, 500mMNaCl, imidazole concentrations of 100mM, 200mM, 250mM, 300mM, pH=7.5) to elute the target protein, and the eluate is subjected to SDS-PAGE electrophoresis to detect the purity of the target protein. The solution is stored at 4°C for use. The results are as follows Figure 3 The results showed that during the imidazole gradient elution process, BP26 protein was found to be eluted starting from the 200mM imidazole eluent, and the 300mM imidazole eluent had the highest BP26 protein content and higher purity. Therefore, the 300mM imidazole eluent was selected for subsequent experiments.

[0085] Example 2

[0086] Example 2 of the present invention prepared an anti-BP26 protein mouse monoclonal antibody, which specifically included the following steps:

[0087] (1) Mouse immunization: Three 6-8 week old SPF grade BALB / c female mice were immunized with recombinant BP26 protein. The immunization dose, immunization method and time are shown in Table 1. The adjuvant and diluted protein were mixed in a ratio of 1:1 and emulsified in an emulsifier for 90 seconds. The mixture was centrifuged and used.

[0088] Table 1 BP26 protein immunization procedure for mice

[0089]

[0090] (2) Determination of titer in immunized mice: After the third immunization, venous blood was collected from the tail vein of mice to separate serum. BP26 protein was diluted to 12.5 μg / mL with 50 mM carbonate buffer (pH = 9.6), coated on ELISA plates overnight at 4°C, and 100 μL was added to each well, i.e., 1.25 μg protein was coated on each well. The ELISA plates were washed 3 times with PBS, blocked with 5% skim milk PBS solution at room temperature for 2 hours, and washed 3 times with 0.05% Tween-20 PBS (PBST) solution; mouse serum diluted with PBST was added and incubated at 37°C for 45 minutes, and washed 3 times with PBST; diluted HRP-labeled goat anti-mouse secondary antibody was added, incubated at 37°C for 45 minutes, and washed 3 times with PBST; then, TMB colorimetric solution (Biyuntian) was added, and color was developed at room temperature in the dark for 10 minutes, and then 2M H2SO4 solution was added to stop color development, and the OD was measured with an enzyme reader. 450 At the same time, negative non-immunized mouse serum was established as a negative control group. The results were determined to be positive with a P / N value ≥ 2.1. As shown in Table 2, the sera from mice No. 1 and No. 3 had the highest ELISA titer of 1:12,800. Based on the P / N value, mouse No. 1 was ultimately selected as a candidate for monoclonal antibody production and subsequently underwent two subsequent surge immunizations.

[0091] Table 2 Determination of serum titer in immunized mice

[0092]

[0093] (3) Preparation of feeder layer cells: BALB / c female mice of about 8 weeks old were selected and killed by blood sampling from their eyeballs using sterilized tweezers. The mice were placed in 0.5% chlorhexidine solution for disinfection for 10-15 minutes. The mice were fixed on a sterile foam board in a clean bench. The abdominal skin was opened with sterile surgical scissors and tweezers to expose the peritoneum. 2.5 mL of RPMI1640 culture medium was aseptically drawn with a 2.5 mL syringe. The mouse peritoneum was pulled up with tweezers and the needle was tilted 45° to penetrate the mouse abdominal cavity. 2.5 mL of RPMI1640 culture medium was gently pushed in. The mouse abdomen was gently massaged 2-3 times with the handle of the tweezers. The peritoneal culture medium was then drawn out with a 2.5 mL syringe. The above operation was repeated 5 times. The peritoneal macrophages of the mice were cleaned. The 12.5 mL of RPMI1640 culture medium containing feeder cells collected 5 times was centrifuged at 400×g for 5 minutes. The supernatant was discarded, the cell pellet was collected, and 5 mL of RPMI1640 was added to wash the cells once. The centrifugation conditions were the same as described above. The precipitated mouse peritoneal macrophages were added to HAT medium (containing 15% fetal bovine serum, 100 μM hypoxanthine (H), 0.4 μM aminopterin (A), 16 μM thymidine (T), penicillin 100 IU / mL, and streptomycin 100 μg / mL) and resuspended to adjust the cell concentration to 10 6 100 μL / well was added to a 96-well cell culture plate for later use.

[0094] (4) Cell fusion: RPMI 1640 cell culture medium, sterile PBS (pH = 7.4), about 1 mL of polyethylene glycol solution (Hybri-Max, Sigma-Aldrich), and RPMI 1640 cell culture medium containing HAT were preheated in a 37°C incubator. Three days after the second shock immunization, mice were taken, blood was collected from the eyeballs, and the mice were killed by cervical dislocation. The mice were immersed in 0.5% chlorhexidine solution for disinfection for 10-15 minutes. The mice were fixed on a sterile foam board in a clean bench, the abdominal cavity was opened aseptically, the spleen was exposed, and the spleen was gently removed with sterile forceps. The spleen of the immunized mouse should be dark red and long, slightly larger than that of the negative mouse. Place the spleen in a sterile cell culture dish and wash it with 5 mL of prewarmed RPMI 1640 medium. Remove any clumps and connective tissue adhering to the spleen. Replace the sterile cell culture dish and add 3-5 mL of RPMI 1640 medium. Gently triturate the spleen tissue using the plunger of a 5 mL syringe and sterile forceps until transparent. Filter through a 200-mesh cell sieve and centrifuge at 450 × g for 5 minutes. Discard the supernatant and wash the cells twice with RPMI 1640. Count the cells for later use. To prepare SP2 / 0 cells, wash actively growing SP2 / 0 cells three times with PBS, gently dislodge the cells with RPMI 1640, and count them for later use. Mix SP2 / 0 cells and spleen cells at a ratio of 1:10 and centrifuge at 450 × g for 8 minutes. Discard the supernatant and aspirate any remaining liquid with a pipette tip. Gently tap the bottom of the centrifuge tube to loosen the pellet. Fusion was performed in a 42°C water bath. Within 30 seconds, 1 mL of fusion agent preheated at 37°C was added, while shaking. After addition, shaking was continued for 90 seconds. Then, preheated RPMI1640 medium was added to terminate the effect of the fusion agent. 1 mL of RPMI1640 was added within the first minute, 2 mL within the second minute, and 7 mL within the next 3 minutes. After standing in a 37°C incubator for 5 minutes, the mixture was centrifuged at 450×g for 5 minutes and the supernatant was discarded. The precipitate was gently suspended in HAT medium, and 100 μL / well was added to the 96-well cell culture plate containing the feeder layer cells prepared in advance in step (3). The cells were cultured in a 37°C, 5% CO2 incubator. The medium of the 96-well cell plate was replaced by half with HAT medium on the third day after fusion, and then the medium was replaced with HT medium (containing 15% fetal bovine serum, 100 μM hypoxanthine (H), 16 μM thymidine (T), penicillin 100 IU / mL, and streptomycin 100 μg / mL) every three days.

[0095] (5) Screening and subcloning of positive hybridoma cells: Hybridoma cell clusters were observed under a microscope on the 7th day after cell fusion. Around the 9th day, when the hybridoma cells grew to more than 10% of the bottom of the cell plate, 100 μL of supernatant was removed from each well for ELISA antibody detection. HT culture medium was added to the wells and culture was continued. The indirect ELISA method was the same as step (2), and the positive clone wells were screened based on the test results. Positive cell clone wells were subcloned using the limiting dilution method as follows: the cells were gently blown to uniformity, counted and appropriately diluted, 230 viable cells were suspended in 4.6 mL of complete RPMI 1640 medium (containing 12% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin), with an average of 5 cells / 100 μL, and inoculated into the first three rows of a new 96-well cell culture plate containing feeder cells, 100 μL / well, for a total of 36 wells; the remaining 1 mL was added with 4 mL of complete RPMI 1640 medium, at this time an average of 1 cell / 100 μL, and inoculated into the middle three rows of a 96-well cell culture plate, 100 μL / well, for a total of 36 wells; 1.4 mL of complete RPMI 1640 medium was added to the remaining 1.4 mL of cell suspension, and 0.5 cells / 100 μL were inoculated into the remaining 24 wells of the 96-well plate. The cell plate was placed in a constant temperature incubator at 37°C and 5% CO2. On the 7th day, the antibodies in the supernatant were detected and the positive well ratio of hybridoma cells was calculated (number of positive wells / number of hybrid cell growth wells). 450 The cell wells with higher values were subcloned again according to the above method until the ELISA positive rate of the wells with cell growth reached 100%, indicating that the cell line could stably and specifically secrete BP26 monoclonal antibody. This cell line was named 2D3 line, and the cells were frozen after expansion culture in 6-well plates.

[0096] (6) Preparation of ascites of BP26 protein monoclonal antibody and titer determination: sterilized liquid paraffin was intraperitoneally injected into 10-week-old BALB / c female mice at a rate of 500 μL / mouse for 7 days. Monoclonal cell lines were revived and 10 5 Cells were injected 7 days later, and after the mouse abdomen had expanded, ascites was collected using an 8-gauge needle. The collected ascites fluid was diluted 2-fold, starting at 1:100, and the titer of the ascites fluid was determined by ELISA. Three replicates were run in each group, with blank wells. The results are shown in Table 3. After a 12,800-fold dilution of the mouse ascites fluid, the P / N value was greater than 2.1. After a dilution of less than 12,800, the P / N value was less than 2.1. Therefore, the ELISA titer of the monoclonal antibody prepared from the ascites fluid was determined to be 1:12,800.

[0097] Table 3 2D3 monoclonal cell line ascites titer identification

[0098] potency 100 200 400 800 1600 3200 6400 12800 25600 51200 Negative Repeat 1 2.710 2.104 1.871 1.454 0.730 0.440 0.258 0.193 0.112 0.101 0.086 Repeat 2 2.600 2.011 1.873 1.453 0.702 0.411 0.241 0.185 0.125 0.094 0.073 Repeat 3 2.640 2.075 1.830 1.403 0.770 0.472 0.287 0.169 0.098 0.097 0.078 average value 2.650 2.063 1.858 1.437 0.734 0.441 0.262 0.182 0.112 0.097 0.079 P / N 33.544 26.118 23.519 18.186 9.291 5.582 3.316 2.308 1.414 1.232 -

[0099] (7) Identification of subtypes of monoclonal antibody 2D3 strain: The subtype of monoclonal antibody 2D3 strain was identified using an antibody subtype classification and identification kit (Southern Biotech) according to the kit instructions. The results are shown in Table 4. Monoclonal antibody 2D3 strain was of IgM subtype, and the light chain was classified as kappa chain.

[0100] Table 4 Identification of subtypes of monoclonal antibodies against BP26 protein

[0101]

[0102] (8) Immunoblotting analysis of the reactivity of monoclonal antibodies with BP26 protein: 50 μg of BP26 protein was added to PBS to make up to 40 μL, and then 10 μL of 5× protein loading buffer was added to mix, and the mixture was boiled in a water bath for 5 minutes. 10 μL of sample was taken for SDS-PAGE electrophoresis, and the colloid was stripped and the recombinant BP26 protein was transferred to the NC membrane. The membrane was washed with PBST 3 times, 5 minutes each time; then, 5% skim milk-PBS was added to block at room temperature for 2 hours, and the membrane was washed 3 times with PBST; 2D3 ascites was diluted 1:2000 times with PBST solution as the primary antibody, and after incubation at room temperature for 1 hour, the NC membrane was washed 3 times with PBST; then HRP-labeled goat anti-mouse antibody was diluted 1:10000 times as the secondary antibody, and incubated at room temperature for 1 hour, and the membrane was washed 3 times with PBST. Finally, ECL color development kit (Shanghai Shenger Biotechnology) was used for color development, and imaging was performed using a chemical imaging system. The results are shown in the figure. Figure 4 As shown, the ascites of the 2D3 monoclonal antibody had a strong reactivity with the BP26 protein, indicating that the monoclonal antibody 2D3 had good reactivity with the BP26 protein.

[0103] Example 3

[0104] Example 3 of the present invention prepared an anti-BP26 protein single-chain antibody, which specifically included the following steps:

[0105] (1) Monoclonal antibody heavy chain variable region (V H ) and light chain variable region (V L ) gene PCR amplification: Trizol method was used to extract total RNA from 2D3 cell lines, and cell cDNA was obtained using Takara reverse transcription kit. Using cDNA as a template, degenerate primers V H F (SEQ ID NO. 3) and V H R (SEQ ID NO.4) was PCR amplified to amplify the monoclonal antibody V H gene; using degenerate primers V L F (SEQ ID NO. 1) and V L R was used for PCR to amplify the monoclonal antibody V L Gene.

[0106] The 50 μL PCR reaction system included 25 μL of 2× PrimeSTARMax Premix, 2 μL each of upstream and downstream primers, 2 μL of cDNA template, and ddH2O to 50 μL. The PCR reaction conditions were: initial denaturation at 98°C for 3 min; 10 cycles of denaturation at 98°C for 15 sec, annealing at 45°C for 20 sec, and extension at 72°C for 30 sec; 20 cycles of denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 72°C for 30 sec; and finally, a final extension at 72°C for 5 min.

[0107] The PCR products were analyzed by 1% agarose gel electrophoresis. Figure 5 As shown, PCR amplification of V H and V L The gene had bands amplified around 300-400bp, which was consistent with the expected results.

[0108] The target gene fragment was recovered by agarose gel recovery kit and V H and V L The fragments were ligated into Tiangen's zero-background T cloning vector pLB and plated onto solid LB plates containing ampicillin. After incubation at 37°C for 10 hours, single colonies were picked and inoculated into liquid LB medium containing ampicillin. Positive bacterial clones were identified by PCR using the T cloning vector sequencing primers pLB-F (SEQ ID NO. 15) and pLB-R (SEQ ID NO. 16). The PCR reaction system consisted of 20 μL, including 10 μL of 2× Taq mix, 1 μL of upstream and downstream primers, 1 μL of bacterial suspension, and ddH2O to 20 μL. The PCR reaction conditions were 35 cycles of initial denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 40 seconds, followed by a final extension at 72°C for 10 minutes.

[0109] The results are as follows Figure 6 As shown in A, the target fragment size of clones 1-8 was about 350 bp and V L The sequence is consistent. Figure 6 As shown in B, only clones 1, 2, 5, and 6 were identified by PCR. The target fragment size was about 350 bp and V H The sequences are consistent. Figure 6 Clone No. 1-8 in A and Figure 6 The recombinant plasmids of clones 1, 2, 5, and 6 in B were sent to Qingke Biotechnology Company for sequencing. The sequences were compared with the known V sequences online using the Blast website. H With V LGene sequences, variable region sequences with greater than 90% similarity were retained, and the consistency was compared with each other, and the fragments containing lethal sequences were discarded. The final V H With V L The nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, and the amino acid sequences encoded by them are shown in SEQ ID NO.7 and SEQ ID NO.8. In the amino acid sequences, the underlined parts are V H and V L The complementarity determining regions (CDR regions) of the antibody are sequenced.

[0110] (2) Overlap PCR amplification of single-chain antibody gene: select V L The correctly sequenced plasmid was used as a template, and primers L2F (SEQ ID NO.17) and L2R (SEQ ID NO.18) were used to amplify V L Gene fragment; select V H The correctly sequenced plasmid was used as a template, and primers H1F (SEQ ID NO.19) and H1R (SEQ ID NO.20) were used to amplify V H Gene fragment; PCR reaction system 50μL, including 2× PrimeSTAR Max Premix 25μL, upstream and downstream primers 2μL each, DNA template 2μL, ddH2O to 50μL. PCR reaction conditions are 98℃ initial denaturation for 3min; 98℃ denaturation for 15sec, 55℃ annealing for 20sec, 72℃ extension for 40sec, and 72℃ final extension for 5min. Results are as follows Figure 7 As shown in A, V L and V H The gene fragment size was approximately 350 bp, consistent with the expected size.

[0111] Agarose gel recovery V L and V H Gene fragments to recover V L and V H The gene fragment was used as a template and L2F and H1R were used as primers to perform overlapping PCR amplification of the single-chain antibody V L -Linker-V H For gene fragments, the PCR reaction system was 50 μL, including 25 μL of 2× PrimeSTARMax Premix, 2 μL each of L2F and H1R primers, and V L and V H 1 μL of each gene fragment was used as template, and ddH2O was added to 50 μL. PCR reaction conditions were: 98°C initial denaturation for 3 minutes, 98°C denaturation for 15 seconds, 55°C annealing for 20 seconds, 72°C extension for 1 minute, and 72°C final extension for 5 minutes.

[0112] The results are as follows Figure 7 As shown in Figure B, the amplified single-chain antibody gene fragment is approximately 750bp in size, and the band size is consistent with expectations. The target fragment was recovered and ligated to the pCold-TF vector digested with BamH I and Hind III using the Novozymes seamless cloning kit. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells, coated with LB solid plates containing ampicillin, and incubated in a 37°C incubator for 10 hours. A single clone was picked and inoculated into LB liquid medium containing ampicillin, incubated at 37°C with shaking at 180 rpm for 10-12 hours, and the plasmid was extracted and sent to Qingke Biotechnology for sequencing. The nucleotide sequence and deduced amino acid sequence of the single-chain antibody gene are shown in SEQ ID NO.9 and SEQ ID NO.10, and the underlined sequence is the encoded linker sequence.

[0113] (3) Expression and purification of recombinant BP26 single-chain antibody: The correctly sequenced plasmid was transformed into BL21 (DE3) competent cells, coated on LB solid plates containing ampicillin, and cultured at 37°C for 10 h. Monoclonal colonies were picked and inoculated into 500 mL of LB liquid medium containing ampicillin, and cultured at 37°C at 180 rpm until the OD 600 The value was 0.6-0.8, IPTG was added to a final concentration of 1 mmol / L, induced at 16°C and 100 rpm for 15 h, centrifuged at 8000 × g for 5 min, the supernatant was discarded, the bacterial pellet was collected and resuspended with 40 mL PBS, and then crushed by high-pressure crusher for 10 min, centrifuged at 10000 × g for 20 min, and the supernatant and pellet were collected for SDS-PAGE analysis. + Affinity chromatography (same steps as in Example 1(5)) was used to obtain relatively pure single-chain antibodies.

[0114] The results are as follows Figure 8 As shown in the figure, the relative molecular weight of the single-chain antibody with TF tag should be 70-75kDa. The single-chain antibody was successfully expressed after IPTG induction. The induced single-chain antibody was expressed in both the supernatant and the precipitate, and most of it was expressed in the bacterial lysis supernatant. + Purification by affinity chromatography revealed that the scFv concentration reached its highest peak in the eluent containing 200 mM imidazole, with almost complete elution. Therefore, the scFv purified by elution with this imidazole concentration was selected for subsequent experiments.

[0115] (4) Immunoblotting to identify the binding ability of the single-chain antibody: BP26 protein was subjected to SDS-PAGE electrophoresis, and 1 μg of protein was added to each well. The steps were the same as those described in Example 2 (7). After the electrophoresis was completed, the protein gel was peeled off and the BP26 protein was transferred to the NC membrane. After the transfer was completed, the same membrane was cut into two parts, one part was incubated with 5 mL of 100 μg / mL single-chain antibody for 2 hours, and the other part was incubated with 5 mL of PBS as a control for 2 hours. After the incubation was completed, the ascites was diluted 1:1000 with PBST as the primary antibody, reacted at 37°C for 1 hour, and the membrane was washed 3 times with PBST. After that, the HRP-labeled goat anti-mouse secondary antibody was diluted 1:10000 with PBST, and the membrane was washed 3 times with PBST. The membrane was developed using an ECL color development kit (Shanghai Shenger Biotechnology) and imaged and photographed using a chemical imaging system.

[0116] The results are as follows Figure 9 As shown in A, lanes 1 and 2 are PBS incubation wells, and lanes 4 and 5 are single-chain antibody incubation wells. It was found that the brightness of the BP26 protein reaction bands in lanes 4 and 5 decreased significantly. To further evaluate the changes in the gray value of the bands, the gray value of the reaction bands was quantitatively analyzed using Image J software. The results are shown in Figure 2. Figure 9 As shown in B, after incubation with the single-chain antibody, the binding ability of the monoclonal antibody 2D3 to the BP26 protein was significantly reduced, indicating that the single-chain antibody has the activity of binding to the BP26 protein and can significantly inhibit the reaction between the monoclonal antibody and the BP26 protein.

[0117] (5) ELISA was used to evaluate the ability of the single-chain antibody to inhibit the binding of monoclonal antibody 2D3 to BP26 protein: BP26 protein was diluted to 12.5 μg / mL in 50 mM carbonate buffer (pH = 9.6), and 100 μL / well was coated on the ELISA plate at 4°C overnight; the ELISA plate was washed 3 times with PBS, 5% skim milk PBS solution was added, and the plate was blocked at room temperature for 2 h; the ELISA plate was washed 3 times with PBST, 30 μg of single-chain antibody solution or PBS solution was added, and the plate was incubated at 37°C for 1 h; the ELISA plate was washed 3 times with PBST, and the monoclonal antibody ascites solution diluted with PBST was added to the single-chain antibody incubation well and the PBS control well, respectively, and two-fold dilution was performed from 100-fold to 3200-fold, for a total of 6 gradients, and the plate was incubated at 37°C for 45 min; the ELISA plate was washed 3 times with PBST, and then TMB colorimetric solution (Biyuntian) was added for color development, 2M H2SO4 was used to stop the color development, and the OD was measured by enzyme reader. 450 According to OD 450 The specific calculation method is: inhibition rate % = OD of the single-chain antibody incubation well 450 Value / OD of PBS control well 450 The result is as follows Figure 10As shown, when the monoclonal antibody ascites solution was diluted 800 times in the case of single-chain antibody incubation, the blocking effect of 30 μg of single-chain antibody was the best, reaching 43.3%. To further verify the binding activity of the single-chain antibody to BP26 protein, the ELISA plate was washed with 5% skim milk PBS solution and PBST, and then incubated with different concentrations of single-chain antibody. The single-chain antibody was diluted with PBS and diluted twice from 30 μg per well to 0.47 μg per well. At the same time, a control well with only PBS was set up; 100 μL of 1:800 diluted monoclonal antibody 2D3 ascites solution was added to the wells pre-incubated with the single-chain antibody, and incubated at 37°C for 45 minutes; the ELISA plate was washed 3 times with PBST, and then TMB colorimetric solution (Biyuntian) was added for color development, 2M H2SO4 was used to stop the color development, and the OD was measured by microplate reader. 450 According to OD 450 The specific calculation method is: inhibition rate % = OD of the single-chain antibody incubation well 450 Value / OD of PBS control well 450 value.

[0118] As the concentration of the single-chain antibody decreased, its ability to inhibit the binding of the monoclonal antibody to the BP26 protein also decreased significantly. The inhibition rate of the single-chain antibody was concentration-dependent. This result further verified that the expressed and purified single-chain antibody of the present invention has obvious reactivity with the BP26 protein.

[0119] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monoclonal cell line 2D3 that specifically secretes BP26 antibodies was deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 4, 2024, and the deposit number is CGMCC NO.46015.

2. A single-chain antibody against Brucella canis BP26 protein, characterized in that: The single-chain antibody against Brucella canis BP26 protein is prepared based on the monoclonal cell line 2D3 that specifically secretes BP26 antibodies according to claim 1.

3. The single-chain antibody against Brucella canis BP26 protein according to claim 2, characterized in that The gene sequence of the single-chain antibody against Brucella canis BP26 protein is shown in SEQ ID NO.9; the amino acid sequence of the single-chain antibody against Brucella canis BP26 protein is shown in SEQ ID NO.

10.

4. A method for preparing a single-chain antibody against Brucella canis BP26 protein according to any one of claims 2 to 3, characterized in that: The steps include: (1) RNA was extracted from the monoclonal cell line 2D3 and reverse transcribed into cDNA. The light chain variable region gene V that expressed the monoclonal antibody was amplified by PCR using degenerate primers. L and heavy chain variable region gene V H ; V L and V H The genes are connected via flexible peptide chains to obtain complete single-chain antibody genes; (2) The single-chain antibody gene obtained in step (1) was connected to the pCold-TF vector and transformed into BL21 competent cells for induced expression to obtain a single-chain antibody against Brucella canis BP26 protein.

5. Use of the single-chain antibody against Brucella canis BP26 protein according to any one of claims 2 to 3 in the preparation of a Brucella canis detection kit.

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