Porcine monoclonal antibody against SzM protein of Streptococcus zooepidemicus

Abstract

The present invention discloses a porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus and its application. Using genetic recombination technology, a murine monoclonal antibody with excellent efficacy in preventing or treating S. zooepidemicus infection was porcine-derived. The resulting chimeric antibody replaces the constant region of the murine antibody with that of the porcine antibody, thus avoiding the common inability of murine antibodies to effectively activate complement and Fc receptor-related immune effects in pigs. This antibody is highly effective in treating S. zooepidemicus infection in pigs, providing a new and effective biological product for the clinical prevention and treatment of S. zooepidemicus infection in pigs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a porcine modified chimeric antibody against the SzM protein of Streptococcus zooepidemicus and its application. Background Art

[0002] Streptococcus equi ssp. Zooepidemicus (SEZ), a member of the Lambda group C Streptococcus, is one of the main pathogens of streptococcal disease in pigs in my country. Infection with SEZ can cause clinical symptoms in pigs, including sepsis, arthritis, and meningitis, and in severe cases, acute death in the host. In recent years, with the restricted use of antibiotics, SEZ has become an epidemic risk in my country and even worldwide. In 2019-2020, North American pig herds experienced their first outbreak of streptococcal disease caused by SEZ infection, resulting in the acute death of thousands of pigs within a week. The main isolates were OH-71905 and TN-74097.

[0003] The SzM protein is a key virulence factor on the surface of Streptococcus zoster (SEZ) bacteria and is closely associated with the pathogenicity of SEZ. The C-terminus of the SzM protein is anchored to the cell wall, while the N-terminus is free. The amino acid sequence of the C-terminal region is highly conserved, while the N-terminal region is highly variable. SEZ strains can be categorized based on differences in the N-terminal variable region. Although the SzM protein homology among SEZ strains is generally low (ranging from 19.66% to 54.66%) due to differences in the variable region, this SzM-type SEZ strain is the predominant cause of streptococcal disease in swine, and its SzM sequence characteristics can be used to distinguish this highly virulent strain prevalent in swine populations from SEZ strains prevalent in other animals. Furthermore, studies have shown that immunization of mice with SzM protein can induce the production of opsonic antibodies and help mice resist challenge with a lethal dose of SEZ, indicating that this protein is a good vaccine candidate.

[0004] Currently, the mainstays of prevention and treatment for Streptococcus suis caused by SEZ are antibiotics and vaccines. However, in recent years, due to the increased bacterial resistance associated with overuse of antibiotics, my country's livestock industry has begun restricting their use. Furthermore, vaccines require time for the animal's body to produce antibodies to combat the pathogen after vaccination. SEZ infection can occur before the immunization process is complete, or even without immunization. Therefore, the development of alternative strategies to prevent and treat SEZ infection is urgently needed. Monoclonal antibodies offer high specificity, rapid onset of action, and immediate passive immune protection upon injection, making them highly valuable for both prevention and treatment. However, a lack of monoclonal antibody-based biological products is currently available to combat SEZ infection.

[0005] Therefore, the development of anti-SEZ porcine monoclonal antibodies that are more suitable for the porcine immune system has important scientific and application value. Summary of the Invention

[0006] One aspect of the present invention is to provide a porcine chimeric antibody that can be used to prevent or treat Streptococcus zooepidemicus infection and targets the SzM protein of Streptococcus zooepidemicus, in response to the clinical situation in the existing pig farming industry where there are few biological products related to the prevention or treatment of Streptococcus zooepidemicus infection in pig herds other than antibiotics.

[0007] Specifically, this application involves the following aspects:

[0008] The first object of the present invention is to provide a porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region:

[0009] The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID No. 2, the amino acid sequence of CDR-H2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR-H3 is shown in SEQ ID No. 4;

[0010] The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID No. 6, the amino acid sequence of CDR-L2 is WAS, and the amino acid sequence of CDR-L3 is shown in SEQ ID No. 7;

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID No. 1, or has at least 95% sequence identity with SEQ ID No. 1;

[0012] The amino acid sequence of the monoclonal antibody light chain variable region is shown in SEQ ID No. 5, or has at least 95% sequence identity with SEQ ID No. 5.

[0013] Furthermore, the monoclonal antibody is a chimeric antibody.

[0014] Furthermore, the chimeric antibody comprises a mouse antibody variable region and a porcine antibody constant region.

[0015] The second object of the present invention is to provide an isolated polynucleotide encoding the aforementioned porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus.

[0016] In a specific embodiment, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID No. 23, and the nucleotide sequence of the light chain variable region is shown as SEQ ID No. 24.

[0017] The third object of the present invention is to provide an expression vector comprising the isolated polynucleotide as described above.

[0018] In a specific embodiment, the basic vector of the expression vector is pcDNA3.4 vector.

[0019] The fourth object of the present invention is to provide a host cell, which comprises the aforementioned polynucleotide or the aforementioned expression vector.

[0020] A fifth object of the present invention is to provide the use of the aforementioned anti-S. zooepidemicus SzM protein swine-derived monoclonal antibody or the aforementioned isolated polynucleotide or the aforementioned expression vector or the aforementioned host cell in the preparation of a drug for preventing and / or treating diseases caused by S. zooepidemicus infection.

[0021] The beneficial effects of the present invention are

[0022] The present invention conducts porcine transformation on the basis of mouse monoclonal antibodies, replacing the mouse antibody constant region with the porcine antibody constant region, which can greatly reduce the side effects caused by the mouse components in the mouse antibody in the pig body, and at the same time avoids the common inability of mouse antibodies to effectively activate complement and Fc receptor-related immune effects in the pig body, thereby improving the therapeutic effect on pigs infected with Streptococcus zooepidemicus. The chimeric antibody of the present invention provides a new biological product for preventing and treating pig herds infected with Streptococcus zooepidemicus in clinical applications.

[0023] Successfully screened monoclonal antibodies with therapeutic effects on Streptococcus zooepidemicus infection, BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The PCR electrophoresis diagram shows the successful amplification of the complete sequence of the pig-mouse chimeric antibody gene using overlap extension PCR, where:

[0025] Figure 1 A is the PCR electrophoresis of the chimeric antibody heavy and light chain variable region gene fragments, M is the DL-2000 marker lane, lane 1 is the mouse antibody heavy chain variable region fragment (333 bp), and lane 2 is the mouse antibody light chain variable region fragment (339 bp);

[0026] Figure 1 B is the PCR electrophoresis diagram of the heavy and light chain constant region gene fragments of the porcine antibody, M is the DL-2000 marker lane, 3 is the porcine antibody heavy chain constant region fragment (1093 bp), and 4 is the porcine antibody light chain constant region fragment (552 bp);

[0027] Figure 1 C is the PCR electrophoresis diagram of the chimeric antibody heavy chain and light chain full-length gene fragments, M is the DL-2000 marker, 5 is the chimeric antibody heavy chain full-length fragment (1426 bp) swimming lane, and 6 is the chimeric antibody light chain full-length fragment (861 bp) swimming lane.

[0028] Figure 2 The double enzyme digestion identification diagram of expression vectors pcDNA3.4-H and pcDNA3.4-L, Lane A: double enzyme digestion identification of expression vector pcDNA3.4-H; Lane B: double enzyme digestion identification of expression vector pcDNA3.4-L; M 1: DL-5000 marker; M 2: DL-15000 marker.

[0029] Figure 3 SDS-PAGE electrophoresis of the purified chimeric antibody.

[0030] Figure 4 Western Blot analysis showed that the chimeric antibody specifically bound to the SzM protein. Lane B: porcine chimeric antibody after transformation; Lane C: mouse monoclonal antibody before transformation; M: marker.

[0031] Figure 5 IFA confirmed that the chimeric antibody had specific binding to SEZ ATCC35246 whole bacteria.

[0032] Figure 6 Diagram showing the evaluation of chimeric antibodies for the treatment and protection of pigs infected with SEZ. McAb-pig 1 and McAb-pig 2 are two pigs treated with chimeric antibodies, and PBS-pig 3 is a pig treated with PBS as a negative control.

[0033] Figure 6 A represents the bacterial load of each major organ of three pigs on the sixth day after challenge (3-5 samples were taken from each organ for statistics);

[0034] Figure 6 B represents the daily changes in pig body temperature after infection. DETAILED DESCRIPTION

[0035] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0036] Unless otherwise specified, the raw materials and chemical reagents used in the examples are conventional commercial products, and the technical means used are conventional means known to those skilled in the art.

[0037] Unless otherwise specified, the materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art and can be obtained through commercial channels.

[0038] The term "chimeric antibody" herein is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody may comprise a murine antibody variable region and a porcine antibody constant region.

[0039] It is well known to those skilled in the art that each heavy chain variable region can be composed of three complementarity determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementarity determining regions (CDRs) and four framework regions (FRs). The complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from N-terminus to C-terminus, both the heavy and light chain variable regions comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0040] In an embodiment of the present application, the CDR sequences are defined and determined using the IMGT system.

[0041] Specific implementation cases:

[0042] Example 1 Obtaining the complete sequence of the pig-mouse chimeric antibody gene

[0043] 1.1 Determination of the variable regions of mouse antibodies

[0044] The hybridoma cell RNA sample was extracted using the Trizol method and then reverse transcribed to obtain cDNA. The nucleotide sequence of the heavy chain variable region HV shown in SEQ ID No. 23 and the nucleotide sequence of the light chain variable region LV shown in SEQ ID No. 24 were amplified and cloned into a vector for sequencing. The sequencing results were compared with the IMGT database, and information such as CDR1 / 2 / 3 was extracted to finally determine the variable region sequence. The mouse antibody heavy chain variable region is 333 bp, and the light chain variable region is 339 bp. The following primers were designed based on the mouse antibody variable region sequence:

[0045] Table 1-1 Primers used to amplify the variable regions of mouse antibodies

[0046]

[0047] 1.2 Amplification of the constant region sequence of porcine antibodies

[0048] 1.2.1 Extraction of porcine lymphocytes from porcine peripheral blood and acquisition of cDNA

[0049] Take fresh anticoagulated porcine whole blood and dilute it with an equal volume of tissue diluent. Slowly add separation solution to the centrifuge tube, ensure that the diluted blood is lightly spread on the separation solution, and centrifuge at 500-1000g / min for 30 minutes. After centrifugation, gently aspirate the lymphocyte layer cells, transfer them to a 15mL clean centrifuge tube, resuspend and wash with 10mL sterile PBS, and then centrifuge at 250g / min for 10 minutes. Remove the supernatant, resuspend and wash the cells again with 5mL sterile PBS, and repeat the centrifugation step. Resuspend the cells with PBS to about 1mL of suspension, divide into 0.5mL per tube, add Trizol reagent, and store in a -80℃ refrigerator. Extract RNA from porcine peripheral blood lymphocytes and perform reverse transcription to obtain cDNA.

[0050] 1.2.2 Design of primers for the constant region of porcine antibodies

[0051] Searching the porcine antibody constant region sequences on the NCBI and IMGT websites revealed that the Genbank accession number for the heavy chain constant region is M81770.1, with an HC size of 1093 bp. The Genbank accession number for the light chain constant region is M59321.1, with an LC size of 522 bp. Primers were designed based on the gene fragments. The primer sequences are shown in Table 1-2:

[0052] Table 1-2 Primers used to amplify the constant region of porcine antibodies

[0053]

[0054] 1.2.3 Linking of constant and variable region sequences by overlap extension PCR

[0055] Based on the obtained mouse hybridoma light and heavy chain variable region gene sequences and the porcine light and heavy chain constant region gene sequences, multiple primer pairs were designed using Snapgene software based on the principle of overlap extension PCR to amplify the full-length sequences of the chimeric antibody light and heavy chains. A start codon and leader sequence were added to the upstream primer cleavage site of the full-length gene, and a stop codon was added to the downstream primer cleavage site of the full-length gene. The expected amplified fragment length for the full-length heavy chain was approximately 1426 bp, and the expected amplified fragment length for the full-length light chain was approximately 861 bp. The PCR system was prepared using the primers designed in Table 1-3 according to the full-length heavy chain amplification system in Table 1-4.

[0056] Table 1-3 Primers used to amplify the full length of the heavy chain

[0057]

[0058]

[0059] Table 1-4 Heavy chain amplification system

[0060]

[0061] Use the primers designed in Table 1-5 and prepare the PCR system according to the light chain full-length amplification system in Table 1-6.

[0062] Table 1-5 Primers used to amplify the full length of the light chain

[0063]

[0064] Table 1-6 Light chain amplification system

[0065]

[0066]

[0067] The prepared heavy chain full-length amplification PCR system and light chain full-length amplification PCR system were reacted according to the PCR program in Table 1-7.

[0068] Table 1-7 PCR program

[0069]

[0070] The PCR electrophoresis diagram of the complete sequence of the pig-mouse chimeric antibody gene successfully amplified by overlap extension PCR is shown in the figure below. Figure 1 As shown, the results show: Figure 1 A is the PCR electrophoresis of the chimeric antibody heavy and light chain variable region gene fragments, M is the DL-2000 marker lane, lane 1 is the mouse antibody heavy chain variable region fragment (333 bp), and lane 2 is the mouse antibody light chain variable region fragment (339 bp); Figure 1 B is the PCR electrophoresis diagram of the heavy and light chain constant region gene fragments of the porcine antibody, M is the DL-2000 marker lane, 3 is the porcine antibody heavy chain constant region fragment (1093 bp), and 4 is the porcine antibody light chain constant region fragment (552 bp). Figure 1 C is the PCR electrophoresis diagram of the chimeric antibody heavy chain and light chain full-length gene fragments, M is the DL-2000 marker, 5 is the chimeric antibody heavy chain full-length fragment (1426 bp) swimming lane, and 6 is the chimeric antibody light chain full-length fragment (861 bp) swimming lane.

[0071] Example 2 Construction and Identification of Expression Vectors pcDNA3.4-H and pcDNA3.4-L

[0072] 2.1 Double enzyme digestion of vector and gene fragment

[0073] The pig-mouse chimeric antibody heavy and light chain genes and the pcDNA3.4 vector were double-digested with EcoRI and BamHI, respectively. The digestion procedure was as follows: incubate in a 30°C water bath for 1 hour, then incubate in a 37°C water bath for 1 hour. After digestion, the digestion products were recovered by gel-gel recovery and ligated into the pcDNA3.4 vector. The digestion system is shown in Table 2-1:

[0074] Table 2-1 Double enzyme digestion system

[0075]

[0076] The specific ligation operation is as follows: After reacting at 37°C for 30 minutes, the sample is immediately placed on ice to cool and obtain the recombinant product. The ligation system is shown in Table 2-2:

[0077] Table 2-2 Connection system

[0078]

[0079] 2.2 Identification of recombinant vectors

[0080] The above ligation products were digested using restriction endonucleases BamHI and EcoRI in a water bath at 30°C and 37°C for 30 min, respectively. The enzyme digestion systems are shown in Table 2-3.

[0081] Table 2-3 Double enzyme digestion system

[0082]

[0083] The results of double enzyme digestion of expression vectors pcDNA3.4-H and pcDNA3.4-L are as follows Figure 2 As shown: Lane A: Double enzyme digestion identification of expression vector pcDNA3.4-H, two bands can be seen in the lane, indicating successful digestion; Lane B: Double enzyme digestion identification of expression vector pcDNA3.4-L, two bands can be seen in the lane, indicating successful digestion; M1: DL-5000 marker; M2: DL-15000 marker.

[0084] Example 3 Preparation and purification of recombinant chimeric antibodies

[0085] 3.1 Transfection-grade plasmid transformation

[0086] 100 ng of the heavy chain or light chain expression plasmid containing the chimeric antibody gene prepared in Example 2 was added to 100 μL of DH5α competent cells. The plasmid-competent cell mixture was heat-shocked in a 42°C water bath for 45 seconds and then immediately cooled on ice for 2 minutes.

[0087] Use the transformed competent cells as host cells, add 900 μL of LB liquid medium, and incubate at 37°C in a shaker at 180 rpm / min for approximately 1 hour. Pipette 100 μL of the competent cell suspension and evenly spread it onto an ampicillin-resistant plate using glass beads. Place the plate in an incubator and incubate overnight at 37°C. The next day, select a single clone and incubate in THB liquid for 16 hours. Collect the cultured liquid by centrifugation, remove the supernatant, and perform plasmid extraction.

[0088] 3.2 Expression of chimeric antibodies in mammalian cells

[0089] The cell density on the day of transfection was controlled at 1.5×10 6 cells / mL. Prepare the DNA-transfection reagent mixture: add transfection-grade plasmid and transfection reagent to the transfection buffer in a certain proportion, mix well and incubate at room temperature for 20 minutes. Add the DNA-transfection reagent mixture dropwise to the cells to be transfected, shaking while adding, and place it in a cell culture incubator for suspension culture at a speed of 120 rpm / min. Record the cell survival rate every day thereafter. About 4-6 days after transfection, when the survival rate drops below 70%, centrifuge the cell suspension at 800 rpm / min and collect the supernatant.

[0090] 3.3 Purification of pig-mouse chimeric antibodies

[0091] The cell culture medium after 5 days of transfection culture was taken and centrifuged at 8000 rpm / min for 20 min in a 4°C centrifuge. The supernatant was filtered with a 0.22 μm filter and then purified using a HiTrap Protein A HP protein purification column.

[0092] The chimeric antibody purification results are as follows Figure 3 As shown: the antibody heavy chain is around 55kD, the antibody light chain is around 25kD, and there are fewer miscellaneous bands, indicating that the chimeric antibody was successfully purified.

[0093] Example 4 Western Blot Identification of Chimeric Antibody Specificity

[0094] The modified porcine chimeric antibody was mixed with 5× Loading Buffer and incubated in a 100°C metal bath for 10 minutes before loading the sample. The protein loading amount per well was 5 μg, 1 μg, and 200 ng, respectively. A negative control was also set up with the unmodified mouse monoclonal antibody. After electrophoresis, the SDS-PAGE gel was transferred to a PVDF membrane using a membrane transfer device. A 5% skim milk solution was prepared and the PVDF membrane was placed in the skim milk and blocked on a shaker at room temperature for 1 hour. The PVDF membrane was removed and HRP-conjugated goat anti-pig IgG (H+L) diluted 1:5000 in 5% skim milk was placed on the membrane and incubated on a shaker at 4°C overnight. The PVDF membrane was washed three times with TBST buffer for 10 minutes each. The color development solution was prepared according to the SuperFemto ECL Chemiluminescence Kit instructions and the color was visualized in the dark.

[0095] Western Blot results are as follows Figure 4 Figure 2: Chimeric antibodies specifically bind to the SzM protein. B: After modification, different concentrations of the porcine chimeric antibody bind to the SzM protein, with a band visible at 55 kD. C: Before modification, different concentrations of the mouse monoclonal antibody (positive control) bind to the SzM protein, with a band visible at 55 kD. M: marker.

[0096] Example 5 IFA Identification of Binding of Antibodies to Whole Strains of Streptococcus Zooepidemicus ATCC35246

[0097] Inoculate SEZ ATCC35246 onto THB plates one day in advance. Scrape colonies, spread evenly onto glass slides, and allow to air-dry. Aspirate 4% paraformaldehyde to cover the bacteria. After 10 minutes, aspirate the 4% paraformaldehyde and cover the colonies with PBS for 5 minutes, repeat three times, and wash off any excess paraformaldehyde. Cover the bacteria with 0.1% Triton-X100. After 10 minutes, cover the colonies with PBS for 5 minutes, repeat three times, and wash off any excess Triton-X100. Prepare a 5% BSA solution to cover the bacteria and block at 37°C for 1 hour. Wash three times with PBST, each for 5 minutes. After washing, incubate the slides with a 1:500 dilution of the pre-modified mouse monoclonal antibody and the post-modified porcine chimeric antibody. Incubate at 37°C for 1 hour, resuspend in PBST, and wash three times. Protect from light. Add Alexa Fluor 488-conjugated secondary antibody at a 1:500 dilution, resuspend, and incubate at room temperature for 45 minutes. Wash three times with PBST, each for 5 minutes. Add DAPI at a 1:40 dilution, incubate at room temperature for 10 minutes, and wash three times with PBST, each for 15 minutes. Allow the slides to air-dry, then add 5 μL of mounting medium, cover with a coverslip, and observe under a fluorescence microscope.

[0098] The results are as follows Figure 5 As shown: The porcine chimeric antibody binds well to the SEZ ATCC35246 bacteria, and obvious green fluorescence is visible in the strain.

[0099] Example 6 Evaluation of the therapeutic and protective effect of chimeric antibodies on SEZ-infected pigs

[0100] Three 2-week-old piglets were selected and, after 3 days of adaptive feeding, 3×10 SEZ ATCC35246 strains were challenged through the ear vein. 3 CFU / pig. Six hours later, two pigs were injected with 20 mg of chimeric antibody via the ear vein, and the remaining pig was given the same volume of PBS. Body temperature was monitored daily after challenge, and on day 6, the pigs were dissected and their internal organs were harvested for bacterial load analysis.

[0101] The results are as follows Figure 6As shown: Figure A shows the bacterial load in organs. No SEZ was detected in the major organs (lungs, liver, kidneys, spleen, heart, cerebrum, cerebellum, and blood) of the two piglets treated with chimeric antibodies. However, the major organs (lungs, liver, kidneys, spleen, heart, cerebrum, and cerebellum) of the piglets treated with PBS all contained high levels of SEZ. Figure B shows the temperature monitoring of piglets after SEZ infection. Although all three piglets experienced abnormally elevated temperatures within 24 hours after challenge, the temperatures of the two piglets treated with chimeric antibodies quickly returned to normal, while the temperatures of the piglets treated with PBS remained abnormally elevated for the next 6 days. This indicates that the porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus described herein has a good therapeutic effect on diseases caused by S. zooepidemicus infection.

[0102] Sequence Listing

[0103] SEQ ID No. 1:

[0104] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNA

[0105] AFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARFDYWGQGTTLTVSS

[0106] SEQ ID No. 2: GFSLTSYG

[0107] SEQ ID No. 3: IWSGGST

[0108] SEQ ID No. 4: ARFDY

[0109] SEQ ID No.5:

[0110] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLKSGNQKNYLTWYQQKPGQPPKLLIFWAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELK

[0111] SEQ ID No.6: QSLLKSGNQKNY

[0112] SEQ ID No.7

[0113] QNDYSYPLT

[0114] SEQ ID No.23:

[0115] CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAA GCACAGACTATAATGCAGCTTTCATATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGATTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0116] SEQ ID No.24:

[0117] GACATTTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAAAAGTGGAAATCAAAAGAACTATTTGACCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTATTGATCTTCTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0118] 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 porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region: The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID No. 2, the amino acid sequence of CDR-H2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR-H3 is shown in SEQ ID No. 4; The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID No. 6, the amino acid sequence of CDR-L2 is WAS, and the amino acid sequence of CDR-L3 is shown in SEQ ID No. 7; The monoclonal antibody is a chimeric antibody; The chimeric antibody comprises a murine antibody variable region and a porcine antibody constant region.

2. The porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus according to claim 1, characterized in that: The amino acid sequence of the monoclonal antibody heavy chain variable region has at least 95% sequence identity with SEQ ID No. 1; The amino acid sequence of the monoclonal antibody light chain variable region has at least 95% sequence identity with SEQ ID No.

5.

3. The porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

5.

4. An isolated polynucleotide, characterized in that The polynucleotide encodes the porcine monoclonal antibody against the SzM protein of Streptococcus zooepidemicus according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector contains the isolated polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell comprises the polynucleotide according to claim 4 or the expression vector according to claim 5.

7. Use of the porcine monoclonal antibody against SzM protein of Streptococcus zooepidemicus according to claim 1, the isolated polynucleotide according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6 in the preparation of a medicament for preventing and / or treating diseases caused by Streptococcus zooepidemicus infection.

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