A monoclonal antibody that can neutralize different serotypes of porcine rotavirus

Abstract

The present invention belongs to the field of immunology, and specifically relates to a monoclonal antibody that can neutralize different serotypes of porcine rotavirus strains. The heavy chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO:1, a CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:3; the light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a CDR2 with an amino acid sequence of RAS, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:5. The monoclonal antibody of the present invention can effectively neutralize different serotypes of PoRV strains and inhibit their infection of cells.

Description

Technical Field

[0001] The invention belongs to the field of immunology, and specifically relates to a monoclonal antibody capable of neutralizing different serotype strains of porcine rotavirus and application thereof. Background Art

[0002] Porcine rotavirus (PoRV) infection mainly causes anorexia, vomiting, diarrhea and dehydration in piglets. Clinically, it is often mixed with multiple porcine enteric coronaviruses or enteric pathogens, resulting in increased mortality and posing a huge threat to the pig industry. Porcine rotavirus can also infect humans and a variety of animals such as cattle, horses and dogs, and has important public health significance and basic research value.

[0003] Rotavirus belongs to the Reoviridae family, Rotavirus genus. The genome is composed of segmented double-stranded RNA, encoding 6 structural proteins (VP1-VP4, VP6, VP7) and 5-6 non-structural proteins (NSP1-NSP5 / NSP6). The virus particles are spherical, without an envelope, with a diameter of about 70nm, and have three layers of shells, of which the inner shell is composed of VP2 protein, the middle shell is composed of VP6 protein, and the outer shell is composed of VP4 protein and VP7 protein. The VP4 trimer is embedded in the middle shell and the outer shell, and protrudes outward to form spikes. The VP4 protein adsorbs receptors and mediates membrane damage, and the VP7 protein binds to Ca 2+ It acts to disaggregate viral particles, and both VP4 and VP7 proteins can induce the production of neutralizing antibodies. According to the different antigenicity of VP6, rotavirus is divided into 10 groups (A to I), among which groups A, B, C, E, and H can infect pigs, and group A porcine rotavirus has the widest distribution and the strongest pathogenicity. According to the different antigenicity of VP7 and VP4, group A rotavirus is divided into different serotypes, called G type and P type, respectively. Currently, 42 G types and 58 P types have been reported, and G type and P type can form a variety of different combinations. In recent years, the positive detection rate of PoRV in pig farms in my country has gradually increased, and there are many different serotypes. Among them, G type is mainly G9, followed by G5, G4 and G3, and P type is mainly P

[23] , followed by P[7] and P

[13] .

[0004] There are many serotypes of PoRV. In clinical practice, mixed infection with different serotypes is common, and the cross-protection between different serotypes is weak, which poses a huge challenge to the effective prevention and control of the disease. At present, there is no specific treatment for porcine rotavirus infection, and vaccination is mainly used for prevention and control. The existing vaccines in China are developed based on the G5 strain, and the protection effect against other strains is not ideal. The acquisition of neutralizing monoclonal antibodies that can identify different serotypes of PoRV can provide useful materials for the effective control of PoRV and the development of diagnostic reagents. Summary of the invention

[0005] The object of the present invention is to provide a monoclonal antibody that can neutralize different serotypes of PoRV strains. The monoclonal antibody can effectively neutralize different serotypes of PoRV strains and inhibit their infection of cells. It is speculated that the monoclonal antibody can be used for the prevention and treatment of PoRV infection.

[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 antibody R-4C2 capable of neutralizing G3, G4, G5 and G9 type PoRV. The heavy chain variable region of the monoclonal antibody R-4C2 includes a CDR1 with an amino acid sequence of GFSLSSYG (SEQ ID NO: 1), a CDR2 with an amino acid sequence of MWSGGSA (SEQ ID NO: 2), and a CDR3 with an amino acid sequence of VRDGGGYYGFAY ​​(SEQ ID NO: 3); the light chain variable region of the monoclonal antibody R-4C2 includes a CDR1 with an amino acid sequence of ESVDTFGDSF (SEQ ID NO: 4), a CDR2 with an amino acid sequence of RAS, and a CDR3 with an amino acid sequence of QQSNEDPRT (SEQ ID NO: 5). The amino acid sequence of the heavy chain variable region of monoclonal antibody R-4C2 is QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVHWVRQPPGKGLEWLGIMWSGGSANYNS TLMSRLSISKDNSKSQVFLKMNSLQSDDTAMYYCVRDGGGYYGFAY ​​(SEQ ID NO: 6), and the amino acid sequence of the light chain variable region of monoclonal antibody R-4C2 is DIVLTQSPASLAVSLGQRATISCRASESVDTFGDSFMHWYQQKPGQPPKLLIYRASNLESGIPV RFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPRT (SEQ ID NO: 7).

[0008] The present invention also provides a hybridoma cell strain R-4C2 which can stably secrete the above-mentioned monoclonal antibody capable of neutralizing G3, G4, G5 and G9 type PoRV, and the hybridoma cell strain R-4C2 has good genetic stability.

[0009] The present invention provides gene sequences encoding the heavy chain variable region and the light chain variable region of the PoRV neutralizing monoclonal antibody R-4C2. The gene sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody R-4C2 are obtained by using the cDNA of the hybridoma cell line R-4C2 as a template and performing PCR amplification using degenerate primers of the heavy chain and light chain of the mouse antibody variable region, respectively. The gene sequence encoding the heavy chain variable region of the monoclonal antibody R-4C2 is 5'-CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTC CATCACTTGCACTGTCTCTGGATTTTCATTAAGCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATAATGTGGTCTGGTGGAAGCGCGAATTAATTCGACTCTCATGTCCAGACTGAGCATCAGTAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAAGTGATGACACAGCCATGTATTATTGTGTCAGAGATGGGGGTGGTTACTACGGGTTTGCTTAC-3', as shown in SEQ ID NO: 8; the gene sequence encoding the light chain variable region of the monoclonal antibody R-4C2 is 5'-GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCA CCATATCCTGCAGAGCCAGTGAAAGTGTTGATACTTTTGGCGATAGTTTCATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGTCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCGGACG-3', as shown in SEQ ID NO: 9.

[0010] The present invention adopts indirect immunofluorescence assay (IFA) to detect and confirm that the monoclonal antibody can produce specific fluorescence reaction with MA-104 cells infected with G3, G4, G5 or G9 type PoRV.

[0011] The invention prepares an ascites monoclonal antibody R-4C2 and purifies it. Neutralization test shows that the neutralization titers of the purified ascites monoclonal antibody R-4C2 against G3, G4, G5 and G9 PoRV are 1:32, 1:128, 1:256 and 1:256, respectively, and the corresponding concentrations are 0.5 μg / mL, 0.125 μg / mL, 0.0625 μg / mL and 0.0625 μg / mL, respectively.

[0012] Therefore, the monoclonal antibody R-4C2 of the present invention, the gene encoding the monoclonal antibody R-4C2, the biological material (expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or host cell) containing the gene encoding the monoclonal antibody R-4C2 or the hybridoma cell line R-4C2 can be used to prepare drugs for preventing or treating PoRV infection, or to prepare PoRV detection reagents or kits, or to be used for basic research on PoRV. The single-chain antibody or antigen-binding fragment obtained by modifying the monoclonal antibody R-4C2 can also be used to prepare drugs for preventing or treating PoRV infection, or to prepare PoRV detection reagents or kits, or to be used for basic research on PoRV.

[0013] The beneficial effects of the present invention are:

[0014] The present invention uses only purified G5 virus particles to immunize mice, and screens out the monoclonal antibody R-4C2 that can neutralize G3, G4, G5 and G9 PoRV.

[0015] The monoclonal antibody R-4C2 of the present invention effectively protects cells from being infected by PoRV by neutralizing G3, G4, G5 and G9 types of PoRV. Therefore, the monoclonal antibody R-4C2 has the clinical effect of treating and preventing pigs from being infected by PoRV. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The SDS-PAGE detection result of the purified G5-type PoRV. Explanation of the reference numerals: M: Protein Marker; 1: PoRV sample before purification; 2: PoRV sample after purification.

[0017] Figure 2 The Western Blot test results of the purified G5-type PoRV. Explanation of the reference numerals: M: Protein Marker; 1: PoRV sample before purification; 2: PoRV sample after purification.

[0018] Figure 3 The results of transmission electron microscopy observation of purified G5-type PoRV.

[0019] Figure 4The results of SDS-PAGE detection of purified monoclonal antibody R-4C2. M: Protein Marker; 1: Purified monoclonal antibody R-4C2.

[0020] Figure 5 IFA detection monoclonal antibody R-4C2 specifically recognizes PoRV. Explanation of reference numerals: 1: MA-104 cells infected with G3-type PoRV; 2: MA-104 cells infected with G4-type PoRV; 3: MA-104 cells infected with G5-type PoRV; 4: MA-104 cells infected with G9-type PoRV; 5: MA-104 cells not inoculated with PoRV.

[0021] Figure 6 IFA detection of monoclonal antibody R-4C2 expressed in HEK-293T cells specifically recognizing PoRV. Explanation of reference numerals: 1: MA-104 cells infected with G3-type PoRV; 2: MA-104 cells infected with G4-type PoRV; 3: MA-104 cells infected with G5-type PoRV; 4: MA-104 cells infected with G9-type PoRV; 5: MA-104 cells not inoculated with PoRV.

[0022] Collection Information

[0023] Porcine rotavirus PoRVRShanD1 strain (G5 type porcine rotavirus):

[0024] Deposit date: June 1, 2024;

[0025] Name of depository: China Center for Type Culture Collection;

[0026] Deposit number: CCTCC NO:V202459;

[0027] Address of depository: Wuhan University, Wuhan, China;

[0028] Classification and nomenclature: Porcine rotavirus PoRV RShanD1 (Porcine rotavirus). DETAILED DESCRIPTION

[0029] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0030] The female BALB / c mice used in the examples were purchased from the Experimental Animal Center of Huazhong Agricultural University; the G3, G4, G5 or G9 PoRV used was stored in the Collaborative Innovation Center for Healthy Pig Breeding of Huazhong Agricultural University (specifically, the samples tested as PoRV positive were homogenized, frozen and thawed, centrifuged and filtered, inoculated with MA-104 cells that grew a monolayer, and isolated and cultured), and the G5 PoRV used as an antigen has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: V202459.

[0031] The antigen, hybridoma cell, monoclonal antibody, etc. in the present invention are described in detail below with reference to specific examples.

[0032] Example 1 Preparation of Antigen

[0033] 1. Proliferation of G5-type PoRV

[0034] 1MOI G5 type PoRV (deposit number: CCTCC NO: V202459) was inoculated into a monolayer of MA-104 cells. Before inoculation, the cells were rinsed twice with serum-free DMEM containing 5μg / mL trypsin, and then adsorbed at 37°C for 1h and replaced with serum-free DMEM containing 5μg / mL trypsin to maintain culture for 24-48h. The pathological changes of the cells were observed under an inverted microscope. When more than 90% of the cells were pathological, they were repeatedly frozen and thawed 3 times, centrifuged at 4000r / min for 10min, and the supernatant virus liquid was collected and stored at -80°C.

[0035] 2. Purification and detection of G5-type PoRV

[0036] The collected G5 PoRV virus solution was filtered through a 0.8 μm filter and concentrated to 1 / 100 of the original volume by ultrafiltration. The virus was purified using the pure chromatography system and Capto Core 700 chromatography column, and the samples with elution peaks were harvested and tested by SDS-PAGE. The results showed that bands appeared at about 90KDa, 40KDa, and 32KDa, which were consistent with the sizes of PoRV VP4 protein, VP6 protein, and VP7 protein, respectively. Figure 1 shown.

[0037] Further, a monoclonal antibody against PoRV VP6 protein (source: BALB / c female mice were immunized with prokaryotically expressed VP6 protein (as shown in SEQ ID NO: 10) as an immunogen, spleen cells of mice with high antibody titer were separated and fused with SP2 / 0 cells, and the monoclonal antibody against PoRV VP6 protein obtained by screening using the limiting dilution method) was used as the primary antibody to perform Western Blot detection on G5-type PoRV before and after purification. The results showed that a specific reaction band appeared at a size of about 40 KDa, as shown in FIG. Figure 2 shown.

[0038] The purified G5 PoRV was dripped onto the copper grid and observed under a transmission electron microscope after negative staining with sodium phosphotungstate. The results showed that the virus particles were about 70 nm in size and were wheel-shaped. Figure 3 shown.

[0039] The above results indicate that the purified G5-type PoRV was obtained and can be used as an antigen to immunize mice for the preparation of monoclonal antibodies.

[0040] Example 2 Preparation of monoclonal antibodies

[0041] 1. Preparation of hybridoma cell line R-4C2

[0042] The purified G5-type PoRV virus solution was mixed with an equal volume of rapid immune adjuvant (purchased from Biolong Immunotechnology Co., Ltd.) and emulsified, and 6-8-week-old female BALB / c mice were immunized by intramuscular injection into the hind limbs. Antibodies against G5-type PoRV in mouse serum were detected by PoRV whole virus indirect ELISA. When the antibody titer reached 1:12800, the purified G5-type PoRV was used for booster immunization. 3-5 days after booster immunization, the mice were killed by orbital bleeding and positive serum was collected. The spleen cells of the immunized mice were aseptically fused with myeloma cells SP2 / 0. After culture in HAT medium and HT medium, the culture supernatant of the fused cells was detected by PoRV whole virus indirect ELISA. The cells in the positive wells were subcloned and screened for 3 rounds. The obtained positive cell lines were expanded and cultured, and the culture supernatant was taken for neutralization test. The results showed that the monoclonal antibody secreted by one hybridoma cell line could neutralize G5-type PoRV, which was named R-4C2.

[0043] 2. Large-scale preparation of monoclonal antibody R-4C2

[0044] Three healthy female BALB / c mice aged 10 weeks were selected and injected intraperitoneally with Freund's incomplete adjuvant (500 μL / mouse). Seven days later, hybridoma cell line R-4C2 (5×10 5 ~1×10 6On the 10th to 14th day, the ascites was collected according to the swelling of the mouse abdomen, centrifuged at 12000r / min for 10min, and the middle yellow clear layer of liquid was collected and stored at -80℃ for later use.

[0045] 3. Purification of Monoclonal Antibody R-4C2

[0046] The ascites monoclonal antibody R-4C2 was purified according to the instructions of the Protein G column (purchased from Fuinde Technology Co., Ltd.), and then the purified monoclonal antibody was detected by SDS-PAGE. The results showed that obvious bands appeared at about 50KDa and 25KDa, which were consistent with the sizes of the antibody heavy chain and light chain, respectively. Figure 4 As shown, it indicates that the monoclonal antibody R-4C2 with high purity is obtained.

[0047] Example 3 IFA detection of the reaction between monoclonal antibody R-4C2 and different G-type PoRV

[0048] 1MOI G3, G4, G5 or G9 PoRV was inoculated into MA-104 cells that had grown into a monolayer. Before inoculation, the cell monolayer was washed twice with serum-free DMEM containing 5μg / mL trypsin. After adsorption at 37℃ for 1h, serum-free DMEM containing 5μg / mL trypsin was replaced to maintain culture. When about 30% of the cells were cytopathic, the samples were collected and fixed, permeabilized and blocked. The monoclonal antibody R-4C2 was used as the primary antibody, and Goat Anti-Mouse IgG H&L (Alexa Fluor 500, 1:2000 dilution) was used as the primary antibody. 488) was used as the secondary antibody for IFA detection, and DAPI dye diluted 1:4000 was added to stain the cell nucleus. The results showed that the monoclonal antibody R-4C2 prepared by the present invention had specific fluorescence reactions with MA-104 cells inoculated with G3, G4, G5 or G9 type PoRV, but had no specific fluorescence reaction with MA-104 cells not inoculated with PoRV, such as Figure 5 This indicates that the monoclonal antibody R-4C2 can specifically recognize G3, G4, G5 and G9 PoRV in IFA.

[0049] Example 4 Neutralization test to detect the neutralizing activity of monoclonal antibody R-4C2 against different G-type PoRV

[0050] G3, G4, G5 and G9 PoRV were diluted to 200 TCID in serum-free DMEM medium containing 5 μg / mL trypsin. 50 / 0.1mL, the purified monoclonal antibody R-4C2 was diluted 2-fold from 4μg / mL to 0.002μg / mL. Monoclonal antibody R-4C2 with different dilutions was mixed with equal volumes of 4 diluted virus solutions and placed at 37℃ for 1h. The MA-104 cell monolayer in the 96-well plate was washed twice with serum-free DMEM culture medium containing 5μg / mL trypsin, and then the incubated antibody-virus mixture was inoculated into the cells. Each antibody-virus mixture was inoculated into 4 wells, 100μL per well, and 200TCID was set at the same time. 50 / 0.1mL, 20TCID 50 / 0.1mL, 2TCID 50 / 0.1mL and 0.2TCID 50 / 0.1mL virus control and cell control without PoRV inoculation. After placing the cell plate in a 37°C, 5% CO2 cell culture incubator for 1 hour, the inoculum was discarded, and the cell monolayer was washed twice with serum-free DMEM culture medium containing 5μg / mL pancreatin, and 100μL of serum-free DMEM culture medium containing 5μg / mL pancreatin was added to each well, and the plate was placed in a 37°C, 5% CO2 cell culture incubator for continued culture. After 96 hours, the cytopathic effect (CPE) was recorded. The neutralization titer of monoclonal antibody R-4C2 against PoRV was calculated according to the Reed-Muench method.

[0051] The results of the neutralization test showed that the neutralization titers of the purified ascites monoclonal antibody R-4C2 against PoRV types G3, G4, G5 and G9 were 1:32, 1:128, 1:256 and 1:256, respectively, and the corresponding concentrations were 0.5μg / mL, 0.125μg / mL, 0.0625μg / mL and 0.0625μg / mL, respectively.

[0052] Example 5 Amplification and analysis of the variable region gene of monoclonal antibody R-4C2

[0053] Twelve upstream degenerate primers and one downstream degenerate primer were designed to amplify the heavy chain variable region gene of monoclonal antibody R-4C2, and 11 upstream degenerate primers and one downstream degenerate primer were designed to amplify the light chain variable region gene of monoclonal antibody R-4C2. Total RNA extracted from hybridoma cell line R-4C2 was used as a template for RT-PCR amplification.

[0054] Table 1. Primer sequences for amplifying the heavy chain variable region

[0055]

[0056] Table 2. Primer sequences for amplifying light chain variable regions

[0057]

[0058]

[0059] The results showed that 2 pairs of primers (MHV5 / MHV6, MHCG1) can amplify the heavy chain variable region gene, and 1 pair of primers (MKV2, MKC) can amplify the light chain variable region gene. The obtained amplified fragments were connected to the T vector and sequenced to obtain two antibody heavy chain variable region gene sequences and one antibody light chain variable region gene sequence, but one of the heavy chain variable region gene sequences contained a stop codon, which was an invalid rearrangement gene. The sizes of the heavy chain variable region and the light chain variable region were 321bp and 303bp, respectively. The gene sequence encoding the heavy chain variable region of the monoclonal antibody R-4C2 is shown in SEQ ID NO:8, and the gene sequence encoding the light chain variable region of the monoclonal antibody R-4C2 is shown in SEQ ID NO:9.

[0060] The amino acid sequence of the heavy chain variable region of monoclonal antibody R-4C2 is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7. The heavy chain variable region of monoclonal antibody R-4C2 includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; the light chain variable region of monoclonal antibody R-4C2 includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, CDR2 with an amino acid sequence of RAS, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0061] Example 6 Expression and verification of the variable region gene of monoclonal antibody R-4C2

[0062] According to the heavy chain variable region and light chain variable region sequences obtained in Example 5 and the constant region sequence of the mouse antibody, primers for amplifying the heavy chain gene and light chain gene of the monoclonal antibody R-4C2 were designed, and RT-PCR amplification was performed using the extracted total RNA of the hybridoma cell line R-4C2 as a template to obtain the full-length gene sequences of the heavy chain and light chain of the monoclonal antibody R-4C2. The full-length gene sequences were inserted into the eukaryotic expression vector pcDNA3.4 to construct recombinant plasmids pcDNA3.4-R-4C2-H and pcDNA3.4-R-4C2-L for expressing the heavy chain and light chain of the monoclonal antibody R-4C2, respectively.

[0063] Table 3. Primer sequences for amplifying heavy chain genes and light chain genes

[0064]

[0065] according to According to the instructions of the transfection reagent (purchased from Polyplus), the recombinant plasmids pcDNA3.4-R-4C2-H and pcDNA3.4-R-4C2-L were co-transfected into HEK-293T cells grown to 70% monolayer. At the same time, a negative control of transfection with empty vector pcDNA3.4 was set up. After 24 hours, the culture supernatant of HEK-293T cells was collected, inactivated at 56°C for 30 minutes, and stored at -80°C for later use.

[0066] According to the method in Example 3, the collected cell culture supernatant was used as the primary antibody, and its reaction with different G-type PoRV was detected by IFA. The results showed that the collected cell culture supernatant had specific fluorescence reaction with MA-104 cells inoculated with G3, G4, G5 or G9 type PoRV, but had no fluorescence reaction with MA-104 cells not inoculated with PoRV, such as Figure 6 This indicates that the monoclonal antibody R-4C2 was expressed in HEK-293T cells, and the expressed monoclonal antibody could specifically recognize G3, G4, G5 and G9 PoRV in IFA.

[0067] The neutralization activity of the monoclonal antibody R-4C2 expressed by HEK-293T cells against different G-type PoRVs was detected by neutralization test according to the method in Example 4. The results of the neutralization test showed that the neutralization titers of the monoclonal antibody R-4C2 expressed by HEK-293T cells against G3, G4, G5 and G9 types of PoRVs were 1:16, 1:64, 1:100 and 1:100, respectively.

[0068] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A monoclonal antibody for neutralizing different serotypes of porcine rotavirus, characterized in that: The heavy chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; the light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a CDR2 with an amino acid sequence of RAS, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

7.

3. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody is a single-chain antibody or an antigen-binding fragment.

4. A gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The monoclonal antibody gene according to claim 4, characterized in that: The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:8, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

9.

6. A biological material containing the gene according to claim 4 or 5, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

7. Use of the monoclonal antibody according to any one of claims 1 to 3, the gene according to claim 4 or 5, or the biomaterial according to claim 6 in any of the following aspects: A: For the preparation of drugs for preventing or treating diseases caused by porcine rotavirus infection; B: For preparing porcine rotavirus detection reagents or kits; The porcine rotavirus is of serotypes G3, G4, G5 and G9.

8. A drug, a detection reagent or a kit comprising the monoclonal antibody according to any one of claims 1 to 3.

Images