CN115725511B - Hybridoma cell strain R2McAb2A1, monoclonal antibody secreted by same and application thereof - Google Patents
Hybridoma cell strain R2McAb2A1, monoclonal antibody secreted by same and application thereof Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain R2McAb2A1, wherein the hybridoma cell strain R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022 and 7 months and 27 days, and the preservation number is CCTCCNO: C2022205. The invention also discloses a monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1. The invention also discloses application of the monoclonal antibody in preparation of a rabbit hemorrhagic disease virus type2 detection kit. The invention successfully prepares a hybridoma cell strain R2McAb2A1 by a hybridoma technology, and the monoclonal antibody secreted by the hybridoma cell strain and rabbit hemorrhagic disease virus type2 (RHDV 2) VP60 protein produce specific immune reaction; and through the detection of an indirect ELISA test, a Westernblot test and a hemagglutination and hemagglutination inhibition test, the monoclonal antibody has high titer and good specificity, and can effectively identify the strain of rabbit hemorrhagic disease virus type 1 (RHDV 1) and RHDV 2.
Description
Technical Field
The invention relates to the technical field of immunity, in particular to a hybridoma cell strain R2McAb2A1, a monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 and application of the monoclonal antibody.
Background
Rabbit hemorrhagic disease virus (Rabbit hemorrhagic disease virus, RHDV) belongs to the genus Rabbit virus (logo virus) of the family Caliciviridae (Caliciviridae), and after infection, causes acute septic infectious diseases in adult rabbits, namely Rabbit hemorrhagic disease (Rabbit hemorrhagic disease, RHD), also known as Rabbit fever. Rabbit plague comprises classical rabbit plague and rabbit plague type2, and mainly causes rabbit respiratory bleeding, solid organ congestion, swelling, bleeding and liver necrosis. Rabbit hemorrhagic disease virus type2 (Rabbit hemorrhagic disease type, RHDV 2), i.e., rabbit fever type 2. The technology for exploring the rapid detection and monitoring methods of classical rabbit fever and rabbit fever type2 has important practical significance, thereby becoming an important research direction and content for preventing and controlling rabbit fever at present.
Unlike classical RHDV strains, RHDV2 is reported to have a broader range of infection, not only to infect all animals of lagomorpha but also to infect and cause death in young rabbits within 30 days, and to be fast in transmission and high in mortality. Therefore, the research of developing a specific diagnosis method of RHDV2 is very urgent as the largest rabbit-raising country and rabbit meat export country in the world.
The RHDV virion capsid consists of 180 chemically identical units, each monomer being the major capsid protein VP60 having a molecular weight of about 60 kD. VP60 itself can assemble to form virus-like particles RHDV VLPs, is a virus immunoprotection antigen, and plays an important role in inducing immune response of an animal body to resist virus infection. Studies have shown that the variability of the RHDV different isoforms VP60 proteins also determines their differences in genetics, antigenic and epidemiological diversity, and immunological response. However, VP60 genes of all isolates of different RHDV subtypes are highly conserved, and the homology of nucleotide and amino acid is over 90%, so that the difficulty of research on a differential diagnosis method is increased. The same is true between classical RHDV (RHDV 1) strain and RHDV2 strain, and in practical production, effective identification is difficult.
Disclosure of Invention
The invention aims to solve the problem that the RHDV type 1 and RHDV type2 are difficult to effectively identify in the prior art, and provides a hybridoma cell strain R2McAb2A1, a monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 and application of the monoclonal antibody.
In order to solve the technical problems, the invention adopts the following technical scheme: a hybridoma cell strain R2McAb2A1, wherein the hybridoma cell strain R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 7 months and 27 days, and the preservation number is CCTCC NO: C2022205.
The invention also provides a monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1.
Preferably, the monoclonal antibody is a specific monoclonal antibody of rabbit hemorrhagic disease virus type 2VP60 protein, and the amino acid sequence of the rabbit hemorrhagic disease virus type 2VP60 protein is shown as SEQ ID N0.1.
The invention also provides application of the monoclonal antibody in preparation of a rabbit hemorrhagic disease virus type2 detection kit.
The invention has the beneficial effects that: the invention successfully prepares a hybridoma cell strain R2McAb2A1 by a hybridoma technology, and the monoclonal antibody secreted by the hybridoma cell strain and rabbit hemorrhagic disease virus type2 (RHDV 2) VP60 protein produce specific immune reaction; and the detection of indirect ELISA test, westernblot test and hemagglutination inhibition test proves that the monoclonal antibody has high titer and good specificity, and can effectively identify the strain of rabbit hemorrhagic disease virus type 1 (RHDV 1) and RHDV 2.
Drawings
FIG. 1 is a diagram showing the expression and purification of recombinant proteins pET-32a-R1VP60 and pET-32a-R2VP 60;
FIG. 2 is a Western blot identification chart of pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins;
FIG. 3 is a chart showing the determination of ascites titer of a monoclonal antibody secreted by hybridoma cell R2McAb2A 1;
FIG. 4 is a diagram showing the purification and identification of monoclonal antibodies secreted by hybridoma cell R2McAb2A 1;
FIG. 5 is an electron micrograph of RHDV1 and RHDV2 virus particles;
FIG. 6 is a Werternblot identification of monoclonal antibodies secreted by hybridoma cell R2McAb2A 1;
FIG. 7 is a chromosome map of SP20 cells and hybridoma cell R2McAb2A 1;
FIG. 8 is a graph showing the results of experiments on the inhibition of RHDV2 hemagglutination by monoclonal antibodies secreted by hybridoma cell R2McAb2A1.
The hybridoma cell strain R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC), and the address is China, wuhan and university of Wuhan; the preservation date is 2022, 7 and 27, the preservation number is CCTCC NO: C2022205, and the hybridoma cell strain R2McAb2A1 is classified and named.
Detailed Description
The invention will be further described with reference to the drawings and the specific examples.
Example 1 establishment of hybridoma cell line R2McAb2A1 and method for preparing monoclonal antibody
RHDV1 in this example is RHDV1 SCH04 (NCBI accession number: KX 844830), and RHDV2 is RHDV2SCCN03 (NCBI accession number: MW 178245). RHDV1 SCH04, RHDV2SCCN03 and SP2/0 myeloma cells were obtained from animal quarantine laboratories of Sichuan university, and 8 female SPF-grade BALB/c mice of about 6-7 weeks old were purchased from Chengdu laboratory animal Co.
HRP-labeled mouse anti-HIS monoclonal antibody, HRP-labeled goat anti-rabbit IgG, HRP-labeled rabbit anti-mouse IgG, ni-NTAHis tag Protein purification kit and Protein G pre-packed gravity column are purchased from Shanghai stock of bioengineering; SDS-PAGE gel preparation kit was purchased from Beijing Soy Bao technology Co., ltd; PEG 1500, 50 XHAT and 50 XHT were purchased from Sigma; mouse monoclonal antibody subclass identification kits were purchased from proteontech.
Prokaryotic expression and purification of pET-32a-R1VP60 and pET-32a-R2VP60
1.1 VP60 amplification primers of RHDV1 and RHDV2 were designed by SnapGene based on VP60 gene sequences of RHDV1 SCH04 strain (accession number: KX 844830) and RHDV2SCCN03 strain (accession number: MW 178245) in NCBI, and SalI and HindIII cleavage sites were added to the upstream and downstream primers, respectively. The primer sequences R1-VP60-F, R1-VP60-R, R2-VP60-F, R2-VP60-R are sequentially shown as SEQ ID N0.3 to SEQ ID N0.6.
1.2 Extraction and reverse transcription of RHDV1 and RHDV2 virus RNA
Grinding liver tissues of rabbits infected with RHDV1 and RHDV2, repeatedly freezing and thawing for three times, adding physiological saline, transferring into a 1.5mL centrifuge tube, extracting total RNA of tissues, and storing cDNA obtained through reverse transcription at-20 ℃ for later use.
1.3 PCR amplification of RHDV 1VP60 gene fragment and RHDV 2VP60 gene fragment
Respectively using the obtained cDNA of RHDV1 and RHDV2 virus RNA as a template, and respectively using the corresponding upstream and downstream primers in the step 1.1 to amplify VP60 genes of RHDV1 and RHDV 2. After the completion of the reaction, the PCR product was detected by 1% agarose gel electrophoresis. PCR reaction system: 2X Taq PCR MasterMix. Mu.L, 2. Mu.L each of the upstream and downstream primers, 2. Mu.L each of cDNA, ddH 2 O19. Mu.L. PCR reaction procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 20s,72 ℃ for 2min,30 cycles; and at 72℃for 10min. VP60 PCR products of RHDV1 and RHDV2 are respectively recovered and stored at-20 ℃ for subsequent enzyme digestion connection tests.
1.4 Construction of pET-32a-R1VP60 and pET-32a-R2VP60 prokaryotic expression vector
The VP60 PCR products of the RHDV1 and RHDV2 purified and recovered in the step 1.3 and the expression vector PET-32a are respectively subjected to double digestion by Sal I and Hind III, and digestion is carried out for 3-5h at 37 ℃. The cleavage system is shown in Table 1.
Table 1 double cleavage reaction System
The RHDV 1VP60 gene fragment and the RHDV 2VP60 gene fragment after the enzyme digestion and recovery are respectively connected with a pET-32a carrier part by using T4 DNA ligase, and the connection system is shown in Table 2.
TABLE 2 prokaryotic expression vector ligation System
The ligation was carried out at 16℃for 12h. 10. Mu.L of each of the two ligation products was used to transform DH 5. Alpha. Competent cells. Colonies grown on the transformed LB ampicillin plates were selected, and inoculated in LB ampicillin liquid medium for about 12 hours. And (3) respectively carrying out PCR amplification by taking the bacterial liquid after the expansion culture as a template, wherein the reaction procedure and the reaction system are the same as those of the VP60 amplification procedure and the reaction system in the step (1.3). Sequencing the bacterial liquid with positive PCR result. Recombinant plasmids with correct sequencing were named pET-32a-R1VP60 and pET-32a-R2VP60, respectively.
Recombinant plasmids pET-32a-R1VP60, pET-32a-R2VP60 and pET-32a empty vector are transformed into BL21 competent cells according to a conventional method, single colonies are respectively picked up and inoculated into LB liquid culture medium containing Amp, shake culture is carried out at 37 ℃ until OD600nm is about 0.5, and IPTG with the final concentration of 1mmol/L is added for continuous culture induction for about 6 hours. And (3) centrifugally collecting thalli, carrying out ultrasonic disruption in PBS, centrifuging the disrupted product for 10min at 8000r/min, collecting supernatant and precipitate, and finally carrying out solubility analysis on recombinant proteins through SDS-PAGE electrophoresis. Purifying the two recombinant proteins according to the specification of the Ni-NTAHis tag protein purification kit, and collecting eluent; and (3) carrying out SDS-PAGE, coomassie brilliant blue staining and decoloration, and then carrying out purity identification on the purified protein. Finally, carrying out amino acid sequencing on the purified recombinant protein, wherein the amino acid sequence of the recombinant protein expressed by pET-32a-R2VP60 is shown as SEQ ID N0.1; the amino acid sequence of the recombinant protein expressed by pET-32a-R1VP60 is shown as SEQ ID N0.2.
The results show that the recombinant proteins expressed by pET-32a-R1VP60 and pET-32a-R2VP60 are both in the form of inclusion bodies. After dissolving the precipitate with Binding buffer containing 8M urea, the two recombinant proteins were purified by nickel column affinity chromatography and concentrated renaturation using ultrafiltration tube. SDS-PAGE analysis shows that the sizes of the two recombinant proteins are 79kD, as shown in FIG. 1, and FIG. 1-A shows the expression of pET-32a-R1VP60 recombinant protein, wherein M: pre-stained protein markers; 1: precipitating pET-32a-R1VP60 thallus; 2: pET-32a-R1VP60 cell supernatant; 3: precipitating pET-32a empty bacterial cells; 4: pET-32a empty cell supernatant. FIG. 1-B shows the purification of pET-32a-R1VP60 recombinant protein, wherein M: pre-stained protein markers; 1: purified pET-32a-R1VP60 protein. FIG. 1-C shows the expression of pET-32a-R2VP60 recombinant protein, wherein M: pre-stained protein markers; 1: pET-32a empty cell supernatant; 2: precipitating pET-32a empty bacterial cells; 3: pET-32a-R2VP60 cell supernatant; 4: pET-32a-R2VP60 bacterial pellet. FIG. 1-D shows the purification of pET-32a-R2VP60 recombinant protein, wherein M: pre-stained protein markers; 1: purified pET-32a-R2VP60 protein. The electrophoresis result shows that the recombinant proteins expressed by the pET-32a-R1VP60 and the pET-32a-R2VP60 are accordant with the expected size and have good purification effect.
Westernblot identification of pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins
And (3) identifying antigenicity of the pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins prepared in the step (1) by using a Western blot test. Performing SDS-PAGE electrophoresis on the purified recombinant protein, transferring the recombinant protein onto a polyvinylidene fluoride membrane, sealing the recombinant protein for 2 hours at normal temperature by using 5% nonfat milk powder, incubating for 2 hours by using a mouse anti-HIS antibody (1:5000) as a primary antibody, incubating for 2 hours by using HRP-labeled rabbit anti-mouse IgG (1:5000) as a secondary antibody, washing three times by using TBST after each incubation, and finally performing DAB chromogenic analysis. The Westrnblot results are shown in FIG. 2, where M: pre-stained protein markers; 1: pET-32a-R1VP60 protein; 2: pET-32a-R2VP60 protein. Westrn blot results show that recombinant proteins of pET-32a-R1VP60 and pET-32a-R2VP60 can react with an HRP-labeled mouse anti-HIS antibody, and the results show that both recombinant proteins are successfully expressed.
3. Immunization of mice
And measuring the concentration of the purified pET-32a-R2VP60 recombinant protein by using a nucleic acid protein instrument, and adjusting the concentration of the pET-32a-R2VP60 recombinant protein to 0.5mg/mL by using PBS for standby. 8 female SPF-class BALB/c mice were divided into a test group and a control group, wherein 5 female SPF-class BALB/c mice were immunized by injecting pET-32a-R2VP60 recombinant protein. The control group was 3, and the feeder layer required for cell fusion was continued without immunization. Mice from the test group were immunized and the immunization procedure is shown in Table 3.
TABLE 3 immunization procedure for mice of the test group
4. Establishment of an Indirect ELISA method
And (2) respectively taking the pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins prepared in the step (1) as antigen coating, and optimizing the conditions of optimal antigen coating concentration, optimal dilution ratio of serum to be detected and the like by using a chessboard method, wherein the primary antibody respectively uses rabbit anti-RHDV 2 serum and RHDV positive serum which are immunized by virus tissue inactivated vaccine, and the secondary antibody is HRP marked goat anti-rabbit IgG. And when the P/N is more than or equal to 2.1, the positive judgment standard is adopted. Antigen was coated overnight at 4deg.C with a coating concentration of 18 μg/mL for pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins, and the serum dilution to be tested was 1: 320. the secondary antibody dilution was 1: at 8000, the P/N value may be highest. The results show that both pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins can react with corresponding RHDV positive serum.
The rabbit anti-RHDV 2 serum and RHDV positive serum after being immunized by virus tissue inactivated vaccine are prepared by adopting a conventional method: rabbits of 2 months of age were immunized with 0.3% formaldehyde-inactivated virus tissue (liver tissue/saline ratio 1/10), and a total of 3 times of subcutaneous multipoint back injections were performed, 2mL each time, for 15 days.
5. Cell fusion and monoclonal screening
Serum is collected from the mice in the step 3 through tail-breaking blood collection after three-way, the immune antibody titer is determined by replacing the secondary antibody with HRP-marked goat anti-mouse IgG diluted by 1:5000 under the reaction conditions established in the step 4, and the mice with the highest titer are selected for cell fusion. Cell fusion is performed using conventional methods. The fused cells were resuspended in 20% hat medium and plated onto two 96-well plates. The medium was changed in time according to the color change of the medium, and after about 15 days, 20% HT medium was changed. When the hybridoma cells grow to occupy about 1/3 of the hole bottom, the cell supernatant is sucked for positive cell strain screening and antibody titer detection, wherein the screening standard is a specific monoclonal antibody cell strain which is combined with the pET-32a-R2VP60 recombinant protein but not combined with the pET-32a-R1VP60 recombinant protein. And selecting a positive cell line meeting the conditions for subcloning, and finally screening a hybridoma cell line capable of stably secreting the specific RHDV2 monoclonal antibody, which is named as R2McAb2A1.
6. Preparation, potency determination and purification of monoclonal antibody McAb ascites
Monoclonal antibody ascites is prepared by selecting a BALB/c mouse of 6-8 weeks according to a conventional method, sensitization is performed by intraperitoneal injection of paraffin, and the ascites is prepared by injecting the hybridoma cell R2McAb2A1 prepared in the step 5 after about one week. Sucking the ascites after about 15d when the abdomen of the mouse swells, centrifuging the ascites to obtain clarified ascites antibody according to the following formula 1:10 by reference to the ELISA procedure in step 5. ELISA detection results are shown in FIG. 3, and ascites titer of the hybridoma cell R2McAb2A1 monoclonal antibody can reach 1:128000. The prepared ascites is purified by Protein G preassembled gravity column, and purity and size are analyzed by SDS-PAGE electrophoresis after purification. The electrophoresis results are shown in FIG. 4, wherein M: pre-stained protein markers; 1: unpurified monoclonal antibodies; 2: the purified monoclonal antibody, in lane 2, has two proteins with molecular weights of about 25kD and 50kD, respectively, corresponding to the heavy chain and the light chain of the monoclonal antibody, which proves that the purity of the monoclonal antibody is higher.
7. Westernblot identification of monoclonal antibodies
Purifying the virus particles of RHDV1 and RHDV2 by sucrose density gradient centrifugation, carrying out virus negative staining on the purified virus particles, and observing whether the virus particles exist or not under an electron microscope. And (3) performing Westernblot analysis on the purified RHDV1, RHDV2, pET-32a-R1VP60 and pET-32a-R2VP60 by performing SDS-PAGE electrophoresis and then transferring the films on a polyvinylidene fluoride film, wherein the primary antibody in the test is the ascites monoclonal antibody (1:5000) purified in the step (6), and the secondary antibody is the HRP-marked rabbit anti-mouse IgG (1:8000). As shown in fig. 5, the purified virus solution is subjected to projection electron microscope observation, a plurality of virus particles can be observed under the mirror, and fig. 5-a is RHDV1 virus particles; FIG. 5-B is RHDV2 virions; the purified virus liquid is proved to contain virus particles, and can be used for the subsequent Westernblot test. As shown in FIG. 6, the Westernblot results show that the monoclonal antibody generated by the hybridoma R2McAb2A1 can react with natural RHDV2 virus particles and pET-32a-R2VP60 proteins, and target protein bands respectively appear at about 60kD and 79kD, so that the target protein bands conform to the expected size; meanwhile, the monoclonal antibody does not react with RHDV1 virus particles and pET-32a-R1VP60, and the monoclonal antibody generated by the hybridoma cell R2McAb2A1 has good specificity.
EXAMPLE 2 subclass identification and hybridoma chromosome analysis of specific RHDV2 monoclonal antibodies
1. Subclass identification and stability analysis of monoclonal antibodies
Hybridoma cell line R2McAb2A1 of example 1, which had been cultured in 96-well cell plates, was passaged and the culture supernatants of 30 consecutive generations were collected, and after measurement by the ELISA method established in example 1, it was found that antibodies were stably secreted and the titer was substantially stable. The ascites fluid purified in example 1 was diluted 1:100000, and subclass identification was performed by using a mouse monoclonal antibody subclass identification kit, and the result shows that the monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 is of the IgG3 subtype, and the light chain is of the Kappa type.
2. Chromosome number analysis
Taking hybridoma cells and SP20 cells which grow well and are in logarithmic growth phase, adding colchicine to a final concentration of 0.2mg/mL, culturing at 37 ℃ for about 2 hours, centrifuging to remove cell supernatant, adding preheated KCL hypotonic solution of 0.075mol/L for about 5mL, and blowing uniformly in a water bath at 37 ℃ for 20 minutes. After centrifugation again to remove the supernatant, fixing with a fixing solution (methanol: glacial acetic acid=3:1), and finally, after tabletting and giemsa staining, observing the shape and number of the chromosomes under a mirror. The results are shown in FIG. 7, 7-A is the SP20 cell chromosome; in the figure, 7-B is the chromosome of the R2McAb2A1 hybridoma, the chromosome number of the R2McAb2A1 cell of the fusion cell is obviously more than that of the SP20 cell, about 100, the chromosome number characteristics of the hybridoma after the fusion of the spleen cell and the SP20 cell of the mouse are met, and the R2McAb2A1 cell is proved to be the fusion cell.
EXAMPLE 3 test for blood coagulation and blood coagulation inhibition
Treatment of liver of RHDV2 infected dying Rabbit according to the prior artDirty, grinding, repeatedly freezing and thawing for three times, centrifuging to obtain supernatant serving as RHDV2 suspension, and detecting the aggregation inhibition effect of RHDV2 by ascites McAb (prepared in example 1) prepared according to the HA and HI test operation in GB/T14926.54 by using 1% of goose fresh red blood cells as an indication system. Meanwhile, healthy rabbit liver grinding fluid is used as RHDV2 negative control. As shown in FIG. 8, the HA titer of the RHDV2 virus for 4 units of working antigen in 8-A was 2 5 Indicating that virus particles are present in the ground liver tissue homogenate and that RHDV2 virus can agglutinate fresh red blood cells of geese; FIG. 8-B shows that ascites McAb purified from R2McAb2A1 completely inhibits RHDV2 agglutination of goose erythrocytes with HI titers of up to 2 10 。
In summary, the invention uses the expressed pET-32a-R2VP60 recombinant protein as an immunogen, uses pET-32a-R2VP60 and pET-32a-R1VP60 recombinant proteins as coating sources to establish an indirect ELISA screening method, adopts the conventional hybridoma technology to prepare the hybridoma of RHDV2, fuses about 12 hybridoma cells, wherein 91.7 percent of the hybridoma cells are cell strains secreting the facultative monoclonal antibodies of RHDV1 and RHDV2, and finally screens out the hybridoma cell strain R2McAb2A1 through an indirect ELISA method, cloning and subcloning. The monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 is only specifically combined with RHDV2 virus particles, but not combined with RHDV1 virus particles, and has good specificity to the RHDV2 virus particles through Western blot test and hemagglutination inhibition test detection. Therefore, the hybridoma cell strain R2McAb2A1 and the monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 can be applied to preparation of a detection kit for the rabbit hemorrhagic disease virus type2, and can effectively identify the rabbit hemorrhagic disease virus type 2.
The specification and figures are to be regarded in an illustrative rather than a restrictive sense, and one skilled in the art, in light of the teachings of this invention, may make various substitutions and alterations to some of its features without the need for inventive faculty, all being within the scope of this invention.
Claims (4)
1. The hybridoma cell strain R2McAb2A1 is characterized in that the hybridoma cell strain R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022 and 7 months and 27 days, and the preservation number is CCTCC NO: C2022205.
2. A monoclonal antibody secreted by the hybridoma cell line R2McAb2A1 of claim 1.
3. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is a specific monoclonal antibody of rabbit hemorrhagic disease virus type 2VP60 protein, and the amino acid sequence of the rabbit hemorrhagic disease virus type 2VP60 protein is shown in SEQ ID N0.1.
4. Use of a monoclonal antibody according to claim 2 or 3 for the preparation of a rabbit hemorrhagic disease virus type2 detection kit.
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