Hybridoma cell strain capable of secreting anti-Senecavirus A monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain secreting an anti-Senecavirus type A monoclonal antibody and application thereof, and belongs to the technical field of hybridoma cell strains. The preservation number of the hybridoma cell strain SVA 1E5 is CCTCC (China Center for Type Culture Collection) NO: C2024263, the preservation date is September 24, 2024, and the preservation unit is China Center for Type Culture Collection. Experiments prove that the titer of the monoclonal antibody secreted by the hybridoma cell strain 1E5 provided by the invention can reach 1: 6553600 times of dilution, the monoclonal antibody can be subjected to specific reaction with VP2 protein and Seneca virus A, and the monoclonal antibody can be applied to research and development of SVA immunodetection products, for example, the monoclonal antibody can be applied to detection of VP2 protein on a colloidal gold platform, and can also be applied to detection of Seneca virus A and VP2 protein. And an immunodetection antibody raw material is provided for rapid on-site detection of the SVA virus.

Description

Technical Field

[0001] The present invention relates to the technical field of hybridoma cell lines, in particular to a hybridoma cell line secreting anti-type A Seneca virus monoclonal antibody and an application thereof. Background Art

[0002] Senecavirus type A (SVA), also known as Seneca Valley virus (SVV), is a virus that has pigs as its primary natural reservoir. SVA infection causes a vesicular disease that is clinically indistinguishable from foot-and-mouth disease, swine vesicular disease, vesicular stomatitis or vesicular rash. Clinical signs in pigs infected with SVA include fluid-filled and ruptured vesicles, ulcerative lesions on the nose and coronary bands, lameness, anorexia, lethargy, skin congestion and fever. SVA infection has also been associated with neonatal piglet deaths (epidemic transient neonatal losses (ETNLs)) in herds of sows infected with SVA. Regardless of the clinical presentation of the disease, SVA infection produces a rapid neutralizing antibody (NA) response in sows and finishing pigs. The lack of an available commercial vaccine and the fact that SVA is not included in the OIE Terrestrial Animal Health Code mean that the incidence of SVA infection in pigs is increasing in different countries. To address this threat, animal husbandry authorities at all levels should monitor the prevalence of SVA, remain vigilant and investigate effective control measures. The diagnosis of SVA can be made by detecting the virus, viral antigens, or viral RNA.

[0003] However, the above detection methods are cumbersome and time-consuming, and require special instruments and technicians, which are not suitable for on-site detection and clinical detection in primary veterinary diagnosis departments. Therefore, a fast, accurate and simple detection method is developed for the detection of SVA antigen, which is conducive to accurately distinguishing infected or vaccinated animals, timely eliminating infected breeding animals, achieving the purpose of population purification, and promoting the healthy development of animal husbandry. Summary of the invention

[0004] The purpose of the present invention is to provide a hybridoma cell line secreting anti-type A Seneca virus monoclonal antibody and its application to solve the problems existing in the above-mentioned prior art. The monoclonal antibody secreted by the hybridoma cell line 1E5 can specifically react with VP2 protein and type A Seneca virus. The monoclonal antibody can be applied to the development of SVA immunoassay products, thereby providing an immunoassay antibody raw material for rapid on-site detection of SVA virus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a hybridoma cell line SVA1E5 that secretes a monoclonal antibody against the VP2 protein of type A Seneca virus. The deposit number of the hybridoma cell line SVA 1E5 (Hybridoma cell line SVA 1E5) is CCTCC NO: C2024263, the deposit date is September 24, 2024, the deposit unit is China Center for Type Culture Collection, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The present invention also provides a monoclonal antibody against type A Seneca virus VP2 protein, wherein the monoclonal antibody is secreted and produced by the hybridoma cell line SVA 1E5.

[0008] Furthermore, the monoclonal antibody specifically binds to the VP2 protein antigen of type A Seneca virus.

[0009] The present invention also provides a method for preparing the monoclonal antibody, comprising the step of utilizing the hybridoma cell line SVA1E5 to secrete and produce the monoclonal antibody.

[0010] The present invention also provides the use of the hybridoma cell line or the monoclonal antibody in preparing a reagent or a kit for detecting type A Seneca virus.

[0011] Furthermore, the method comprises using the monoclonal antibody to detect Seneca virus type A by colloidal gold immunochromatography.

[0012] The present invention also provides the use of the hybridoma cell line or the monoclonal antibody in preparing antibodies or vaccines against type A Seneca virus.

[0013] The present invention also provides a reagent or a kit for detecting type A Seneca virus, comprising the monoclonal antibody.

[0014] The present invention also provides an antibody or vaccine against type A Seneca virus, comprising the monoclonal antibody.

[0015] The present invention discloses the following technical effects:

[0016] The invention utilizes molecular biological technology and adopts an Escherichia coli prokaryotic expression system to successfully express and purify a recombinant SVA VP2 protein.

[0017] The present invention uses recombinant SVA VP2 protein to immunize mice, and obtains hybridoma cell line 1E5 that secretes anti-SVA VP2 protein monoclonal antibodies through cell fusion and clone screening. The monoclonal antibody secreted by the hybridoma cell line 1E5 has a titer of up to 1:6553600 times dilution, can specifically react with VP2 protein and type A Seneca virus, and can be applied to the development of SVA immunoassay products. For example, it can be used to detect VP2 protein on a colloidal gold platform, thereby providing an immunoassay antibody raw material for rapid on-site detection of SVA virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0019] Figure 1 The purification results of recombinant SVA VP2 protein; 1-5 are: loading flow-through, 50mM imidazole elution, 100mM imidazole elution, 200mM imidazole elution, 300mM imidazole elution; M: protein molecular weight standard;

[0020] Figure 2 The antibody titer is the anti-SVA VP2 protein monoclonal antibody 1E5;

[0021] Figure 3 The purity of the monoclonal antibody 1E5 against SVA VP2 protein; M: protein molecular weight standard, 1E5: monoclonal antibody 1E5;

[0022] Figure 4 Western blot was used to identify the reactivity of monoclonal antibody 1E5; 1: recombinant SVA VP2 protein; 2: SVA virus; 3: PK15 cells; M: protein molecular weight standard;

[0023] Figure 5 The results of monoclonal antibody 1E5 applied to the colloidal gold detection platform; 1: negative control protein BSA; 2: recombinant SVA VP2 protein. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1 Preparation of recombinant SVA VP2 protein

[0030] 1.1 Gene synthesis

[0031] Primers were designed using the gene sequence of SVA VP2 protein (gene accession number: MH316116.1) as a template, and the primer sequences are shown in Table 1. Among them, F1 is the upstream primer, R1 is the downstream primer, and Nde I and Xho I restriction sites are added to the upstream and downstream 5' ends of the primers, respectively (the underlined part is the restriction site, and the wavy part is the HIS tag sequence).

[0032] Table 1 Primers for amplifying the SVA VP2 protein coding sequence

[0033]

[0034] 1.2 Plasmid construction and prokaryotic expression

[0035] The synthesized SVA VP2 gene sequence was amplified. The reaction system was: 4 μL of SVA VP2 gene fragment, 2 μL of upstream and downstream primers, 25 μL of PrimeSTAR HS (Premix), and sterile water was added to 50 μL. Amplification program: 95°C for 5 min; 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; cooled to 10°C for storage. After the PCR product was identified by agarose gel electrophoresis, the target gene fragment was purified and recovered using a gel recovery kit.

[0036] The expression vector pET-30a(+) was digested with restriction endonucleases Xho I and Nde I and then connected to the SVA VP2 gene gel recovery product. The competent cells of E. coli Trans10 were transformed and the vector construction was identified by PCR. The strains identified as positive by PCR were sequenced to ensure the correct connection of the recombinant gene sequence. Sequencing was performed by Beijing Liuhe BGI Technology Co., Ltd.

[0037] The correctly connected recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, and the cells were inoculated into LB liquid medium. After culturing at 37℃ for 1 hour, the cells were plated onto LB solid medium containing kanamycin and cultured at 37℃ overnight. The colonies were picked and transferred to LB liquid medium containing kanamycin and cultured at 37℃ for 4 hours. After PCR verification, the positive bacterial liquid was screened and inoculated into LB liquid medium for expansion and cultured until OD 600 nm When the pH value was 0.8, isopropylthiogalactoside (IPTG) was added at a final concentration of 0.1 mmol / L to induce expression. The culture was carried out at 20°C for 18 hours, and the bacterial solution and precipitate were collected by centrifugation. The precipitate was resuspended in a resuspension buffer (Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4), and then ultrasonically disrupted in an ice bath.

[0038] 1.3 Purification and identification of recombinant proteins

[0039] After the SVA VP2 was broken, the bacterial solution was centrifuged, and the supernatant was filtered with a 0.45 μm filter membrane, and the His-tagged protein was purified using a Ni-NTA Resin affinity chromatography column. The protein was eluted using 50 mmol / L, 100 mmol / L, 200 mmol / L, and 300 mmol / L imidazole buffers, and the elution peak was collected. The purified protein was concentrated and dialyzed into 1× PBS to completely remove the residual imidazole, thereby obtaining the purified recombinant SVA VP2 protein.

[0040] After ultrafiltration and concentration, the recombinant SVA VP2 protein was subjected to SDS-PAGE electrophoresis to identify the purity of the protein, and the concentration of the purified recombinant protein was determined according to the instructions of the Bradford protein quantitative detection kit. The results are as follows: Figure 1 As shown, it can be seen that 100mmol / L imidazole has the best elution effect.

[0041] Example 2 Preparation of mouse anti-SVA VP2 protein monoclonal antibody

[0042] 2.1 Immune process

[0043] The developed recombinant SVA VP2 protein was used as an antigen to immunize mice. The antigen was appropriately diluted with physiological saline, mixed and emulsified with an equal amount of Freund's complete adjuvant, and injected subcutaneously at multiple points on the back of the mice. A total of 3-4 immunizations were performed, 50 μg / mouse, with an interval of 2 weeks each time. For the second and third immunizations, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant; one week after the third immunization, the tail blood of the mice was taken to test its titer. The results are shown in Table 2. Mice with higher titers that met the fusion requirements were selected, and 50 μg of antigen was injected intraperitoneally for booster immunization 3 days before the expected cell fusion date.

[0044] Table 2 The titer data of mice after three immunizations

[0045] 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:25600 1:51200 1:102400 1:204800 blank Negative 0.4112 0.2737 0.1828 0.1272 0.1065 0.0961 0.0766 0.0750 0.0687 0.1058 0.1397 0.0625 1# 3.5632 3.5461 3.4862 3.3212 3.2075 2.7049 1.8008 1.1413 0.6689 0.3850 0.2471 0.0651 2# 3.0751 3.1852 3.2204 3.2262 3.1251 2.7650 1.9649 1.2856 0.8182 0.4050 0.2914 0.0729 3# 3.3748 3.4353 3.4675 3.3669 3.3329 2.9721 2.5508 1.5478 0.9255 0.3476 0.3513 0.1020

[0046] 2.2 Antibody Preparation

[0047] Resuscitate SP 2 / 0 cells and culture them in an incomplete DMEM culture medium at 37°C, 5% CO2 incubator. When the cells are in the logarithmic growth phase and in good condition, and when they have reached a certain number, blow off the cells, centrifuge at 1000rpm for 10 minutes, and discard the supernatant. Resuspend and mix with an appropriate amount of culture medium, take a small amount of the resuspension and count under a microscope for later use. Prepare feeder cells according to routine operations, resuspend with an appropriate amount of HAT, and plate in a 96-well plate.

[0048] Take the mouse with the highest serum titer after immunization, and take eyeball blood as a positive control. Take the mouse spleen according to routine operations, use incomplete DMEM culture medium to prepare spleen cell suspension, centrifuge at 1000rpm for 10min, discard the supernatant, repeat washing and centrifugation. Take a small amount of cell suspension, count under a microscope and set aside.

[0049] Mix spleen cells and SP2 / 0 at a ratio of (5-10):1, centrifuge at 1000rpm for 10min, and discard the supernatant. After breaking up the cells, slowly add 0.8mL PEG, let stand for about 30s, and then quickly add DMEM culture medium. Centrifuge at 1000rpm for 10min and discard the supernatant. Suspend the cells with HAT selection medium, mix well, and drop into a 96-well plate containing feeder cells, and culture in a 37℃, 5% CO2 incubator.

[0050] Observe the growth of fused cells 7 days after cell fusion, replace the culture medium with HT selection medium, and detect positive clones the next day. 450 nm The cells with high values ​​and monoclonal clones were subcloned by limiting dilution method until a single hybridoma cell line that could secrete antibodies was screened out. A total of 12 hybridoma cell lines including 1E5 were obtained. The 1E5 cell line was selected and subcloned to a fixed strain. 5 The antibody was injected into the peritoneal cavity of mice at a density of 1.54 g / mouse. The ascites was collected on days 7 to 14 and affinity purified with Protein A to obtain the 1E5 antibody.

[0051] Among them, the hybridoma cell line SVA 1E5 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: C2024263.

[0052] 2.3 Antibody titer and purity identification

[0053] The recombinant SVA VP2 protein was used as the detection antigen, and the antibody titer was determined by indirect ELISA. The antigen was diluted with carbonate buffer to a concentration of 5 μg / mL, and 100 μL was coated on each well of a 96-well plate and left to stand at 4°C overnight. The coating solution was discarded, and 200 μL of 10% calf serum was added to each well for blocking, and incubated at 37°C for 1h or at 4°C overnight. The blocking solution was discarded and washed three times with PBST. 1 μg of 1E5 antibody was diluted in multiples and 100 μL was added to a 96-well plate, incubated at 37°C for 60min, and washed three times with PBST. 100 μL of HRP-labeled goat anti-mouse IgG antibody was added to each well, incubated at 37°C for 30min, and washed three times with PBST. 100 μL of TMB colorimetric solution was added to each well and incubated at 37°C for 15min. 50 μL of stop solution was added to each well, and the OD was read using an enzyme reader. 450 nm The antibody purification effect was identified using conventional SDS-PAGE.

[0054] like Figure 2 As shown, the titer of 1E5 antibody can reach 1:6553600 times dilution. Figure 3 ), 1E5 antibody showed obvious bands near the relative molecular mass of 50kDa and 25kDa, corresponding to the heavy chain and light chain of the antibody, respectively, with good purity.

[0055] Example 3 Characterization of monoclonal antibody 1E5

[0056] 3.1 Western blot detection of the reaction characteristics of monoclonal antibody 1E5

[0057] The reactivity of monoclonal antibody 1E5 with viruses and antigens was detected by Western blot. The purified recombinant SVAVP2 protein, concentrated SVA virus, and PK15 cells were mixed with the loading buffer, boiled for 10 minutes, and then subjected to SDS-PAGE. After completion, they were transferred to a nitrocellulose membrane and blocked with 5% skim milk powder at 4°C overnight. Purified monoclonal antibody 1E5 (1:1000 dilution) was added, incubated at room temperature for 2 hours, and washed 3 times with PBST. Goat anti-mouse IgG HRP secondary antibody was added, incubated at room temperature for 1 hour, and washed 3 times with PBST. Color development solution was added for color development. According to Figure 4 It can be seen that monoclonal antibody 1E5 specifically reacts with recombinant protein SVA VP2 and SVA virus.

[0058] 3.2 Reaction characteristics of monoclonal antibody 1E5 on colloidal gold platform

[0059] The reactivity of monoclonal antibody 1E5 with viral antigen (purified recombinant SVA VP2 protein) was detected by colloidal gold colorimetric test. The purified recombinant SVA VP2 protein and negative control protein (BSA) were coated on the NC membrane, where the C line (quality control line) was coated with goat anti-mouse IgG at a coating concentration of 0.8 mg / mL, and the T line (detection line) was coated with purified recombinant SVA VP2 protein and negative control protein (BSA), respectively, at a coating concentration of 1 mg / mL. Then the 1E5 monoclonal antibody was labeled with colloidal gold, and the test strips were assembled after labeling. The gold label pad and sample pad were sequentially adhered to the coated NC membrane. After cutting the strips, the gold label antibody was applied to the gold label pad at a volume of 4 μL / cm. After the gold label pad was dried, the test was performed, and PBS was dripped onto the sample pad of the assembled test strips in sequence for colorimetric reaction. Figure 5 It can be seen that monoclonal antibody 1E5 can react with viral antigen (purified recombinant SVA VP2 protein), but has no reaction with negative control protein (BSA), thus indicating that monoclonal antibody 1E5 can recognize recombinant SVA VP2 protein and has good reactivity.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A hybridoma cell line SVA 1E5 secreting a monoclonal antibody against the VP2 protein of Seneca virus type A, characterized in that: The deposit number of the hybridoma cell line SVA 1E5 is CCTCC NO: C2024263.

2. A monoclonal antibody against Senecavirus VP2 protein type A, characterized in that: The monoclonal antibody is secreted and produced by the hybridoma cell line SVA 1E5 according to claim 1.

3. The monoclonal antibody according to claim 2, characterized in that The monoclonal antibody specifically binds to the VP2 protein antigen of type A Seneca virus.

4. The method for preparing a monoclonal antibody according to claim 2 or 3, characterized in that: The method comprises the step of utilizing the hybridoma cell line SVA 1E5 of claim 1 to secrete and produce the monoclonal antibody.

5. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to any one of claims 2 to 3 in the preparation of a reagent or a kit for detecting type A Seneca virus.

6. The use according to claim 5, characterized in that The invention comprises using the monoclonal antibody to detect type A Seneca virus by colloidal gold immunochromatography.

7. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to any one of claims 2 to 3 in the preparation of antibodies or vaccines against type A Seneca virus.

8. A reagent or kit for detecting type A Seneca virus, characterized in that: Comprising the monoclonal antibody according to claim 2 or 3.

9. An antibody or vaccine against Seneca virus type A, characterized in that: Comprising the monoclonal antibody according to claim 2 or 3.

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