CN112359021B - Hybridoma cell line for expressing classical swine fever virus NS3 protein monoclonal antibody, antibody and kit - Google Patents
Hybridoma cell line for expressing classical swine fever virus NS3 protein monoclonal antibody, antibody and kit Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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Abstract
The invention belongs to the field of cell engineering and immunology, and particularly relates to a hybridoma cell line for expressing a swine fever virus NS3 protein monoclonal antibody, an antibody and a kit. The hybridoma cell line provided by the invention is prepared by recombinant classical swine fever virus NS3 antigen, immunized mice and fused, screened for specific hybridoma cell strains and other processes. The hybridoma cell line provided by the invention has good antigenicity and strong specificity, and the prepared antibody has good affinity with natural protein, and the kit provided by the invention contains a monoclonal antibody tightly combined with NS3 antigen, and the antibody can be specifically combined with swine fever wild virus, but does not react with swine fever lapinized attenuated vaccine strain, and plays an important role in differential diagnosis of swine fever vaccine strain and wild strain.
Description
Technical Field
The invention belongs to the field of cell engineering and immunology, and particularly relates to a hybridoma cell line for expressing a swine fever virus NS3 protein monoclonal antibody, an antibody and a kit.
Background
Classical Swine Fever (CSF) is a highly contagious disease caused by Classical Swine Fever Virus (CSFV), which is characterized mainly by fever and bleeding, and has a wide prevalence and a high incidence and mortality. Causing serious economic losses to the swine industry, which OIE lists as an infectious disease that must be reported. CSF is still the disease most threatening the global swine industry at present. In China, the hazard of swine fever is very serious, and the swine fever is one of four major epidemic diseases of the swine in China.
CSFV is single strand positive strand RNA virus, the genome is about 12.3kb, the two ends are non-coding regions 5-NCR and 3-NCR, an open reading frame exists in the middle, and the sequence from the N end to the C end is: npro, C, E0, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B. Classical swine fever virus nonstructural protein NS3 is highly conserved, has serine protease, nucleoside triphosphatase and RNA helicase activities, and is an essential protein for virus replication and proliferation. NS3, E0 and E2 are main antigen proteins of CSFV, can induce organisms to generate antibodies, and can reflect the infection condition of CSFV in vivo by detecting the level of NS3 antibody in pigs. In order to adapt to the rapid development of the genetically engineered vaccines of classical swine fever virus E2 and E0, a set of matched serological detection methods for identifying and diagnosing wild virus infection and immunizing vaccinated pigs needs to be established. If the pig is immunized only to express E2 and E0 proteins, but not whole virus, an ELISA detection kit aiming at NS3 recombinant protein can be used for distinguishing the whole virus (including vaccine virus and wild virus) from antibodies generated by a swine fever recombinant vaccine-stimulated organism.
In the prior art, the research on the NS3 monoclonal antibody is very little, and most of the NS3 monoclonal antibodies adopt affinity column chromatography to purify proteins, so that the preparation cost of the monoclonal antibody is increased, the production scale is small, and meanwhile, the existing monoclonal antibody has poor affinity with natural proteins.
Chinese patent application CN110669134A discloses an IgM-FC fragment, an IgM-FC antibody, a preparation method and an application thereof, and provides an IgM-FC antibody, wherein pentameric protein is selected, and the fragment is obtained through the processes of crude separation treatment, protein impurity removal and the like, and is used as immunogen to immunize animals to generate antibodies; although the antibody prepared by the method can be specifically combined with IgM, the specificity is not high, and the affinity chromatography is adopted, so that the production cost is increased.
Meanwhile, Chinese patent CN102435733B discloses an AFP-IgM detection kit and a detection method, the kit comprises immunoglobulin IgM, AFP antibodies, horseradish peroxidase markers and the like, the kit can improve liver cancer diagnosis accuracy and is high in sensitivity, however, the kit needs to spend a large amount of time for detection during detection, false positive problems are easy to occur, and the production cost is high.
In conclusion, the prior art generally has the defects of poor specificity, poor affinity with natural protein, long time consumption, high preparation cost and the like.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a hybridoma cell line for expressing a swine fever virus NS3 protein monoclonal antibody, an antibody and a kit; the cell line provided by the invention can specifically secrete classical swine fever virus NS3 protein monoclonal antibody, and the antibody can be specifically combined with classical swine fever wild viruses, but does not react with classical swine fever lapinized attenuated vaccine strains, and plays an important role in differential diagnosis of classical swine fever vaccine strains and wild strains.
In order to achieve the purpose, the invention adopts the technical scheme that:
a hybridoma cell line CSFV-NS3H for expressing a classical swine fever virus NS3 protein monoclonal antibody is prepared by the following method:
s1, preparation of recombinant classical swine fever virus NS3 antigen: fully mixing the classical swine fever virus NS3 protein with a Freund's complete adjuvant and a Freund's incomplete adjuvant for 10-20 min to prepare a recombinant NS3 antigen;
s2, immunizing a mouse by using the recombinant NS3 antigen prepared in the step S1, and carrying out cell fusion to obtain a fusion cell;
s3, screening the hybridoma capable of secreting the classical swine fever virus NS3 protein monoclonal antibody in the fused cells obtained in the step S2, and marking the hybridoma as CSFV-NS 3H.
Preferably, the adding amount of the classical swine fever virus NS3 protein in the step S1 is 100-300 mg/mL, and the volume ratio of Freund 'S complete adjuvant to Freund' S incomplete adjuvant is 1-3: 5-8 of the additive.
Preferably, the specific procedures for immunizing mice with the recombinant NS3 antigen in step S2 are as follows: uniformly mixing the recombinant antigen obtained in the step S1 with equivalent volume of Freund' S complete adjuvant, and injecting a BALB/c mouse into an abdominal cavity; 14 and 28 days after the first immunization, equal amounts of recombinant antigen and Freund's incomplete adjuvant are mixed, and multiple subcutaneous injections are performed on the same mice for two and three times, and 7 days after each immunization, tail collection is performedBlood, and the level of immune response was measured by ELISA. Collecting blood from tail vein of mouse 14 days after three-immunization, coating with recombinant antigen, detecting serum titer by ELISA, and collecting titer greater than 5 × 106Fusing the spleen cells of the mice with myeloma cells; and (3) screening the fusion cells by using a DMEM/F-12 culture medium containing 15% fetal calf serum.
Preferably, the specific operation of screening the fused cells in step S3 is: screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 3-5 times by a limiting dilution method until the cell positive rate reaches 100%, screening 10-15 strains to obtain a cell strain capable of stably secreting the swine fever virus NS3 protein monoclonal antibody, and reacting antibodies expressed by the screened cells with a swine fever standard virulent phylum strain, a plurality of wild strains and a swine fever rabbit attenuated vaccine strain respectively to obtain a cell strain which can only be combined with the swine fever standard virulent phylum strain and the wild strains but does not react with the swine fever rabbit attenuated vaccine strain and is marked as CSFV-NS 3H.
The hybridoma cell line CSFV-NS3H provided by the invention is preserved in China center for type culture Collection, the address is Wuhan, Wuhan university, the name (classification name) of the culture is hybridoma cell strain CSFV-NS3H, the preservation number is CCTCC NO: C2020227, the preservation date is 11 months and 11 days in 2020,
the invention also provides an NS3H monoclonal antibody of the classical swine fever virus NS3 protein, which is expressed by the CSFV-NS3H hybridoma cell strain.
Preferably, the monoclonal antibody is a subtype of IgM and the light chain is a lambda chain.
The invention also provides an indirect immunofluorescence detection kit for classical swine fever virus, which contains the monoclonal antibody of classical swine fever virus NS3 protein NS 3H.
Preferably, the kit further comprises fibrinogen and a sodium chloride solution with the mass fraction of 0.88%.
The invention also provides a method for expressing the monoclonal antibody by using the hybridoma cell line CSFV-NS3H, which comprises the following steps: culturing CSFV-NS3H hybridoma cell without serum, collecting supernatant, purifying monoclonal antibody by fusion label with self-cutting self-aggregation function, and determining the purity of monoclonal antibody by SDS-PAGE.
Preferably, the serum-free culture adopts HL-1TM complete serum-free culture medium to culture the cells.
When the recombinant classical swine fever virus NS3 antigen is prepared, a Freund's complete adjuvant and a Freund's incomplete adjuvant are added at the same time, and the two are mixed with NS3 protein according to a certain proportion, so that the prepared antigen has strong activity, and the specificity and the sensitivity of the antibody can be obviously improved when the antibody is further prepared. Moreover, the inventor also unexpectedly finds that when the detection kit is prepared, the fibrinogen and the sodium chloride solution with the mass fraction of 0.88% are added, so that the activity of the antibody protein can be improved, the specific combination of the antibody protein and the antigen is accelerated, the detection time is effectively shortened, and the production cost is greatly reduced.
Compared with the prior art, the hybridoma cell line for expressing the classical swine fever virus NS3 protein monoclonal antibody, the antibody and the kit provided by the invention have the following advantages:
(1) the cell line provided by the invention adopts mammalian cell expression to obtain protein for immunization, the protein is close to the natural conformation and modification processing of virus protein, the antigenicity is good, and the affinity of the prepared monoclonal antibody and the natural protein is good;
(2) the fibrinogen and sodium chloride solution are added into the kit provided by the invention, so that the activity of the antibody protein is improved, the affinity reaction between the antibody protein and the antigen is accelerated, the detection time is reduced, and the production cost is saved.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Classical swine fever virus NS3 protein expressed by mammalian cells is produced and stored by Guangzhou Bernitz Biotechnology, Inc.; the hog cholera lapinized attenuated vaccine strain and the wild strain are provided by the military veterinary research institute of the military medical academy of sciences; bovine serum albumin and agarose were purchased from Amresco; diaminobenzidine was purchased from maurel chemical technologies, inc; dimethyl sulfoxide 0.25% trypsin, DMEM/F-12 culture medium and fetal bovine serum are products of Sigma company; commercial products were used for both Freund's complete adjuvant and Freund's incomplete adjuvant.
Example 1A hybridoma cell line CSFV-NS3H expressing classical swine fever virus NS3 protein monoclonal antibody
The hybridoma cell line is prepared by the following method:
s1, preparation of recombinant classical swine fever virus NS3 antigen: fully mixing the classical swine fever virus NS3 protein with Freund's complete adjuvant and Freund's incomplete adjuvant for 10min to prepare a recombinant NS3 antigen; the adding amount of the classical swine fever virus NS3 protein is 100mg/mL, and the volume ratio of Freund's complete adjuvant to Freund's incomplete adjuvant is 1: 5, adding;
s2, mixing the recombinant antigen obtained in the step S1 with equivalent volume of Freund' S complete adjuvant, and injecting a BALB/c mouse into an abdominal cavity; 14 and 28 days after the first immunization, equal amounts of recombinant antigen and Freund's incomplete adjuvant are taken and mixed, the same mice are injected subcutaneously for two times and three times, blood is collected statically at the tail 7 days after each immunization, and the level of immune response is detected by adopting an ELISA method. Collecting blood from tail vein of mouse 14 days after three-immunization, coating with recombinant antigen, detecting serum titer by ELISA, and collecting titer greater than 5 × 106Fusing the spleen cells of the mice with myeloma cells; and (3) screening the fusion cells by using a DMEM/F-12 culture medium containing 15% fetal calf serum.
S3, screening a hybridoma cell capable of secreting the classical swine fever virus NS3 protein monoclonal antibody in the fusion cell obtained in the step S2, and marking the hybridoma cell as CSFV-NS3H to obtain the swine fever virus monoclonal antibody; the screening process comprises the following steps: screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 3 times by a limiting dilution method until the cell positive rate reaches 100%, screening 10 cell strains capable of stably secreting the swine fever virus NS3 protein monoclonal antibody, reacting the antibodies expressed by the 10 cell strains with a swine fever standard virulent phylum strain, a plurality of wild strains and a swine fever rabbit attenuated vaccine strain respectively, and finally obtaining 3 cell strains which can only be combined with the swine fever standard virulent phylum strain and the wild strains but do not react with the swine fever lapinized attenuated vaccine strain and are marked as CSFV-NS 3H.
Example 2 a hybridoma cell line CSFV-NS3H expressing a monoclonal antibody against classical swine fever virus NS3, the hybridoma cell line CSFV-NS3H, prepared by the following method:
s1, preparation of recombinant classical swine fever virus NS3 antigen: fully mixing the classical swine fever virus NS3 protein with Freund's complete adjuvant and Freund's incomplete adjuvant for 20min to prepare a recombinant NS3 antigen; the adding amount of the classical swine fever virus NS3 protein is 300mg/mL, and the volume ratio of Freund's complete adjuvant to Freund's incomplete adjuvant is 3: 8 adding
S2, mixing the recombinant antigen obtained in the step S1 with equivalent volume of Freund' S complete adjuvant, and injecting a BALB/c mouse into an abdominal cavity; 14 and 28 days after the first immunization, equal amounts of recombinant antigen and Freund's incomplete adjuvant are taken and mixed, the same mice are injected subcutaneously for two times and three times, blood is collected statically at the tail 7 days after each immunization, and the level of immune response is detected by adopting an ELISA method. Collecting blood from tail vein of mouse 14 days after three-immunization, coating with recombinant antigen, detecting serum titer by ELISA, and collecting titer greater than 5 × 106Fusing the spleen cells of the mice with myeloma cells; screening the fused cells by using a DMEM/F-12 culture medium containing 15% fetal calf serum to obtain fused cells;
s3, screening a hybridoma cell capable of secreting the classical swine fever virus NS3 protein monoclonal antibody in the fusion cell obtained in the step S2, and marking the hybridoma cell as CSFV-NS3H to obtain the swine fever virus monoclonal antibody; the screening process comprises the following steps: screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 5 times by a limiting dilution method until the cell positive rate reaches 100%, screening 12 cell strains capable of obtaining a cell strain capable of stably secreting the swine fever virus NS3 protein monoclonal antibody, reacting antibodies expressed by the 12 cell strains with a swine fever standard virulent phylum strain, a plurality of wild strains and a swine fever rabbit attenuated vaccine strain respectively, and finally obtaining 4 cell strains which can only be combined with the swine fever standard virulent phylum strain and the wild strains but do not react with the swine fever lapinized attenuated vaccine strain and are marked as CSFV-NS 3H.
Example 3 a hybridoma cell line CSFV-NS3H expressing a monoclonal antibody against classical swine fever virus NS3, the hybridoma cell line CSFV-NS3H, prepared by the following method:
s1, preparation of recombinant classical swine fever virus NS3 antigen: fully mixing classical swine fever virus NS3 protein with Freund's complete adjuvant and Freund's incomplete adjuvant for 15min to prepare recombinant NS3 antigen; the adding amount of the classical swine fever virus NS3 protein is 200mg/mL, and the volume ratio of Freund's complete adjuvant to Freund's incomplete adjuvant is 2: 7, adding;
s2, mixing the recombinant antigen obtained in the step S1 with equivalent volume of Freund' S complete adjuvant, and injecting a BALB/c mouse into an abdominal cavity; 14 and 28 days after the first immunization, equal amounts of recombinant antigen and Freund's incomplete adjuvant are taken and mixed, the same mice are injected subcutaneously for two times and three times, blood is collected statically at the tail 7 days after each immunization, and the level of immune response is detected by adopting an ELISA method. Collecting blood from tail vein of mouse 14 days after three-immunization, coating with recombinant antigen, detecting serum titer by ELISA, and collecting titer greater than 5 × 106Fusing the spleen cells of the mice with myeloma cells; screening the fused cells by using a DMEM/F-12 culture medium containing 15% fetal calf serum to obtain fused cells;
s3, screening a hybridoma cell capable of secreting the classical swine fever virus NS3 protein monoclonal antibody in the fusion cell obtained in the step S2, and marking the hybridoma cell as CSFV-NS3H to obtain the swine fever virus monoclonal antibody; the screening process comprises the following steps: screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 4 times by a limiting dilution method until the cell positive rate reaches 100%, screening 15 cell strains capable of obtaining cell strains capable of stably secreting the swine fever virus NS3 protein monoclonal antibody, reacting the antibodies expressed by the 15 cell strains with a swine fever standard virulent phylum strain, a plurality of wild strains and a swine fever rabbit attenuated vaccine strain respectively, and finally obtaining 6 cell strains which can only be combined with the swine fever standard virulent phylum strain and the wild strains but do not react with the swine fever rabbit attenuated vaccine strain and are marked as CSFV-NS 3H.
Example 4 potency assay
1. And (3) measuring the immune serum titer: by indirect ELISAAnd (5) determining the titer of immune serum. 100 ug of the recombinant classical swine fever virus NS3 protein was dissolved in 10mL of 0.05M pH9.6 carbonate buffer, coated with a 96-well microplate, 100 ul/well, overnight at 4 ℃. The following day, the plates were washed three times with PBS (containing 0.05% (V/V) Tween-20), mice were bled at the orbital 7 days after each immunization, and the mouse immune sera were treated with 10mMPBS containing 1% BSA 10mMPBS at 10-1-10-8Diluting, adding 96-well plate, washing at 37 deg.C for 1 hr with PBS (containing 0.05% (V/V) Tween-20) for three times, adding 1: 1000-time diluted horseradish peroxidase labeled goat anti-mouse IgG secondary antibody at 37 deg.C for 1 hr, washing with the plate, developing with TMB, 100. mu.L/well, keeping the plate away from light at room temperature for 10min, adding 100. mu.L/well 2M H2SO4Stopping reaction, measuring 450nm absorption value, taking the serum of the mouse before immunization as negative control, and taking the ratio of the measured value to the control value to be more than or equal to 2.1 as positive to judge the titer of the immune serum.
2. Titer determination of purified antibody: mu.g of recombinant CSFV NS3 protein was dissolved in 10mL of 0.05M carbonate coating buffer pH9.6, added to a 96-well plate at 100. mu.l/well and coated overnight at 4 ℃. PBST washing plate three times, with 1% BSA blocking solution PBS150 u L/hole, 37 degrees C blocking for 1 h; washing the plate three times with PBST, adding 100 μ L purified antibody into each well, incubating at 37 deg.C for 1h, washing the plate three times with PBST, adding horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, washing the plate three times with 100 μ L/well at 37 deg.C for 1h, developing with TMB, 100 μ L/well, keeping the plate dark at room temperature for 15min, adding 100 μ L/well 2M H2SO4The reaction was stopped and the absorbance at 450nm was measured.
Example 5 an indirect immunofluorescence assay kit for hog cholera virus
The detection kit adopts the following method: adding 0.15 mu g of fibrinogen into a sodium chloride solution with the mass fraction of 0.88% and the volume of 5 mu L, then uniformly mixing with 45 mu L of Modifier reagent, adding 50 mu L of purified NS3 protein, uniformly mixing, adding the mixed liquid into an FITC freeze-dried powder tube containing 120 mu g, processing for 4h at 25 ℃ in the dark, finally adding 50 mu L of a Quencher reagent, uniformly mixing, standing for 1h, subpackaging, and storing at-80 ℃ for later use.
Comparative example 1 hybridoma cell line CSFV-NS3H expressing classical swine fever virus NS3 protein monoclonal antibody
The hybridoma cell line was prepared in a similar manner to example 3;
the difference from example 3 is that the Freund's complete adjuvant and Freund's incomplete adjuvant in comparative example 1 were mixed in a volume ratio of 1:1, preparing a composition; finally, 1 cell line was selected.
Comparative example 2 Swine fever virus indirect immunofluorescence detection kit
The indirect immunofluorescence detection kit for the hog cholera virus is similar to that in example 5;
the difference from example 5 is that comparative example 2 does not contain fibrinogen and sodium chloride solution.
Test example 1 monoclonal antibody purity determination
1. Test samples: hybridoma cell lines obtained in examples 1 to 3 and comparative example 1;
2. the test method comprises the following steps: the cells prepared in examples 1-3 and comparative example 1 were cultured in HL-1TM complete serum-free medium for 12h, the supernatant was collected, then the pellet was thoroughly resuspended in an equal volume of cleavage buffer B2(20mmol/LTris-HCl, 500mmol/LNaCl, 1mmol/LEDTA, 40mmol/L DTT, pH8.5), and the pellet was placed in a 4 ℃ rotary homogenizer to induce cleavage for 24 h. After induction cutting, centrifuging at 8000rpm for 20min at 4 ℃, collecting supernatant, and determining the purity of the monoclonal antibody by SDS-PAGE; the antibody concentration was measured using a Smart Spec plus protein analyzer manufactured by BIO-RAD; the subtype of hybridoma cell lines was identified using a murine monoclonal antibody subtype identification kit from Hbt.
3. And (3) test results: through detection, the monoclonal antibody is an IgM subtype, the light chain is a lambda chain, and the specific test results of concentration and purity are shown in Table 1.
TABLE 1 comparison of purity and concentration of monoclonal antibodies prepared by different methods
| Test sample | Concentration of monoclonal antibody/mg/mL | Purity/% of monoclonal antibody |
| Example 1 | 1.08 | 96.7％ |
| Example 2 | 1.12 | 97.3％ |
| Example 3 | 1.18 | 98.5％ |
| Comparative example 1 | 0.76 | 78.9％ |
Therefore, the purity of the monoclonal antibody secreted by the cell strains screened by the methods shown in examples 1-3 of the present application is above 96%, especially the purity of the monoclonal antibody in the group of example 3 is 98.5%, so the group of example 3 is the best example of the present invention; while comparative example 1 changed the ratio of the two adjuvants, affecting the antigenic activity, eventually reducing the single antibody purity to below 80%.
Test example 2 hog cholera virus test
1. Test samples: the cells obtained in examples 1 to 3 and comparative example 1; the kits selected in example 5 and comparative example 2;
2. the test method comprises the following steps:
2.1 sensitivity test: using PKWRL cells at 2X 104Laying a 96-well plate at a density of one well and 5% CO at 36 deg.C2Culturing in an incubator. Diluting hog cholera virus 16h after cell adherence, and inoculating into 96-well plate, wherein each well is provided with80 μ L, at 36 ℃ and 5% CO2Culturing in an incubator for 48 h. Discarding the culture solution, washing with PBS for 2 times, fixing the cells with precooled 4% paraformaldehyde, and fixing at room temperature for 1 h; washing with PBS for 2 times, adding 0.1% Triton, 40 μ L/well, and penetrating membrane for 20 min; and equally dividing into 8 parts, respectively diluting FITC-labeled classical swine fever virus E2 protein monoclonal antibody into 20, 100, 500, 1000, 2000, 4000, 6000 and 8000 times, respectively adding into the cell fluid, respectively placing in a 37 ℃ incubator for incubation for 1h, washing with PBS for 3 times, and observing fluorescence intensity under a fluorescence microscope.
2.2 determination of specificity: bovine viral diarrhea virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus standard virulent phylum strain and lapinized attenuated strain are taken, and immunofluorescence experiments are respectively carried out by using the kits described in example 5 and comparative example 2 to detect the specificity of the monoclonal antibody.
2.3 lapinized low virulent strain and wild virulent strain of hog cholera: taking a classical swine fever virus standard virulent phylum Strain (SM), wild strains (comprising SM, GD53-2.1c, GD176-2.1b, GD2-2.1c, GD49-2.1j and HY-11-2.1h) and a lapinized low-virulent strain (HCLV), carrying out an immunofluorescence experiment, and detecting the specificity of the monoclonal antibody.
3. And (3) test results: the specific test results are shown in Table 2.
TABLE 2 test results of different kits
As can be seen from Table 2, the antigenicity and sensitivity of the groups of examples 1-3 of the present invention are both very high, and fluorescence can still be emitted after being diluted to more than 6000 times, especially, the fluorescence intensity of the group of example 3 is still very strong when being diluted to 6000 times, and is reduced only when being diluted to 8000 times, so that example 3 is the best example of the present invention; in addition, the kit provided in the comparative example 2 does not affect the detection result of the specificity of the monoclonal antibody by removing fibrinogen and a sodium chloride solution, but the sensitivity to the monoclonal antibody is obviously reduced, the kit provided in the comparative example 2 is adopted, the fluorescence intensity is greatly reduced, the proportion of an adjuvant is changed in the comparative example 1, the antigen activity is changed, and the identification result of the monoclonal antibody in different strains can be affected.
Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A hybridoma cell line CSFV-NS3H for expressing a classical swine fever virus NS3 protein monoclonal antibody is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2020227, and is characterized by being prepared by the following method:
s1, preparation of recombinant classical swine fever virus NS3 antigen: fully mixing the classical swine fever virus NS3 protein with a Freund's complete adjuvant and a Freund's incomplete adjuvant for 10-20 min to prepare a recombinant NS3 antigen;
s2, immunizing a mouse by using the recombinant NS3 antigen prepared in the step S1, and carrying out cell fusion to obtain a fusion cell;
s3, screening the hybridoma capable of secreting the classical swine fever virus NS3 protein monoclonal antibody in the fused cells obtained in the step S2, and marking the hybridoma as CSFV-NS 3H.
2. The hybridoma cell line CSFV-NS3H according to claim 1, wherein the classical swine fever virus NS3 protein is added in an amount of 100 μ g/individual for the primary immunization, 50 μ g/individual for the secondary and tertiary immunization, and complete freund 'S adjuvant and incomplete freund' S adjuvant are added in a volume ratio of 1-3: 5-8 of the additive.
3. The hybridoma of claim 1The cell line CSFV-NS3H, wherein the specific operation process of immunizing the mouse with the recombinant NS3 antigen in step S2 is as follows: uniformly mixing the recombinant antigen obtained in the step S1 with equivalent volume of Freund' S complete adjuvant, and injecting a BALB/c mouse into an abdominal cavity; 14 and 28 days after the first immunization, mixing equivalent recombinant antigen and Freund's complete adjuvant, injecting the same mouse for two and three times by multiple points subcutaneously, collecting blood 7 days after each immunization, and detecting the immune response level by adopting an ELISA method; collecting blood from tail vein of mouse 14 days after three-immunization, coating with recombinant antigen, detecting serum titer by ELISA, and collecting titer greater than 5 × 106Fusing the spleen cells of the mice with myeloma cells; and (3) screening the fusion cells by using a DMEM/F-12 culture medium containing 15% fetal calf serum.
4. The hybridoma cell line CSFV-NS3H of claim 1, wherein the fused cells are selected in step S3 by: screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, continuously cloning for 3-5 times by a limiting dilution method until the cell positive rate reaches 100%, screening 10-15 strains to obtain a cell strain capable of stably secreting the swine fever virus NS3 protein monoclonal antibody, and reacting antibodies expressed by the screened cells with a swine fever standard virulent phylum strain, a plurality of wild strains and a swine fever rabbit attenuated vaccine strain respectively to obtain a cell strain which can only be combined with the swine fever standard virulent phylum strain and the wild strains but does not react with the swine fever rabbit attenuated vaccine strain and is marked as CSFV-NS 3H.
5. An NS3H monoclonal antibody of classical swine fever virus NS3 protein, which is expressed by the CSFV-NS3H hybridoma cell line of claim 1; the preservation number of the CSFV-NS3H hybridoma cell line is CCTCC NO: C2020227.
6. The NS3H monoclonal antibody of classical swine fever virus NS3 protein of claim 5, wherein the monoclonal antibody is of an IgM subtype and the light chain is a lambda chain.
7. An indirect immunofluorescence detection kit for classical swine fever virus, comprising the monoclonal antibody of classical swine fever virus NS3 protein NS3H according to claim 5 or 6.
8. The kit of claim 7, further comprising fibrinogen and 0.88% by weight sodium chloride solution.
9. A method for expressing a monoclonal antibody using the hybridoma cell line CSFV-NS3H according to claim 1, comprising the steps of: culturing CSFV-NS3H hybridoma cell without serum, collecting supernatant, purifying monoclonal antibody by fusion label with self-cutting and self-aggregating function, and determining the purity of monoclonal antibody by SDS-PAGE;
the preservation number of the CSFV-NS3H hybridoma cell line is CCTCC NO: C2020227.
10. The method for expressing monoclonal antibodies of claim 9, wherein said serum-free culture comprises culturing the cells in HL-1TM complete serum-free medium.
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