CN108329378B - Senakava virus VP1 protein, coding gene, hybridoma cell strain and monoclonal antibody and application thereof - Google Patents
Senakava virus VP1 protein, coding gene, hybridoma cell strain and monoclonal antibody and application thereof Download PDFInfo
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- CN108329378B CN108329378B CN201810200884.3A CN201810200884A CN108329378B CN 108329378 B CN108329378 B CN 108329378B CN 201810200884 A CN201810200884 A CN 201810200884A CN 108329378 B CN108329378 B CN 108329378B
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Abstract
The invention relates to a Seneca Valley virus VP1 protein, a coding gene, a hybridoma cell strain, a monoclonal antibody and application thereof, belonging to the technical field of viruses. The Sagnac Valley virus Hubei strain VP1 protein is used as the antigen protein of Sagnac Valley virus in the application of immunoassay. The invention provides a hybridoma cell strain 2G6 secreting monoclonal antibody against the protein of the Seneca Valley virus Hubei strain VP1 of claim 1, wherein the preservation number of the hybridoma cell strain is CCTCC NO: C2017226. The monoclonal antibody is secreted by the hybridoma cell strain 2G 6. The monoclonal antibody is applied to immunological binding of the protein of Hubei strain VP1 of Seneca valley virus. The invention provides application of the monoclonal antibody in a kit for blocking cells infected by Hubei strain of Seneca valley virus.
Description
Technical Field
The invention belongs to the technical field of viruses, and particularly relates to a Seneca Valley virus VP1 protein, a coding gene, a hybridoma cell strain, a monoclonal antibody and application thereof.
Background
The Seneca Valley Virus (SVV) genome is single-stranded, positive-stranded and non-segmented RNA, has no envelope, has the particle diameter of 25-30 nm, and is the only member of Seneca Virus of picornaviridae. Can cause blister and ulcer of oral mucosa, nose and hoof tissues of sows and fattening pigs to further cause anorexia, high fever and lameness; the piglets develop infectious vesicular diseases such as sudden death, severe or occasional fatal diarrhea, dehydration and lethargy.
C6 cell culture, identified as a potential oncolytic virus for tumors of neuroendocrine origin, was occasionally discovered by a genetic therapy company in the us in 2002, and was designated SVV-001, which was not genome-wide sequenced until 2005. Since 9 months in 2015, the swine herds in multiple areas of the middle and western part of the United states have vesicular epidemic situation, which is spread, the nasal kisses and hoof-crown-shaped zones of infected pigs have vesicular lesions, occasionally have diarrhea symptoms, the disease condition is rapid, and the fatality rate of newborn piglets is more than 30-70%. The etiology differential diagnosis of the Meidao exotic animal disease laboratory (FADDL) eliminates important exotic animal epidemic diseases such as foot-and-mouth disease, swine vesicular disease, vesicular stomatitis and the like, and is finally diagnosed as Seneca Valley virus infection.
Since 2015, the reports of SVV infected swinery appear in China in Guangdong, Hubei, Guangxi, Anhui provinces and other provinces in China in succession, so that the pig industry in China faces new threats and challenges, and important attention should be paid in view of higher pathogenicity of piglets. Although SVV is not as dangerous as FMDV and does not cause serious public health consequences and economic losses, the prognosis is good after infection in most adult swine. However, as a member of the same viral family as FMDV, the prevalence of SVV may lead swine farmers to acquire a constant habit of pigs with similar foot and mouth disease symptoms in the future, and further neglect the possibility of foot and mouth disease. The United states is worried about the influence of SVV on pork and beef exports, and the price of live pigs is calculated to drop by 40-60% due to export restriction.
Because the clinical symptoms of SVV infected pigs are very similar to those of FMDV, SVDV, VSV and VEV infections, and are difficult to distinguish, the diagnosis can be confirmed by laboratory diagnosis. The electron microscope, immunohistochemistry, RT-PCR, fluorescence quantitative Real time PCR and virus neutralization experiments can be used for pathogen detection and diagnosis of SVV.
Disclosure of Invention
In view of the above, the present invention aims to provide a Seneca Valley virus VP1 protein, a coding gene, a hybridoma cell line, a monoclonal antibody and applications thereof, wherein the monoclonal antibody can effectively prevent Seneca Valley virus from infecting cells, thereby achieving the effect of preventing Seneca Valley virus.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a protein of Hubei strain VP1 of Seneca valley virus, which has an amino acid sequence shown in a sequence table SEQ ID No. 1.
The invention provides a coding gene of the protein of the Hubei strain VP1 of Seneca valley virus, which has a nucleotide sequence shown in the sequence table SEQID No. 2.
The invention provides application of the Sagnac Valley virus Hubei strain VP1 protein as a Sagnac Valley virus antigen protein in an immunoassay kit.
The invention provides a hybridoma cell strain 2G6 secreting monoclonal antibody against the protein of the Seneca Valley virus Hubei strain VP1 of claim 1, wherein the preservation number of the hybridoma cell strain is CCTCC NO: C2017226.
The invention provides a monoclonal antibody secreted by hybridoma cell strain 2G 6.
The invention provides application of the monoclonal antibody in a kit for immunodetection of the protein of Hubei strain VP1 of Seneca valley virus.
The invention provides application of the monoclonal antibody in a kit for blocking cells infected by Hubei strain of Seneca valley virus.
Preferably, the titer of the monoclonal antibody is not less than 1:28。
Preferably, the cell comprises a BHK-21 cell or a 293T cell.
The invention provides a protein of Hubei strain VP1 of Seneca valley virus, which has an amino acid sequence shown in a sequence table SEQ ID No. 1. The protein of the Hubei strain VP1 of the Seneca Valley virus contains more epitopes of the virus, and the existence of the pathogen of the Hubei strain of the Seneca Valley virus can be effectively reflected by detecting the antibody of the VP1 protein.
The invention provides a hybridoma cell strain 2G6 secreting monoclonal antibody against the protein of Hubei strain VP1 of Seneca Valley virus, and the preservation number of the hybridoma cell strain is CCTCC NO: C2017226. The chromosome number of the hybridoma cell 2G6 provided by the invention is about 100, the chromosome number of SP2/0 cell is about 60, the chromosome number of normal mouse spleen cell is about 40, and the chromosome number of the hybridoma cell 2G6 is about the sum of the chromosome numbers of SP2/0 cell and normal spleen cell, which indicates that the hybridoma cell strain 2G6 is formed by fusing mouse spleen cell and myeloma cell. The hybridoma cell 2G6 genetic stability analysis shows that the hybridoma cell 2G6 has better genetic stability.
The invention provides a monoclonal antibody secreted by hybridoma cell strain 2G 6. The monoclonal antibody has a good neutralizing and protecting effect on the Hubei strain of Seneca Valley virus; meanwhile, the plaque test shows that the number of virus plaques of the 2G6 experimental group is obviously reduced compared with that of a virus control group and an IgG control group, and the monoclonal antibody prepared by the invention can effectively prevent the Hubei strain of the endo-Valley virus from adsorbing and entering cells.
The invention provides application of the monoclonal antibody in a kit experiment for blocking cells infected by Hubei strain of Seneca valley virus. Through indirect immunofluorescence analysis of monoclonal antibody, the monoclonal antibody prepared by the invention is specifically combined with Seneca Valley virus, and shows a strong fluorescence signal. The result of the plaque test shows that compared with the result of a virus control group and an IgG control group, the 2G6 experimental group has obviously reduced virus plaque number, which indicates that the monoclonal antibody prepared by the invention can better neutralize Hubei strain of Seneca Valley virus, thereby reducing the virus amount invading cells and achieving the effect of protecting cells. Meanwhile, the virus copy number in 293T cells of an experimental group and a control group which are adsorbed and enter the experiment by Taq man real-time fluorescence quantitative PCR detection is found, and the SVV virus copy number measured by the 2G6 experimental group is 102.70Copy/. mu.L, IgG controlThe determined SVV virus copy number is 102.78Copy/. mu.L, SVV virus copy number determined for virus control group was 102.83And the copy/. mu.L shows that the virus copy number of the experimental group is lower than that of the IgG control group and the virus control group, and the monoclonal antibody can be combined with a virus surface receptor so as to block the virus from being adsorbed into cells.
The invention provides application of the monoclonal antibody in a kit for immunodetection of the protein of Hubei strain VP1 of Seneca valley virus. Detection by a Western blotting method finds that: the monoclonal antibody can perform specific binding reaction with the protein of the Sailka valley virus Hubei strain VP1 without any effect on the unloaded pET-28a, which indicates that the monoclonal antibody has the characteristic of resisting the protein of the Sailka valley virus Hubei strain VP 1.
Drawings
FIG. 1 is a map of a universal commercial plasmid pET-28a vector used in the present invention;
FIG. 2 is a map of recombinant plasmid pET-28a-SVV-VP1 constructed in the present invention;
FIG. 3 shows the results of the serum titers of mice after triple immunization in example 2 of the present invention;
FIG. 4 is the recombinant VP1 protein of nuclear-expressed Seneca valley virus Hubei strain in example 3, in which FIG. 4-1 is pET-28a empty vector control; FIG. 4-2 shows recombinant VP1 protein;
FIG. 5 shows the results of detection using indirect immunofluorescence in example 3, wherein FIG. 5-A shows the Hubei strain of Seneca Valley virus expressed by BHK-21 cells; FIG. 5-B is a control of BHK-21 cells;
FIG. 6 shows the results of the neutralization protection assay for monoclonal antibodies of example 4, wherein FIG. 6-A is a control group of Hubei strain of Seneca Valley virus; FIG. 6-B is an IgG control group, and FIG. 6-C is an experimental group of 2G6 monoclonal antibody;
FIG. 7 is the result of measurement of genetic stability of hybridoma cells in example 5, in which FIG. 7-A is a graph showing the number of chromosomes 2G6 of hybridoma cells; FIG. 7-B is a graph showing the number of chromosomes of myeloma cells (SP 2/0).
Biological material preservation information
The hybridoma cell strain 2G6 is preserved in China Center for Type Culture Collection (CCTCC) with the address of Wuhan, Wuhan university, the preservation time of 11 months and 21 days in 2017 and the preservation number of CCTCC NO of C2017226.
Detailed Description
The invention provides a protein of Hubei strain VP1 of Seneca valley virus, which has an amino acid sequence shown in a sequence table SEQ ID No. 1. The protein of Hubei strain VP1 of Seneca Valley virus comprises 264 amino acid residues. The protein VP1 of Hubei strain of Seneca Valley virus is a structural protein of Seneca Valley virus, and comprises more antigen epitopes of Hubei strain of Seneca Valley virus. The preparation method of the Saibokagu virus Hubei strain VP1 protein is preferably prepared by a method of directly amplifying virus by using sensitive cell BHK-21 and performing ultra-separation and purification to obtain virus particles (containing VP1 protein).
The invention provides a coding gene of the protein of the Hubei strain VP1 of Seneca valley virus, which has a nucleotide sequence shown in the sequence table SEQID No. 2. The nucleotide sequence of the coding gene is shown as 1-792 base, namely CDS region. The method for obtaining the coding Gene comprises the steps of separating the virus, sequencing the whole genome, and then comparing and dividing coding regions according to published Gene sequences in a Gene Bank. The coding gene is preferably expressed in the recombinant plasmid pET-28a-SVV-VP1 (FIG. 2).
The invention provides application of the Sagnac Valley virus Hubei strain VP1 protein as an antigen protein of the Sagnac Valley virus Hubei strain in an immunoassay kit.
In the present invention, the immunoassay includes ELISA assay, colloidal gold immunochromatography assay or immunohistochemical assay. The method of the immunoassay is not particularly limited in the present invention, and an immunoassay method well known in the art may be used. The blocking ELISA method is established by taking the Hubei strain VP1 protein as an antigen coating, taking immune pig positive serum as standard positive serum and uninfected pig serum as standard negative serum as primary antibody, and taking HRP-labeled monoclonal antibody which resists VP1 protein and has blocking activity as a secondary antibody to compete and combine with VP1 antigen.
The invention provides a hybridoma cell strain 2G6 secreting monoclonal antibody against the protein of Hubei strain VP1 of Seneca Valley virus, and the preservation number of the hybridoma cell strain is CCTCC NO: C2017226.
The invention provides a monoclonal antibody secreted by hybridoma cell strain 2G 6. The method for isolating the monoclonal antibody of the present invention is not particularly limited, and any method known in the art may be used. The ascites titer of the monoclonal antibody is 1:213×100。
The invention provides application of the monoclonal antibody in a kit for immunodetection of the protein of Hubei strain VP1 of Seneca valley virus. In the invention, the detection of the protein VP1 of Hubei strain of Seneca Valley virus is preferably Westernblotting. The Western blotting method is not particularly limited, and the Western blotting scheme known in the art can be adopted.
The invention provides application of the monoclonal antibody in a kit for blocking cells infected by Hubei strain of Seneca valley virus. In the present invention, the cells preferably include 293T cells or PK-15 cells. In the present invention, the monoclonal antibody achieves protection against cell infection by neutralizing seneca virus strain Hubei. The method for neutralizing Sabei strain of Seneca Valley virus is preferably performed by using a fixed virus-diluted antibody method. The titer of the monoclonal antibody obtained by the neutralization test result is preferably not less than 1:28. The source of the seneca valley virus strain Hubei is seneca valley virus isolated from Hubei province. The monoclonal antibody is presumed to have the effect of treating the swine infected with the Hubei strain of Seneca archaea virus.
The Seneca Valley virus VP1 protein, encoding gene, hybridoma cell line and monoclonal antibody provided by the present invention and their uses will be described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of antigens
1. Mass amplification of Saibocar virus strain in Hubei
(1) BHK-21 cells were passaged to 175cm2In a culture flask, DMEM + 10% FBS + 1% PS medium at 37 deg.C and 5% CO2Culturing under the condition until the fullness is 80%.
(2) The medium was replaced with serum-free DMEM, and SVV-CH-HB2016 virus in an MOI of 0.1 was added thereto at 37 ℃ with 5% CO2Infection was carried out for 1h under these conditions.
(3) Removing supernatant, changing into DMEM + 2% FBS + 1% PS medium, and culturing at 37 deg.C with 5% CO2After culturing for about 24h, 50-60% of cells are spherical and float, but the cells can not be lysed.
2. Purification of Saiboga virus Hubei strain whole virus particles
(1) Cell debris removal: after the SVV virus is amplified by the cells (cytopathic effect), the mixed solution of the cells and the supernatant is subjected to preliminary centrifugation at 4 ℃ and 10000rpm for 30min, and cell debris is removed.
(2) Superdetached virus supernatant: virus supernatants were ultracentrifuged at 170000g for 3h at 4 ℃ in SW45Ti, the bottom of the tube was primed with 20% sucrose (filtered through a 0.22 μm filter) (50mL virus supernatant +15mL sucrose), and the pellet was resuspended in 2mL1 × Tris + NaCl or PBS after ultracentrifugation (where 1mL of the tube bottom was washed).
(3) The resuspended sample is kept overnight (fully dissolved and precipitated) at 4 ℃ on a turntable, centrifuged (5-10 min at 12000rpm at 4 ℃ on the next day) before loading, different sucrose gradients of 6mL 60% (3.6g), 45% (2.7g), 35% (2.1g) and 20% (1.2g) are prepared in advance, 1 × Tris + NaCl or PBS is used for preparation, 2-3 mL Buffer is added to dissolve the sucrose, the mixture is shaken on the turntable to accelerate dissolution (about 4 ℃ and 3-6 h, the higher the concentration is, the more insoluble the solution is), the corresponding Buffer is added to the volume of 6mL after dissolution, a 0.22 mu m filter is used for filtration, and the virus solution 20% sucrose is filtered from low concentration to high concentration.
(4) Spreading a sucrose gradient: laying 60%, 45%, 35% and 20% of sucrose from top to bottom in sequence, laying 2.5mL of 65% of sucrose, slowly adding 2.5mL of 45% of sucrose along the edge of the liquid level, avoiding overlarge force and breaking up the boundary of two layers (if sucrose containing a long and thin needle head is used, high-concentration sucrose is laid in four layers in sequence, then the four layers are kept stand overnight in a refrigerator at 4 ℃ to form a natural gradient), and so on, keeping the four layers at 4 ℃ for overnight after the four layers are laid (the four layers of sucrose are allowed to naturally settle and fully expand to form the natural gradient), and avoiding bubbles in the adding process.
(5) Sucrose gradient centrifugation: centrifuging the sample the next day, adding the sample into the uppermost layer of the laid sucrose along the liquid surface by the same method, performing sucrose gradient centrifugation, and centrifuging at 220000g for 2.5-6 h at 4 ℃. (size position of the bands marking the corresponding sucrose gradient on the centrifuge hose).
(6) Sampling: after the centrifugation is finished, the indoor lamp is turned off, the lamp illumination of the mobile phone is used for observing whether bright bands exist and the bright band position (the centrifugal tube is vertically placed on the illumination position), 500-600 mu L of liquid is sucked by a liquid transfer gun from top to bottom and placed in a 1.5mL EP tube (the approximate position of solid virus particles is the position of the 14 th-19 th tube) and the marking is carried out.
(7) Removing sucrose: combining the better tube number after electron microscope observation, adding the tube number into a centrifugal tube of SW45Ti, adding a corresponding Buffer for dilution, centrifuging for 2h at 4 ℃ at 170000g, inverting the centrifugal tube after centrifugation, draining the liquid of the tube opening and the tube body (the liquid can be lightly wiped by filter paper), resuspending the precipitate (about 400 mu L) by the corresponding Buffer, standing overnight at 4 ℃ on a rotating disc (fully dissolving the precipitate), detecting by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and observing by an electron microscope to determine the purity of the virus particles, and subpackaging for later use.
The purity of the virus particles was determined to be 90%.
Example 2
Preparation of monoclonal antibodies
(I) an immunoassay
Immunization test Using 5-week-old female BALB/c mice (purchased from the center of laboratory animals, university of agriculture, Huazhong, abbreviated as mice), whole virus particles purified and prepared in example 1 were used as antigens, and the immunization procedure was as shown in Table 1:
table 1 mouse immunization procedure
Ten days after the third immunization, blood is collected by breaking the tail, the serum titer of the mice is detected by an indirect ELISA method, and the mice with the highest serum titer are selected for subsequent experiments.
The indirect ELISA method detects serum titers as follows:
1. establishment of indirect ELISA detection method
Determining the optimal coating concentration of the antigen and the optimal dilution multiple of serum by a square matrix titration method:
(1) diluting the purified SVV virus particles and BHK-21 cell contrast from 4 mu g/mL to 0.125 mu g/mL by using a coating solution, longitudinally coating a 96-well enzyme label plate, coating 100 mu L/well, and coating overnight at 4 ℃;
(2) discarding the coating solution the next day, washing with PBST for 5min, repeating for 3 times, adding sealing solution, sealing at 37 deg.C for 2 hr at 100 μ L/hole;
(3) discarding the blocking solution, washing with PBST for 5min, repeating for 3 times, diluting the positive serum from 1:200 times to 1:25600, diluting for 8 times, respectively and transversely adding into 96-well enzyme label plate at 100 μ L/well, setting the negative mouse serum as negative control, diluting to the same multiple by the same method, and incubating at 37 deg.C for 1 h;
(4) discarding positive serum and negative serum, washing with PBST for 5min, repeating for 3 times, adding HRP-labeled goat anti-mouse IgG diluted at ratio of 1:5000, 100 μ L/well, and incubating at 37 deg.C for 1 h;
(5) discarding the secondary antibody, washing with PBST for 5min, repeating for 3 times, adding 50 μ L of substrate solution A and 50 μ L of substrate solution B into each hole, standing at room temperature in a dark place for reaction for 10min, adding 50 μ L of stop solution to terminate the reaction, and measuring OD value with an enzyme-labeling instrument.
2. Antibody level detection in immune serum
After the immunization of the mouse is completed, the tail vein is broken and the blood is collected, the blood is placed at 37 ℃ for 30 min-1 h, then the refrigerator is kept overnight at 4 ℃, the next day is centrifuged at 4 ℃ and 5000rpm for 10min, and the supernatant is separated to obtain the positive serum to be detected. Coating SVV virus particles and BHK-21 cells according to the optimal antigen coating concentration, sequentially diluting positive and blank mouse negative serum obtained by separation from 1:100 times to 1:12800 times in a ratio of 2 times as primary antibody, and detecting the titer of the mouse serum by using an established indirect ELISA method. When the OD ratio (P/N) of the serum to be detected and the negative serum is more than or equal to 2.1, and the maximum dilution multiple of the OD value step of the serum to be detected is more than or equal to 0.4 is the serum titer of the immune mouse.
Results
1. The optimal coating concentration of the antigen obtained by the square matrix titration method is 1 mu g/mL, and the optimal dilution multiple of the serum is 1: 6400.
2. As can be seen from FIG. 3, the serum titers of 3 mice immunized with SVV virus particles all reached 1:12800, wherein the immunization effect of the No.2 mouse is the best and superior to that of the other 2 mice.
(II) cell fusion
Three days before fusion, the 2 # mouse with the highest serum titer is boosted, and 40 mu g of antigen is injected into the abdominal cavity.
1. Preparation of SP2/0 cells
SP2/0 cells were prepared from solid tumors in mice. The method comprises the following specific steps:
1) SP2/0 cells frozen in a recovery laboratory are cultured in a six-well plate, when the cell state is good, the cells are resuspended by 1640 basic culture solution (purchased from Wuhan Feiyi technology Co., Ltd., brand name Hyclone), BALB/c mice are injected subcutaneously, and tumors grow out on the backs of the mice after 13 days.
2) The mice were sacrificed by dislocation of cervical vertebrae and soaked in 75% alcohol for 5 min.
3) Taking the tumor on an ultraclean workbench under an aseptic state, placing the tumor in an aseptic homogenizer, adding 5mL of 1640 basic culture solution, fully grinding, then adding 10mL of 1640 basic culture solution, uniformly mixing, standing for 5min, sucking the upper cell suspension in a centrifuge tube for later use after a larger tissue block is settled at the bottom of the tube, then adding 10mL of 1640 basic culture solution to resuspend the tissue, repeating the washing twice, centrifuging the cell suspension at 1000rpm for 10min, and then taking the sediment to resuspend with 15mL of 1640 basic culture solution.
4) And adding 20mL of lymphocyte separation liquid into another 50mL centrifuge tube, lightly adding the cell suspension on the lymphocyte separation liquid, centrifuging at 1000rpm for 10min, sucking the white cell layer positioned on the interface by using a suction tube, washing once by using 10mL1640 basic culture liquid, and counting for later use.
2. Preparation of immune spleen cells
Taking one BALB/c mouse which is finally boosted to be immunized, bleeding the orbit to be killed, collecting blood and separating serum to obtain positive serum.
The mice were sacrificed by pulling their necks, and after 5min of immersion in 75% alcohol, the mice were transferred to a clean bench and placed on a dissecting plate, with forelimb fixation and hindlimb cross (left hindlimb cross) fixation. Firstly, shearing and tearing skin to expose peritoneum, changing a set of scissors forceps, shearing the peritoneum to expose spleen, taking out the spleen under aseptic condition, putting the spleen into a homogenizer, adding 5mL of 1640 basic culture medium for grinding, then adding 10mL of 1640 basic culture medium, uniformly mixing, and standing for 5 min; gently sucking the upper liquid into the centrifuge tube, supplementing 10mL1640 basic culture medium into the homogenizer, uniformly mixing and standing for 5min, repeatedly washing for 2 times in this way, centrifuging at 1000rpm for 10min, discarding the supernatant, and resuspending the splenocytes with a proper amount of 1640 basic culture medium for later use.
3. Preparation of feeder cells
One uninmmunized BALB/c mouse was sacrificed by orbital bleeding, blood was collected and serum was isolated to obtain negative serum. Soaking the mouse in 75% alcohol for 5 min; feeder spleen cells were prepared according to the preparation method of immune spleen cells in the above step (2), and the finally obtained feeder spleen cells were resuspended in an appropriate amount of HAT medium (purchased from Sigma) and then evenly plated in 96-well cell culture plates for use, at 100. mu.L/well.
4. In vitro fusion of myeloma cells with immune splenocytes
1) Suspending SP2/0 myeloma cells (1-2 × 10)7cells) and immune spleen cell suspension (1 × 10)8Cells) are evenly mixed in a 50mL centrifuge tube and centrifuged at 1000rpm for 10 min;
2) emptying the supernatant (which can be dried by sterilized filter paper), and lightly knocking the bottom of the centrifugal tube to loosen the cells;
3) placing the centrifuge tube filled with the cell mixture in a 37 ℃ water bath, slowly adding 0.8mL of 50% polyethylene glycol (PEG) preheated to 37 ℃ within 1min, and gently stirring by using a pipette tip while adding;
4) stirring for 1 min;
5) slowly adding 10mL of 1640 basic culture medium preheated to 37 ℃ within 5min, and slowly and gently stirring when adding; finally, slowly adding 30mL of 1640 basic culture medium preheated to 37 ℃;
6) centrifuging at 1000rpm for 10min, discarding supernatant, and standing at 37 deg.C for 7 min;
7) with appropriate amount of HAThe T medium was resuspended and plated evenly in 96-well feeder cells-plated cell culture plates at 150. mu.L/well at 37 ℃ in 5% CO2Culturing in an incubator.
5. Screening for Positive hybridoma cells
Adding 50 mu L of fresh HAT culture medium on the 4 th day after fusion, removing all the culture medium after 8-10 days, changing the culture medium into an HT (purchased from sigma company) (namely 1640 basic culture medium + 20% fetal calf serum + 1% streptomycin + 2% HT (hypoxanthine + thymidine)), and carrying out ELISA detection when the size of the colony formed by the fused cells reaches 1/4 and the cell supernatant turns yellow.
6. Hybridoma cell supernatant ELISA detection
Detecting the supernatant of the hybridoma cells by adopting a conventional indirect ELISA method, which comprises the following specific steps:
1) the ultraseparated and purified SVV whole virus particles and BHK-21 cells (used as negative control) are respectively coated on a 96-well enzyme label plate and are kept at 4 ℃ overnight.
2) After coating, PBST buffer (PBS buffer + 0.5% Tween) washing three times, adding 2% blocking solution (2g bovine serum albumin dissolved in 100mL PBST buffer) at 37 degrees C temperature 2h incubation.
3) Washed three more times with PBST, hybridoma cell culture supernatant was added and incubated at 37 ℃ for 1 h.
4) PBST was washed three times, 100. mu.L of HRP-labeled goat-anti-mouse (Wuhan Dr. Biotech Co., Ltd.) was added, and the mixture was incubated at 37 ℃ for 1 hour.
5) After PBST was washed three times, a substrate solution (purchased from animal Biometrics Ltd. before Wuhan Ked) and a color developing solution (purchased from animal Biometrics Ltd. before Wuhan Ked) were added, and after 10min, the color developing reaction was observed and a stop solution (purchased from biological Ltd. before Wuhan Ked) was added, and the OD value was measured at a wavelength of 630 nm. Meanwhile, positive serum of the immunized mouse is measured as a positive control, and SP2/0 cell culture supernatant is set as a negative control.
6)OD630Samples 2 times greater than the negative control were positive. Selecting the single colony cell growth hole which has positive reaction with the antigen coated plate and negative reaction with the negative control plate and has good cell growth vigorous shape for further subcloning.
7. Subcloning of Positive hybridoma cells
Preparing feeder cells before cloning (the specific steps are the same as those in step 3 and preparing feeder cells), and performing limited dilution on hybridoma cells which are detected to be positive by ELISA so that 1-2 hybridoma cells exist in each cell culture hole. Then observing the growth condition of cells in the holes at regular time and recording; when the cells grow to about 1/3-1/2 of the culture hole after 8-10 days of cloning, detecting by using an indirect ELISA method (the specific steps are the same as the step (6)); single colony and positive wells were selected and cloning was resumed in the same manner to further purify the cell line until all wells were 100% positive. Finally, the established strain cells are expanded for use.
The hybridoma cell strain which stably secretes anti-Sabei strain VP1 protein of Seneca virus and has a certain neutralizing protection monoclonal antibody is obtained by the method, the applicant names the hybridoma cell strain as 2G6 and delivers the hybridoma cell strain to China, Wuhan university China type culture Collection in 2017 at 11/21, and the preservation number is CCTCC NO: c2017226).
8. Mass preparation and purification of monoclonal antibodies
Taking 5 BALB/c mice of 8 weeks old, and injecting 0.5mL of incomplete Freund's adjuvant into the abdominal cavity; collecting the hybridoma cell line 2G6 after 7-10 days, and injecting the hybridoma cell line into the abdominal cavity of a mouse (10)5～106cells/cell), ascites is collected after 7-10 days, the mixture is centrifuged at 2000rpm for 10min, and the supernatant is taken for standby.
The ascites fluid obtained by the above centrifugal crude extraction was purified according to the protocol of an IgG antibody purification kit (purchased from Thermo Co.) by the following specific steps:
1) removing the cap at the bottom of the antibody purification column, placing the purification column on a 1.5mL EP tube, adding 300 μ LBinding Buffer into the column, covering the purification column with a cover, reversing, mixing, centrifuging at 5000rpm for 1 min;
2) discarding the liquid in the lower layer of the centrifugal tube, and repeating the steps for 2 times;
3) adding 100 μ L of ascites fluid of monoclonal antibody to be purified into the purification column, reversing the upper part and mixing the ascites fluid evenly for 10min, centrifuging the mixture for 1min at 5000rpm, taking the liquid contained in 1.5mL of EP tube as eluent, and marking;
4) washing the purification column with Binding Buffer for 3 times, each time at 5000rpm, and centrifuging for 1 min;
5) adding 300 mu L of eluent into a purification column, reversing the eluent up and down, uniformly mixing, placing the column on a 1.5mL EP tube into which 40 mu L of neutralizing solution is added in advance, centrifuging at 5000rpm for 1min, collecting the liquid in the 1.5mL EP tube, namely the monoclonal antibody obtained by the first purification, and marking;
6) repeating the steps 1) to 5), respectively obtaining antibodies obtained by the second and third purification, and marking;
7) washing the purification column with Binding Buffer for 3 times, each time at 5000rpm, and centrifuging for 1 min;
8) washing the purification column with storage solution (provided by the kit) for 3 times, centrifuging at 5000rpm for 1 min;
9) adding 300 mu L of preservation solution into the purification column, uniformly mixing, and then placing the kit back to 4 ℃ for preservation.
Example 3
Detection of biological Activity of monoclonal antibodies
(1) Ascites titer determination: the titer of monoclonal antibodies in the ascites fluid was determined by indirect ELISA. After the enzyme label plate is coated by the SVV whole virus particles prepared by the invention, ascites is treated by the method of mixing the ascites with the enzyme label plate according to the volume ratio of 1:21× 100 lean by 1:216× 100 was used as a primary antibody, and HRP-labeled goat anti-mouse IgG (purchased from Strobile GmbH, Wuhan Dynasty) was used as a secondary antibody for indirect ELISA detection, and nonimmune mouse serum was used as a negative control.
And (3) test results: the ascites titer of the monoclonal antibody is 1:213×100。
(2) Expression of the recombinant protein pET-28a-SVV-VP1
First, the gene sequence encoding the wild-type SVV VP1 was analyzed by the gene software MacVector 7.2 to find out the codon usage preference, and the sites in the wild-type gene sequence that differ from the codon usage preference of E.coli were found out. For E.coli, preferred codons were used, and E.coli-preferred codons were substituted in the wild-type geneWith different codon bias, a codon optimized VP1 gene was then designed. The codon-optimized VP1 gene (VP1-opt) was synthesized by Shanghai Biotech, and the recombinant plasmid PUCK/VP1-opt was constructed by adding restriction sites BamHI and XhoI to both ends of the gene sequence, respectively, and loading the resulting mixture into a backbone vector PUCK (containing Amp resistance gene). Then, the recombinant plasmid PUCK/VP1-opt and the prokaryotic expression vector are respectively recovered by double enzyme digestion and are connected to obtain a recombinant plasmid pET-28a-VP1, the recombinant plasmid pET-28a-VP1 is transformed into escherichia coli competence BL21(DE3) after the sequencing is correct, the escherichia coli competence BL21 is evenly coated on a prepared LB plate, and the prepared LB plate is placed in a 37 ℃ incubator for inverted culture for 12 hours. Aseptically picking white single colony, inoculating into 5mL LB liquid culture medium containing Kan antibiotic, shake culturing at 37 deg.C and 180r/min overnight, adding overnight cultured bacterial liquid into LB liquid culture medium containing corresponding antibiotic at a ratio of 1:100, shake culturing at 37 deg.C and 180r/min for about 3 hr to OD600And (3) adding IPTG (isopropyl-beta-thiogalactoside) to a final concentration of 0.1mmol/L, inducing at 37 ℃ and 180rpm for 3h, collecting thalli after induction is finished, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis and Westernblotting detection on an induced expression product.
(3) Western blotting detection of monoclonal antibodies
1) Carrying out SDS-PAGE electrophoresis on recombinant protein pET-28a-SVV-VP1 induced to express and a no-load pET-28a sample thereof, wherein the concentration of separation gel is 12 percent, and the concentration of concentrated gel is 5 percent;
2) after electrophoresis is finished, transferring the film to a PVDF film by a semi-dry transfer method, and transferring the film for 1h at 20V;
3) sealing 5% skimmed milk at room temperature for 2 hr;
4) adding a proper amount of 2G6 hybridoma cell supernatant serving as a primary antibody, and incubating for 2h at room temperature;
5) then, an appropriate amount of HRP-labeled goat anti-mouse IgG (1:5000) was added as a secondary antibody, and the mixture was incubated at room temperature for 1 hour.
6) Finally, adding a proper amount of SuperSignal Chemicals primers color developing solution (1:1), and developing in a BIO-RAD light-shielding manner on a membrane illuminator.
As shown in FIG. 4, the antibody secreted by the hybridoma of strain 2G6 reacts specifically with recombinant VP1 protein of Hubei strain of Seneca Valley virus in Westernblotting reaction, but does not react with the unloaded pET-28 a. Based on the Westernblotting detection method, the monoclonal antibody is prepared into a detection kit, and the components also comprise a secondary antibody (HRP-labeled goat anti-mouse IgG).
(4) Indirect immunofluorescence assay for monoclonal antibodies
BHK-21 cells (stored in the laboratory) were cultured in a 24-well cell culture plate, and when the cells grew to 70% -80%, SVV-CH-HB2016 virus with an MOI of 0.1 was added, while non-virus-inoculated cells were used as negative controls.
After 10h, the cell culture supernatant was discarded, fixed with 100% methanol (-20 ℃ precooled for 30min) for 10min, and then washed three times with PBS for 5 min/time.
And (3) sealing: blocking with 5% Bovine Serum Albumin (BSA) for 1h, and washing with PBS three times for 5min each.
Primary antibody incubation: a monoclonal antibody sample to be detected (ascites dilution 1000 times prepared by hybridoma cell 2G 6) is added, and simultaneously, a blank mouse serum which is not immunized is diluted by 1:50 and ascites dilution 1:1000 prepared by SP2/0 cell is set as two negative controls. Incubate at 37 ℃ for 1h and wash three times with PBS, 5min each.
And (3) secondary antibody incubation: fluorescein Isothiocyanate (FITC) labeled goat anti-mouse IgG (sigma 1:1000) was added, incubated at 37 ℃ for 1h, washed three times with PBS, 5 min/time.
And (5) observing the result under a fluorescence microscope.
The results show that: the antibody secreted by the selected hybridoma cell 2G6 can react with specific IFA of Hubei strain of Seneca Valley virus.
The test results are shown in fig. 5, the experimental group results show obvious fluorescence signals, while the control group has no fluorescence reaction, which indicates that the monoclonal antibody prepared by the invention can be specifically combined with Seneca Valley virus.
Example 4
Detection of neutralizing protective action of monoclonal antibody
1. Antibody neutralization assay (fixed virus-diluted antibody method)
(1) Adding 50 mu L of blank 1640 culture medium into each well of 1-10 rows of a 96-well culture plate, adding 50 mu L of 2G6 monoclonal antibody (4 times of repetition) into the 1-4 rows, and sequentially diluting the monoclonal antibody to 1:28 (finally 50 mu L of liquid in each well) by 2 times;
(2) respectively store 200 TCIDs50The SVV-CH-HB2016, SVV-GX-2016 and SVV-AH01-2017 virus liquid is diluted 10 times to 0.2 TCID5050 μ L of 200 TCIDs were added to each well of columns 1 to 45050 μ L of 200/20/2/0.2 TCID was added to the virus solutions (experimental groups) of columns 5 to 8, respectively50Virus solution (virus control group), and 50 μ L1640 culture medium (cell control group) is added to the 9 th to 10 th columns; wherein SVV-CH-HB2016, SVV-GX-2016 and SVV-AH01-2017 viruses are three local strains of Hubei, Guangxi and Anhui of Seneca Valley virus in turn;
(3) mixing the above materials at front, back, left and right, gently (without shaking the liquid to other wells), placing the culture plate at 37 deg.C and 5% CO2Acting in incubator for 90 min.
(4) After the induction is completed, 100. mu.L of BHK-21 cell suspension is added into each well, and the mixture is continuously placed at 37 ℃ and 5% CO2Culturing in an incubator, observing and recording results day by day, generally observing for 3-4 days, and calculating the result according to a Reed-Muench two-handed method or a Karber method.
And (3) test results: the monoclonal antibody 2G6 strain has better neutralization protection effect on the Hubei strain of Seneca Valley virus, wherein the neutralization protection titer is 1:28。
2. Plaque test
Measuring the IgG concentration of the supernatant of the culture medium of the hybridoma cell line 2G6 by using a spectrophotometer, and taking the supernatant with the IgG total amount of 2mg and 2 × 106A TCID50In SVV-CH-HB2016 virus solution at 37 deg.C and 5% CO2The incubator is used for 1h, and 4 × 10 is added5And (3) uniformly mixing 293T cells on a rotating disc at 4 ℃ by shaking for 1h, centrifuging at low temperature and low speed to remove supernatant, washing for 4 times by using precooled PBS buffer solution, respectively adding 100 mu L serum-free DMEM for repeated freeze thawing and virus harvesting to perform plaque detection or adding 1mL Trizol lysis cells to extract RNA for qPCR detection, and simultaneously setting a virus control group (only containing virus) and an IgG control group (containing non-neutralization IgG).
(1) Sensitive cells are cultured as monolayers in plates or 6-well plates.
(2) The culture medium was aspirated and washed 1-2 times with an appropriate amount of PBS (pH 7.4).
(3) The virus liquid is continuously diluted by 10 times by serum-free culture medium, a virus suspension with proper dilution is selected to inoculate a monolayer of cells in the culture hole, the inoculation amount is about 1/2 of the original culture liquid, so that the cell monolayer is covered, and 2-3 holes are inoculated in each dilution.
(4) The plates were incubated at 37 ℃ with 5% CO2The incubator was allowed to adsorb for 1.5h, the virus solution was aspirated off, and the wells were washed 3 times with an appropriate amount of PBS (pH7.4), taking care to blot the liquid in the wells dry, and also taking care not to dry the cells.
(5) 2% low melting agarose (melted at 72 ℃ C. and placed in an incubator) was mixed with an equal amount of phenol red-free 2 XDMEM containing 2% serum and a diabody, and injected into a cell culture plate (note: the addition process was light, no air bubbles were generated, and the temperature in the chamber was low and rapid, preventing rapid solidification).
(6) Standing at room temperature until agarose is solidified, or standing in refrigerator at 4 deg.C for several minutes until agarose is solidified, and then standing at 37 deg.C and 5% CO2The incubator continues to culture.
(7) Cytopathic conditions were observed under a microscope every day.
(8) After the plaque appears (2-5 days), fixing with 10% formaldehyde, and acting at room temperature for 3-4 h.
(9) And (3) after discarding the agarose gel, dyeing with a crystal violet dyeing solution, acting at room temperature for 2-4 h (paying attention to the fact that dyeing time is not too long and cannot be washed away), discarding, washing with clear water, airing, counting, and counting.
The test results are shown in fig. 6, and compared with the virus control group and the IgG control group, the 2G6 test group results have obviously reduced virus plaque number, which indicates that the monoclonal antibody prepared by the invention can effectively prevent the Hubei strain of the endo-Valley virus from adsorbing and entering cells. The monoclonal antibody is based on the specific binding of the monoclonal antibody and the virus to prevent the virus from entering the cell, thereby protecting the cell from infection.
Taqman method Real time PCR
(1) The 293T cells of the experimental group and the control group are respectively taken to extract total RNA, then 0.5 mu G of RNA is taken to be reversely transcribed into cDNA, and the virus copy number absorbed into the 293T cells after the 2G6 monoclonal antibody and SVV-CH-HB2016 virus are acted is detected by adopting the real-time fluorescence quantitative PCR method of Seleneckin Valley virus established by Chenxin in the laboratory.
(2) The plasmid pEASY-Blunt-5' UTR plasmid was used as a standard, and the copy number concentration of the plasmid was determined and calculated (formula (6.02 × 10)23)×(ng/μl×10-9) V (DNA length × 660) ═ copy/. mu.l), a series of 10-fold dilutions were made to 103Copy number/. mu.L. In the experiment, samples and standards are detected, and each sample is subjected to three repeated experimental holes with two ddH2And (4) performing O control. The measurement primers are shown in Table 2, the reaction system is shown in Table 3, and the reaction sequence is shown in Table 4. The whole reaction process is carried out on a fluorescence quantitative PCR instrument (ABI), and the copy number content in the detection sample is calculated according to a standard curve established by the standard substance.
Table 2 primer design:
TABLE 3 reaction System
TABLE 4 reaction amplification conditions
And (3) test results: the SVV-CH-HB2016 virus copy number measured by the 2G6 experimental group is 102.70Copy/. mu.L, IgG control group determined viral copy number 102.78The number of virus copies measured in the virus control group was 10 copies/. mu.L2.83Copies/. mu.L. The virus copy number of the experimental group is lower than that of the IgG control group and the virus control group, which indicates that the monoclonal antibody can be accepted on the surface of the virusThe body binds, thereby blocking the adsorption of the virus into the cell.
Example 5
Hybridoma cell 2G6 genetic stability analysis
(1) The hybridoma cell 2G6 with good growth state is treated by colchicine to make the cell stay in the metaphase: taking cells cultured on 24-well cell plate, adding colchicine to final concentration of 0.4 μ g/mL, 37 deg.C, and 5% CO2And (5) performing incubator culture for 3 h. Meanwhile, myeloma cells were treated identically as controls.
(2) Hypotonic treatment, swelling of cells: 0.075mol/L KCl pre-warmed at 37 ℃ (formulation: 0.28g KCl added to 50mL ddH)2O, mix) hypotonic solution, 37 deg.C, 5% CO2Incubate for 35 min.
(3) Collecting colchicine treated cells: the colchicine treatment solution was decanted and the cells were blown up after addition of 1640 basal medium. The cell pellet was collected by centrifugation at 1600rpm for 5 min.
(4) Hypotonic treatment, swelling of cells: the cell pellet was suspended in 600. mu.L of 0.075mol/L KCl hypotonic solution pre-warmed at 37 ℃ and mixed well before incubation at 37 ℃ for 35 min.
(5) Fixing: adding 600 μ L of newly prepared stationary liquid (methanol: glacial acetic acid), mixing, centrifuging at 1600rpm for 5min, and removing supernatant. Then, 800. mu.L of the stationary liquid was added, the mixture was gently mixed, the mixture was left to stand for 30min, and the mixture was centrifuged at 1600rpm for 5min, and the supernatant was discarded. Adding 800 μ L of the fixing solution again, mixing gently, standing for 30min, centrifuging at 1600rpm for 5min, and removing supernatant to obtain cell precipitate.
(6) Cell resuspension and concentration detection: resuspending the cells with an appropriate amount of fixative, pipetting a drop of the cell suspension onto a clean slide pre-cooled at 4 deg.C, gently blowing the drop to spread the cell suspension onto the slide, and naturally drying at room temperature.
(7) Dyeing: the cells were stained with 10% Giemsa stain for 15 min. Washing with running water, and naturally drying.
(8) And (3) observation and counting: the slides were counted under a microscope oil microscope.
The results showed that the number of chromosomes of hybridoma 2G6 prepared according to the present invention was about 100, that of SP2/0 cell was about 60, and that of normal mouse spleen cell was about 40, and that hybridoma 2G6 prepared according to the present invention was about the sum of the number of chromosomes of SP2/0 cell and normal spleen cell (see FIG. 7). The hybridoma cell line 2G6 of the present invention is a fusion of mouse spleen cells and myeloma cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> Seneca Valley virus VP1 protein, coding gene, hybridoma cell strain and monoclonal antibody and application thereof
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Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr Asp
1 5 10 15
Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Ser Asn His Thr Asn
20 25 30
Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val
35 40 45
Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Ala Thr Gly
50 55 60
Thr Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro Thr
65 70 75 80
Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Val Ser Pro Ser
85 90 95
Asp Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser
100 105 110
Leu Ala Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val
115 120 125
Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser Gly
130 135 140
Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro
145 150 155 160
Tyr His Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly
165 170 175
Lys Val Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro
180 185 190
Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu
195 200 205
Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Ile Pro Arg Pro
210 215 220
Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Val Arg
225 230 235 240
Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg Ser
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Tyr Lys Gln Lys Met Leu Met Gln
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tccaccgaca acgccgagac tggggttatt gaggcgggta acactgacac cgatttctct 60
ggtgaattgg cggctcctgg ctctaaccac actaatgtca agttcctgtt tgaccgatct 120
cgattactga atgtaattaa ggtactggag aaggacgccg tcttcccccg ccctttcccc 180
acagcaactg gtacacaaca ggacgatggt tacttttgtc ttctaacacc ccgcccaaca 240
gtcgcctccc gacccgccac tcgtttcggc ctgtacgtca gtccgtctga cagtggcgtt 300
ctcgccaaca cttcactgga tttcaatttt tacagcttgg cctgtttcac ttactttaga 360
tcagaccttg aagtcacggt ggtctcactg gagccagatc tggaattcgc tgtagggtgg 420
ttcccctctg gcagtgagta ccaggcttcc agctttgtct acgaccaact gcatgtaccc 480
taccacttta ctgggcgcac tccccgcgct ttcgccagca agggtgggaa ggtatctttc 540
gtgctccctt ggaactctgt ctcatccgtg cttcccgtgc gctggggggg cgcttccaag 600
ctttcttctg ccacgcgggg tctgccggct catgctgact gggggactat ttacgccttt 660
atcccccgtc ccaacgagaa gaaaagcacc gctgtaaagc atgtggccgt gtacgttcgg 720
tacaagaacg cgcgtgcctg gtgccccagc atgcttccct ttcgcagcta caagcagaag 780
atgctgatgc aa 792
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aaccggctgt gtttgctaga g 21
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<213> Artificial Sequence (Artificial Sequence)
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gaactcgcag accacaccaa 20
<210>5
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atgcccagtc cttcctttc 19
<210>6
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caggcagtat ccaaagcacg 20
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ccaaaggtgt tagcgcaccc aaacg 25
Claims (6)
1. A hybridoma cell strain 2G6 secreting a monoclonal antibody of a Hubei strain VP1 protein of Seneca Valley virus with an anti-amino acid sequence shown as a sequence table SEQ ID No.1 is characterized in that the preservation number of the hybridoma cell strain is CCTCCNO: C2017226.
2. A monoclonal antibody against protein VP1 of Hubei strain of Seneca Valley virus, which has an amino acid sequence shown in sequence table SEQ ID No.1, is characterized by being secreted from hybridoma cell strain 2G6 as claimed in claim 1.
3. The use of the monoclonal antibody of claim 2 in the preparation of a kit for the immunodetection of the protein of VP1 of Hubei strain of Seneca valley virus.
4. Use of the monoclonal antibody of claim 2 for the preparation of a kit for blocking infection of cells by the Hubei strain of Seneca virus.
5. The use as claimed in claim 4, characterized in that the titer of the monoclonal antibody is not less than 1:28。
6. The use of claim 4, wherein said cells comprise BHK-21 cells or 293T cells.
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