CN102776152B - Monoclonal antibody against BTV (bluetongue virus) VP7 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain - Google Patents
Monoclonal antibody against BTV (bluetongue virus) VP7 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody against BTV (bluetongue virus) VP7 protein, a hybridoma cell strain capable of secreting the monoclonal antibody and application of the hybridoma cell strain. A BALB/c mouse is immunized by using prokaryotically expressed BTV-VP7 protein, spleen cells of the immunized mouse are separated and are enabled to be fused with SP2/0 cells, prokaryotically expressed BTV-VP7 protein is used for detecting hybridoma cells capable of secreting corresponding monoclonal antibodies, a limiting dilution method is adopted for separating and purifying the hybridoma cells obtained through screening, finally the hybridoma cell strain capable of stably secreting the monoclonal antibodies against the BTV-VP7 protein, and the monoclonal antibodies which are secreted by the hybridoma cell strain are named as BTV-4H7. According to IFA (immunofluorescence assay) experiment results, specific reaction can occur between the BTV-4H7 and type-1 to type-24 BTV. A C-ELISA (competitive enzyme-linked immunosorbent assay) which is established by using the monoclonal antibodies can rapidly and accurately detect type-1 to type-24 BTV infected animal positive serum.
Description
Technical field
The present invention relates to the monoclonal antibody of a strain of hybridoma strain and secretion thereof, relate in particular to a strain and secrete the hybridoma cell strain of anti-blue tongue virus VP7 protein monoclonal antibody and secreted monoclonal antibody thereof.The invention still further relates to the application in preparation diagnosis blue tongue virus infection animal reagent of above-mentioned hybridoma cell strain and monoclonal antibody, belong to the prevention and control field of bluetongue.
Background technology
Bluetongue (Bluetongue disease, BT) be by blue tongue virus (Bluetongue disease virus, BTV) cause that the ruminating animal viral infectious of propagating by hematophagous bug is changed to feature with oral cavity, nasal cavity and gastrointestinal tract mucosa generation ulcerative inflammation.BTV is double-stranded rna virus, and its genome comprises 10 sections, and size is about 19kb.BTV is one of member of Reoviridae (Reoviridae) Orbivirus (Orbivirus) blue tongue virus subgroup (Bluetingue virus subgroup), Orbivirus has 14 subgroups, and wherein blue tongue virus subgroup and deer haemorrhagic virus subgroup have stronger cross reactivity.BT is early than the sheep that is found in South Africa for 1876, and because morbidity sheep continues high heat, ulcerative lesions appears in oral cavity, and oral mucosa and tongue turn blue, and therefore, proposes to name as " bluetongue " in 1906, and the BT of ox is found in nineteen forty-three.BTV almost can infect all ruminating animals, comprises domestic and wild ox, goat and some wildlife.The wild ruminating animals such as ox, goat, deer and antelope can be with poison for a long time, and are playing the part of the role of virus host in the disease stream intermittent phase.And hematophagous bug, particularly storehouse midge are its main communication medias.Before 1940, this disease only limits to the African continent on the south the Sahara, has spread some countries and regions to the Middle East, for example: Cyprus, Syria, Iraq, Turkey, Israel and Palestine to the forties.Within 1948, the U.S. reports this disease.This disease of the later stage seventies is distributed widely in the torrid zone, subtropics country.1956～nineteen fifty-seven, especially Spain and Portugal were popular in Europe.1978 are separated to BTV in Australian storehouse midge body.At present, all PI BTV or closely-related virus with it of the susceptible animal of subtropical and tropical zones overwhelming majority of countries.First China found this disease and isolated BTV Yunnan master of great learning and integrity in 1979, thereby determined the existence at home of this disease, in Hubei, 29 provinces such as (1983), Anhui (1985), Sichuan (1988), Gansu (1990), Shanxi (1991) all detect the positive livestock of BTV serology subsequently.In recent years, along with global warming, BT breaks out in succession in many countries, and distribution range constantly expands.In August, 2006, German, Belgian, French, Dutch etc. find first cattle infected BT, in July, 2007, this disease is broken out in the states such as Britain, France, Italy.OIE (OIE) circular, in March, 2008～April, BT has been broken out in the state such as French, Italian, in January, 2009～December, BT is successively broken out in the states such as Britain, France, Italy, Australia, Greece, Israel, Denmark, Czech, Sweden, Norway, Spain, Germany, Austria, Portugal, Hungary, Holland, Oman, Algeria, Morocco and Tunisia.
Because BTV has 24 serotypes, every country serotype distribution heterogeneity, up to the present, does not have effective vaccine and prevents effecting a permanent cure disease, so, early stage Accurate Diagnosis, early prevention, is the most effectual way that prevents the outburst of this disease.In view of China detects this disease in most laboratories and area, in addition the risk that in international trade, this disease exists, still state is badly in need of strengthening the research to this disease, carries out the tachnical storage that can carry out to it prevention and control and detection, and the laboratory diagnostic method of particularly setting up BT is particularly important.
VP7 is a kind of extremely hydrophobic protein, has 349 amino acid, and the relatively primary structure of VP7 albumen finds that in each serotypes B TV-VP7 albumen, more than 94% amino acid is conservative, comprises the Methionin of the 255th.The conservative of aminoacid sequence determined that VP7 is this feature of group specific antigen.Existing experiment has at present confirmed that VP7 albumen exists absolute conservative region, what is more important, VP7 is the main exposure albumen on nucleoid particle, has at least two epi-positions to be exposed to virus surface, and these characteristics all show that VP7 can be used as the desirable target antigen that detects BT.
Summary of the invention
One of object of the present invention is to provide a strain can secrete the hybridoma cell strain of anti-blue tongue virus VP7 protein monoclonal antibody;
Two of object of the present invention is to provide one can there is specific reaction with 24 serotype blue tongue viruss (BTV), and the monoclonal antibody not reacting with IBAV, CV, AKAV, BVDV, IBRV, BRV, RV, BEV and FMDV;
Three of object of the present invention is to provide the antibody of above-described hybridoma cell strain and secretion thereof and diagnoses or detect the application in blue tongue virus (BTV) infection animal reagent in preparation.
The present invention is achieved through the following technical solutions:
The present invention adopts TRIZOL method to extract the total RNA of BTV-12 C-type virus C, and then utilize reverse transcription technology to clone VP7 gene cDNA, this cDNA is inserted to prokaryotic expression carrier pMAL-c4X, utilize pMAL-c4x prokaryotic expression carrier, by TB1 competent cell, BTV-VP7 gene is carried out to prokaryotic expression, the expressed recombinant VP7 protein antigen in this laboratory carries maltose binding protein (MBP) label, the amylose resin post providing in pMAL expressing fusion protein and purification kit is provided, the VP7 albumen of amalgamation and expression is carried out to purifying.Using the solubility BTV-VP7 albumen after a part of purifying as immunogen, immune BALB/c mouse, gets its splenocyte and SP2/0 myeloma cell and merges.VP7 albumen after another part purifying is as detection antigen, set up indirect ELISA detection method screens the anti-BTV-VP7 protein monoclonal antibody of final acquisition one strain stably excreting hybridoma cell strain to positive hybridoma cell, called after BTV-4H7, its Classification And Nomenclature is: the hybridoma cell strain of secretion BTV-4H7 monoclonal antibody, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC No.5715, preservation date is on January 16th, 2012.
In addition, the present invention also provides a kind of monoclonal antibody by described hybridoma cell strain secretion, and by monoclonal antibody called after BTV-4H7 secreted this hybridoma cell strain.
IiT detected result shows, BTV-4H7 can specific reactions occur with 24 serotypes B TV, and does not react with IBAV, AKAV, CV, BVDV, IBRV, BRV, BEV, RV and FMDV.
Therefore, the present invention has proposed again described hybridoma cell strain in preparation diagnosis or has detected the application in blue tongue virus (BTV) infection animal reagent.And
Described monoclonal antibody is in preparation diagnosis or detect the application in blue tongue virus (BTV) infection animal reagent.
In sum, the present invention prepares and has identified the monoclonal antibody of a species specificity for BTV group specific antigen VP7 albumen.For the serological diagnostic method of setting up BT is laid a good foundation.
Brief description of the drawings
Fig. 1 is BTV-VP7 gene RT-PCR amplification;
1:DL5000 DNA Marker; 2,3:VP7 gene RT-PCR amplification.
Fig. 2 is that the enzyme of recombinant plasmid pMAL-c4X-VP7 is cut qualification;
1:DL1500 DNA Marker; 2:EcoR I and Sal I enzyme are cut pMAL-c4X result; 3,4:EcoR I and Sal I enzyme are cut pMAL-c4X-VP7 result.
Fig. 3 is the SDS-PAGE analytical results of recombinant expressed BTV-VP7;
1: not induced product of recon pMAL-c4X-VP7; 2: recon pMAL-c4X-VP7 induces 5 hours expression products; 3: recon pMAL-c4X-VP7 induces 10 hours expression products; 4: expression product in 24 hours thalline of recon pMAL-c4X-VP7 induction; 5: expression product in 24 hours bacterium liquid of recon pMAL-c4X-VP7 induction; 6: protein molecule standard (14.4-116KD); 7: empty carrier pMAL-c4X induces 24 hours expression products.
Fig. 4 is recombinant protein pMAL-c4X-VP7 purification result.
1: not purification of recombinant proteins; 2: the recombinant protein after purifying; 3: protein molecular weight standard (10-200KD).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Main experiment material and source
1. albumen, cell and virus
Procaryotic cell expression purifying BTV12-VP7 albumen, TB1 competent cell, BHK-21 mouse source nephrocyte, SP2/0 myeloma cell, BTV1～24 C-type virus C strain, IBAV, CV and AKAV preserve by Harbin veterinary institute, provide; BVDV and IBRV are provided by Harbin veterinary institute Xue Fei researcher; BRV, RV, BEV and FMDV are provided by Harbin veterinary institute Yu Li researcher.
2. main agents and medicine
Foetal calf serum (FBS), DMEM and L-glutaminate are purchased from GIBCO company; The goat anti-mouse IgG of freund's adjuvant, 50%PEG, 50X HAT, 50X HT, 8-anaguanine (8-AG) and fluorescein isothiocyanate (FITC) mark is purchased from Sigma company; The goat anti-mouse IgG of horseradish peroxidase (HRP) mark is the import packing of Bioisystech Co., Ltd of Golden Bridge; SBA ClonotypingTM System/HRP Subclass of antibody identification kit is purchased from Southern Biotechnology company; PMAL
tMprotein Fusion and Purification System expressing fusion protein and purification kit are purchased from New ENGLAND Biolabs(NEB) company; Restriction enzyme EcoR I and Sal I ThermoScript II, T4 ligase enzyme and LA Taq polysaccharase are purchased from precious biotechnology (Dalian) company limited.Glue reclaims test kit purchased from Shanghai Hua Shun company.
3. laboratory animal
6 week age, BALB/c mouse was provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
Prokaryotic expression and the purifying of embodiment 1 BTV-VP7 albumen
1. design of primers
According to BTV-12 S7 gene order (sequence number is: AY263377) the design pcr amplification primer logging on GenBank, sequence is as follows:
BTVP7-EcoR I-18F:5'-GAC
gAATTCaTGGACACTATCGC-3', the EcoR I restriction enzyme site of line part for introducing.
BTVP7-SalI-1067R:5'-TAA
gTCGACtTACACATAGGCGGC-3', the SalI restriction enzyme site of line part for introducing.
2.BTV-12 the extraction of viral RNA and reverse transcription
Utilize Trizol method to extract virus genome RNA as template from the BHK-21 cell of infection BTV-12, as reverse transcription primer, carry out the synthetic viral cDNA of reverse transcription with BTVP7-Sal I-1067R primer.
Trizol method is extracted RNA step: results infect BHK-21 cell 1-5 × 10 of BTV-12
7individual, add 1mL Trizol to mix, room temperature leaves standstill 5min, add 0.2mL chloroform, firmly jolting 15s, incubated at room 2-3min, 12000g, 4 DEG C of centrifugal 15min, centrifugal rear liquid divides three layers, the careful upper strata colourless liquid that takes out, add the Virahol of equal-volume precooling, mix rear incubated at room 10min, 12000g, 4 DEG C of centrifugal 10min, abandon supernatant, precipitation adds the ethanol (preparation of DEPC water) of 1mL 75%, 15s gently shakes, 7500g, 4 DEG C of centrifugal 5min, carefully abandon most supernatant, settling chamber's warm air is done 3-5min, add 20-30 μ L DEPC water dissolution,-20 DEG C of preservations.
Carry out the synthetic cDNA of reverse transcription by the virus total RNA of extracting, system is as follows:
In reaction process, first virus total RNA and primer are hatched to 10min in 95 DEG C, the cooling 5min of ice bath, then adds all the other reagent, mixes, and hatches 60min for 42 DEG C, hatches 15min deactivation for 70 DEG C.
3.BTV-12VP7 gene amplification and purifying
The cDNA obtaining taking reverse transcription is as template, utilize designed BTVP7-EcoR I-18F as upstream primer, BTVP7-Sal I-1067R as downstream primer, amplification BTV-12 C-type virus C VP7 gene fragment.
(50 μ L) is as follows for PCR reaction system:
Reaction conditions is 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 54 DEG C of annealing 1min, 72 DEG C are extended 1.5min, cyclic amplification 30 times; 72 DEG C are extended 10min.
Then PCR product glue is reclaimed; whole pcr amplification products are carried out to agarose gel electrophoresis; and under ultraviolet lamp, cutting the blob of viscose that contains goal gene, the VP7 gene BTV-VP7 gene RT-PCR amplification going out according to Shanghai Hua Shun biotechnology company limited glue recovery test kit specification sheets recovery pcr amplification is afterwards as shown in Figure 1.The nucleotide sequence of VP7 gene is as shown in SEQ ID NO.2.
4.BTV-VP7 gene is connected with expression vector
Glue is reclaimed to the VP7 gene obtaining and carry out enzyme and cut, it is as follows that enzyme is cut system:
Meanwhile, prokaryotic expression carrier pMAL-c4X is carried out to enzyme and cut, it is as follows that enzyme is cut system:
Above-mentioned endonuclease reaction condition is 37 DEG C of water-baths, and be 4h action time, afterwards whole enzymes is cut to product and is carried out agarose gel electrophoresis and glue recovery.
VP7 gene and the pMAL-c4X prokaryotic vector of double digestion glue recovery purifying are connected, and linked system is as follows:
Condition of contact is to connect in instrument 16 DEG C at DNA, spends the night.
5. transform and choose bacterium
The connection product obtaining in 4 is all proceeded in E.coli DH5 α competent cell, and ice-water bath is placed 30min, 42 DEG C of water-bath thermal stimulus 1.5min afterwards, then be placed on rapidly in ice-water bath, leave standstill 2min.Xiang Guanzhong adds 250 μ L LB liquid nutrient mediums, and wave and culture 1h in 37 DEG C of shaking tables coats 100 μ L bacterium liquid on the LB flat board containing penbritin (100 μ g/mL) 37 DEG C of overnight incubation.The single bacterium colony of random picking from flat board, is inoculated into respectively 37 DEG C of shaking culture in 5mL LB (Amp+, 100 μ g/mL) liquid nutrient medium.
6. the enzyme of recombinant plasmid is cut qualification and order-checking
1) utilize the in a small amount extraction agent box of plasmid of AXYGEN company, in the bacterium liquid of cultivating according to the operation of test kit specification sheets, extract plasmid from 5, extracted plasmid is carried out together with pMAL-c4X vector plasmid to agarose gel electrophoresis, select doubtful recombinant plasmid.
2) doubtful recombinant plasmid is carried out to enzyme and cut qualification, it is as follows that enzyme is cut system:
Enzyme tangent condition is 37 DEG C of water-baths, effect 2h.
3) above-mentioned enzyme is cut to product and carry out agarose gel electrophoresis, according to electrophoresis result preliminary judgement positive plasmid.
4) by through tentatively concluding that the bacterium liquid that contains positive plasmid delivers Nanjing Genscript Biotechnology Co., Ltd. and carry out sequencing, will be by sequencing, identify correct recombinant plasmid called after pMAL-c4X-VP7.The enzyme of recombinant plasmid pMAL-c4X-VP7 is cut qualification result as shown in Figure 2.
7.BTV-12VP7 the prokaryotic expression of gene and purifying
According to method for transformation described in 5, recombinant plasmid pMAL-c4X-VP7 is transformed into prokaryotic expression TB1 competent cell, then taking out 100 μ L bacterium liquid coats on the LB flat board that contains penbritin (100 μ g/mL), 37 DEG C of overnight incubation, the isolated bacterium colony of white on picking LB plate culture medium, is inoculated in 37 DEG C of overnight incubation in the LB liquid nutrient medium that 5mL contains penbritin (100 μ g/mL).The condition that induced expression condition adopts pMAL expressing fusion protein and purification kit specification sheets to recommend, concrete operations are as follows: get in the LB substratum that 1mL recombinant bacterium bacterium liquid joins 100mL 37 DEG C of concussions and be cultured to OD600nm and be about at 0.5 o'clock, adding IPTG is 0.5mmol/L to final concentration, 16 DEG C of induction 24h.
This is tested expressed recombinant VP7 protein antigen and carries maltose binding protein (MBP) label, and the amylose resin post providing in pMAL expressing fusion protein and purification kit is provided, and can carry out purifying to the VP7 albumen of amalgamation and expression.Purification step reference reagent box specification sheets carries out, specific as follows: by the 500mL bacterium liquid 4000g through induction, the centrifugal 10min of room temperature, collects bacterial precipitation.With the resuspended bacterial precipitation of 25mL Column Buffer (pH7.0), and resuspended bacterium is carried out to ultrasonication.By the bacterium liquid 9000g after ultrasonication, 4 DEG C of centrifugal 20min.Supernatant after centrifugal is crossed after post (2mL) 5 times in amylose resin post, with Column Buffer (pH7.0) the cleaning resin column of 12 times of column volumes, finally Column Buffer (pH7.0) the wash-out fusion rotein containing 10mM maltose with 5mL, be the BTV VP7 albumen of expression and purification, as shown in Figure 3, after recombinant protein pMAL-c4X-VP7 purifying, SDS-PAGE analytical results as shown in Figure 4 for the SDS-PAGE analytical results of recombinant expressed BTV-VP7.The aminoacid sequence of expressed BTV VP7 albumen is as shown in SEQ ID NO.1.
The preparation of embodiment 2 monoclonal antibodies
1. mouse immune
With 5 of prokaryotic expression recombinant VP7 protein antigen immunity after purifying female BALB/c mouse in 6 week age, immunity 3 times altogether, one exempts from recombinant VP7 protein antigen and isopyknic Freund's complete adjuvant mixing and emulsifying, two exempt to exempt from recombinant VP7 protein antigen and isopyknic Freund's incomplete adjuvant mixing and emulsifying with three, immunizing dose is 50 μ g/, and immunization route is peritoneal immunity.Respectively two exempt to exempt from three after one week to the mouse blood sampling of docking, separation of serum (4 DEG C are centrifugal, 3000g, 10min), detects antibody horizontal with indirect ELISA.In cytogamy first 3 days, the BALB/c mouse of good immune effect is carried out to booster immunization again, every mouse peritoneal is injected 50 μ g immunizing antigens (not adding adjuvant).
2. cytogamy
Merge and prepare feeder layer cells the day before yesterday, get BALB/c mouse peritoneal macrophage according to ordinary method and be laid in 96 porocyte culture plates stand-by.Disconnected neck is put to death the mouse of spleen to be got, and aseptic spleen the separating Morr. cell got, carries out cytogamy in the ratio of splenocyte and SP2/0 myeloma cell 4:1 with PEG, and the cell after fusion is laid on accurate good feeder layer cells.
3. the screening of positive hybridoma cell strain and clone
Utilize the prokaryotic expression BTV12-VP7 albumen after purifying to set up the strain of indirect ELISA detection method screening positive hybridoma cell, to the hybridoma enlarged culturing of reacting positive, carry out the clone of hybridoma with limiting dilution assay simultaneously, clone 3 takes turns, and the positive hybridoma cell of having cloned is frozen in time.And finally obtain the hybridoma that a strain can the anti-BTV-VP7 protein monoclonal antibody of stably excreting, called after BTV-4H7, its Classification And Nomenclature is: the hybridoma cell strain of secretion BTV-4H7 monoclonal antibody, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.5715, preservation date is on January 16th, 2012.
4. a large amount of preparations of monoclonal antibody
Give the healthy BALB/c mouse abdominal injection Freund's incomplete adjuvant about 10 week age, only, within 1 week, pneumoretroperitoneum injects 1 × 10 to 0.5mL/
6individual hybridoma extracts ascites after 7d～10d in the time of mouse web portion extreme expansion, takes out once every 2d, by the centrifugal 10min of ascites 10000g/min extracting, removes upper strata grease and precipitation, and supernatant packing is stored in-20 DEG C.
The qualification of embodiment 3 monoclonal antibodies
1. the subgroup identification of monoclonal antibody
SBA ClonotypingTM System/HRP Subclass of antibody identification kit carries out subgroup identification to the prepared monoclonal antibody of embodiment 1, and concrete grammar carries out with reference to process specifications.
Result shows that the heavy chain of monoclonal antibody BTV-4H7 of the present invention is IgG
1, light chain is κ chain.
2.IFA test
The BHK-21 cell that grows to 70%～80% is met to poison 1～24 type BTV.Infect 4 ° of C of cold ethanol after 48 hours and fix 30 minutes, the BTV-VP7 ascites sense that 1:100 doubly dilutes is done 1 hour, and then the anti-37 ° of C sense of the sheep anti mouse two of the FITC mark of 1: 128 times of dilution is done 1 hour.Last fluorescence microscope result.
The specificity of reacting with BTV in order to confirm the prepared monoclonal antibody of the embodiment of the present invention 1, IBAV, AKAV, CV, BVDV, IBRV, BRV, BEV, RV and FMDV contrast have also been set in this experiment.
Test-results confirmation, the prepared monoclonal antibody BTV-4H7 of the present invention can react and not react (table 1) with IBAV, AKAV, CV, BVDV, IBRV, BRV, BEV, RV and FMDV with 1～24 type BTV.
Table 1 IFA qualification monoclonal antibody 4H7 specificity
Test example 1 monoclonal antibody BTV-4H7 of the present invention detects BTV infection animal serum
1 materials and methods
1.1 reagent
Except special instruction, all reagent is analytical pure.
The sheep anti-mouse igg of HRP mark is purchased from company of Zhong Shan Golden Bridge.
BTV-VP7 protein monoclonal antibody blocking-up/competitive ELISA serotype diagnostic kit is purchased IDEXX company.
PBS damping fluid: sodium-chlor (NaCl) 8g, Repone K (KCl) 0.2g, Sodium phosphate dibasic (Na
2hPO
4) 1.44g, potassium primary phosphate (KH
2pO
4) 0.24g, be dissolved in 950mL deionized water, adjust pH value to 7.4, be settled to 1000mL, 121 DEG C of autoclaving 20min, room temperature preservation.
PBST:1000mL PBS damping fluid adds 500 μ L tweens (Tween) 20, matching while using.
Confining liquid: the skimming milk that the final concentration that PBS damping fluid dissolves is 5%, matching while using.
Stop buffer: with the 2M H of deionized water dilution
2sO
4solution.
Coated damping fluid: sodium carbonate (Na
2cO
3) 1.59g, sodium bicarbonate (NaHCO
3) 2.93g, be dissolved in 950mL deionized water, adjust pH value to 9.6, be settled to 1000mL, 121 DEG C of autoclaving 20min, room temperature preservation.
Substrate buffer solution: citric acid 2.1g, Na
2hPO
4.12H
2o 71.6g, is dissolved in 950mL deionized water, adjusts pH value to 5.0, is settled to 1000mL, 121 DEG C of autoclaving 20min, room temperature preservation.
Substrate nitrite ion: substrate buffer solution 10mL, OPD 5mg, 30%H
2o
215 μ L.
Serum: BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-6, BTV-7, BTV-8, BTV-11, BTV-14, BTV-15, BTV-16, BTV-17, BTV-18, BTV-22 standard positive serum, 25 parts of known background positive serums, 25 parts of known background negative serums and AKAV positive serum are preserved by this laboratory; IBAV positive serum, BRV positive serum and FMDV positive serum are provided by Harbin veterinary institute Yu Li researcher; 322 parts of lowlenthal serum samples pick up from Guangxi.
1.2 method
1.2.1 C-ELISA detection method operating process
With the coated elisa plate of the recombinant VP7 protein antigen that is diluted to 10 μ g/mL, 1000ng/100 μ l/ hole, 4 DEG C of coated spending the night.Then discard coating buffer, add confining liquid, 100 μ L/ holes, 37 DEG C are sealed 2 hours, PBST washing 3 times, each 3 minutes, thieving paper patted dry elisa plate.MAb ascites is done to 1000 times of dilutions, the MAb ascites of having diluted and former times of serum (comprising yin and yang attribute serum and serum to be checked) are added in elisa plate simultaneously, the each 50 μ L in every hole, 37 DEG C of effects are after 2 hours, and PBST washes plate three times, each 3 minutes.The sheep anti-mouse igg of HRP mark is done to 1:4000 and doubly dilute, 100 μ L/ holes add elisa plate, and 37 DEG C act on 2 hours, and PBST washs after 3 times, and thieving paper pats dry elisa plate.Every hole adds 50 μ L substrate nitrite ions, and 37 DEG C of lucifuges develop the color 10 minutes, 2M H
2sO
4after termination reaction, by the microplate reader detection OD of 492nm place value.In the OD at 492nm place value, calculate blocking-up rate according to each sample.Utilize the C-ELISA detection method of setting up, measure 25 parts of positive lowlenthal serums and 25 parts of negative lowlenthal serums, determine sample criterion.
1.2.2 specific test
Utilize the C-ELISA detection method of setting up, detect respectively IBAV, AKAV, BRV, FMDV and BTV-12 C-type virus C positive serum, measure each serum blocking-up rate.
1.2.3 replica test
The competitive ELISA detection method of 3 bit manipulation persons to set up, detects above-mentioned 50 parts of known background lowlenthal serums respectively, relatively the result difference between different operating person.
1.2.4 ELISA chief component composition preservation period test
The coated elisa plate of recombinant VP7 protein antigen of purifying, 4 DEG C of preservations of vacuum packaging; MAb, 4 DEG C of preservations; The sheep anti-mouse igg of HRP mark ,-20 DEG C of preservations.Respectively after preservation the 3rd, after 6,9 months, utilize the C-ELISA detection method of setting up to verify its stability.
1.2.5 known sample coincidence rate experiment
Utilize the C-ELISA detection method of the prepared MAb foundation of example 1 of the present invention and the BTV-VP7 protein monoclonal antibody blocking-up/competitive ELISA serotype diagnostic kit that IDEXX company produces, detect BTV standard positive serum and 50 parts of known background lowlenthal serums of 15 kinds of different serotypes simultaneously, then calculate both coincidence rates.
1.2.6 terrain sample detection test
The C-ELISA detection method of utilizing the prepared MAb of example 1 of the present invention to set up, BTV-VP7 protein monoclonal antibody blocking-up/competitive ELISA serotype diagnostic kit with the production of IDEXX company, detect 322 parts of terrain lowlenthal serum samples that pick up from Guangxi province simultaneously, detected result is carried out to statistical study.
2 test-results
Determining of 2.1 criterion
By the detection to 50 parts of known background lowlenthal serums, determine that utilizing the criterion of the C-ELISA detection method that MAb embodiment 1 of the present invention sets up is that the negative sample of blocking-up rate≤30%, the positive sample of blocking-up rate >=40%, 30% < sample blocking-up rate < 40% are suspicious specimen.
2.2 specific test
The C-ELISA detection method of utilizing the prepared MAb of the embodiment of the present invention 1 to set up, detects IBAV positive serum, AKAV positive serum, BRV positive serum and FMDV positive serum, and result shows that above each serum blocking-up rate is all less than 30%, is judged to be feminine gender.And the blocking-up rate of BTV-12 type positive serum is greater than to 40%, be judged to be the positive.The C-ELISA detection method that proof utilizes the prepared MAb of the embodiment of the present invention 1 to set up has good specificity (table 2).
Table 2 specific test result
2.3 replica test
3 bit manipulation persons detect 50 parts of known background lowlenthal serums, and detected result is basically identical, prove that the C-ELISA detection method of utilizing the prepared MAb of the embodiment of the present invention 1 to set up has satisfactory stability and repeatability.
2.4 chief component compositions are preserved test
Vacuum-packed ELISA Sptting plate and BTV-4H7 MAb preserved after 9 months, through C-ELISA checking, reactive without changing.
2.5 known sample coincidence rate tests
Utilize the C-ELISA detection method of the prepared MAb foundation of the embodiment of the present invention 1 and the test kit that IDEXX department produces, the BTV standard positive serum and the 50 parts of known background lowlenthal serums that detect 15 kinds of anti-different serotypes carry out simultaneous test simultaneously, result shows, two kinds of method detected result coincidence rates are 100%(table 3).
The test of table 3 known sample coincidence rate
2.6 terrain sample detection tests
Utilize the C-ELISA detection method of the prepared MAb foundation of the embodiment of the present invention 1 and the BTV-VP7 protein monoclonal antibody blocking-up/competitive ELISA serotype diagnostic kit that IDEXX company produces, detect 322 parts of lowlenthal serum samples that pick up from different areas, Guangxi, result shows that the C-ELISA detection method of this research foundation and the test kit detected result coincidence rate of IDEXX company production are up to 98%(table 4 simultaneously).
Table 4 terrain sample detection test-results
The above results explanation, the C-ELISA detection method of utilizing the prepared MAb of the embodiment of the present invention 1 to set up, can detect the antibody in BTV infection animal serum fast and accurately, is applicable to clinical diagnosis needs.
Claims (4)
1. the hybridoma cell strain of anti-blue tongue virus VP7 protein monoclonal antibody is secreted in a strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCC No.5715.
2. a monoclonal antibody of being secreted by hybridoma cell strain claimed in claim 1.
3. the application of hybridoma cell strain claimed in claim 1 in preparation diagnosis or detection blue tongue virus infection animal reagent.
4. the application of monoclonal antibody claimed in claim 2 in preparation diagnosis or detection blue tongue virus infection animal reagent.
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| CN103305472A (en) * | 2013-06-18 | 2013-09-18 | 中国农业科学院哈尔滨兽医研究所 | Bluetongue virus serum 1 (BTV1) VP2 protein monoclonal antibody (BTV1-3E8), B-cell epitope polypeptide identified by BTV1-3E8 and application of BTV1-3E8 |
| CN103305473B (en) * | 2013-06-18 | 2015-01-07 | 中国农业科学院哈尔滨兽医研究所 | Bluetongue virus serum 1 (BTV1) VP2 protein monoclonal antibody (BTV1-4B6), B-cell epitope polypeptide identified by BTV1-4B6 and application of BTV1-4B6 |
| CN106190986B (en) * | 2016-07-07 | 2019-05-17 | 东北农业大学 | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody |
| CN108384889B (en) * | 2018-03-23 | 2021-11-26 | 重庆海关技术中心 | Multiple RT-PCR kit for genotyping and identifying bluetongue virus and detection method thereof |
| CN109254157A (en) * | 2018-10-26 | 2019-01-22 | 中国检验检疫科学研究院 | A kind of colloidal gold immune chromatography test and the preparation method and application thereof |
| CN112390862B (en) * | 2020-10-27 | 2022-03-22 | 中国检验检疫科学研究院 | Protein for detecting bluetongue, coding gene and soluble preparation method thereof |
| CN115057925B (en) * | 2022-06-22 | 2024-03-26 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
| CN116041447A (en) * | 2022-12-29 | 2023-05-02 | 北京亿森宝生物科技有限公司 | Kit for detecting African horse sickness virus and application thereof |
| CN116836270B (en) * | 2023-08-03 | 2024-03-26 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody of anti-bluetongue virus VP7 protein, preparation method and application |
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