CN102776152A - 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 a strain of hybridoma strain and excretory monoclonal antibody 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 through blood sucking insects is propagated is changed to characteristic with oral cavity, nasal cavity and gastrointestinal tract mucosa generation ulcerative inflammation.BTV is a 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 was found in South Africa in 1876, because morbidity sheep continues high heat, ulcerative lesions appears in the oral cavity, and oral mucosa and tongue turn blue, and therefore, proposes to name to be " bluetongue " that the BT of ox is found in nineteen forty-three in 1906.BTV almost can infect all ruminating animals, comprises domestic and wild ox, goat and some wildlife.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 blood sucking insects, particularly storehouse midge are its main communication medias.This disease only limits to the African continent on the south the Sahara before 1940, has spread some countries and regions to the Middle East to the forties, for example: Cyprus, Syria, Iraq, Turkey, Israel and Palestine.The U.S.'s this disease of report in 1948.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 the midge body of Australian storehouse.At present, all PI BTV or closely-related with it virus of the torrid zone and the susceptible animal of subtropical zone overwhelming majority of countries.The master of great learning and integrity at first found this disease and isolates BTV in Yunnan in 1979 in China; Thereby confirmed the existence at home of this disease, 29 provinces such as (1983), Anhui (1985), Sichuan (1988), Gansu (1990), Shanxi (1991) all detect the positive livestock of BTV serology in Hubei subsequently.In recent years, along with global warming, BT breaks out in many countries in succession, and distribution range constantly enlarges.In August, 2006, Germany, Belgium, France, Holland etc. find cattle infected BT first, in July, 2007, states such as Britain, France, Italy this disease of outburst.OIE (OIE) circular; In March, 2008～April; BT has been broken out in states such as France, Italy; In January, 2009～December, BT is successively broken out in 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, up to the present each national serotype distribution heterogeneity, does not have effective vaccine and prevents effecting a permanent cure disease, so, early stage accurately 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; The risk that this disease exists in the international trade in addition; Still state is badly in need of strengthening the research to this disease, carries out the tachnical storage that can carry out prevention and control and detection to it, 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 relatively the proteic primary structure of VP7 finds that the amino acid more than 94% is conservative in each serotypes B TV-VP7 albumen, comprises the 255th Methionin.The conservative VP7 that determined of aminoacid sequence is this characteristic of group specific antigen.At present existing experiment confirm VP7 albumen have absolute conservative region; What is more important; VP7 is the main exposure albumen on the nucleoid particle, has at least two epi-positions to be exposed to virus surface, and these characteristics show that all VP7 can be used as the desirable target antigen that detects BT.
Summary of the invention
One of the object of the invention provides the hybridoma cell strain that a strain can be secreted anti-blue tongue virus VP7 protein monoclonal antibody;
Two of the object of the invention provides and a kind ofly can specific reaction take place with 24 serotype blue tongue viruss (BTV), and the monoclonal antibody that does not react with IBAV, CV, AKAV, BVDV, IBRV, BRV, RV, BEV and FMDV;
Three of the object of the invention provides above-described hybridoma cell strain and the application of excretory antibody in preparation diagnosis or detection blue tongue virus (BTV) infection animal reagent thereof.
The present invention realizes through following technical scheme:
The present invention adopts the TRIZOL method to extract the total RNA of BTV-12 C-type virus C; And then utilize the reverse transcription technology to clone the VP7 gene cDNA; This cDNA is inserted prokaryotic expression carrier pMAL-c4X; Utilize the pMAL-c4x prokaryotic expression carrier BTV-VP7 gene to be carried out prokaryotic expression through the TB1 competent cell; The expressed reorganization VP7 albumen in this laboratory carries maltose binding protein (MBP) label, utilizes the amylose resin post that provides in pMAL expressing fusion protein and the purification kit, and the VP7 albumen of amalgamation and expression is carried out purifying.As immunogen, immune BALB/c mouse is got its splenocyte and SP2/0 myeloma cell and is merged with the solubility BTV-VP7 albumen behind a part of purifying.VP7 albumen behind another part purifying is used antigen as detecting; Set up indirect ELISA detection method screens the anti-BTV-VP7 protein monoclonal antibody of final acquisition one strain stably excreting to positive hybridoma cell hybridoma cell strain; Called after BTV-4H7, its called after of classifying: the hybridoma cell strain of secretion BTV-4H7 monoclonal antibody is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address No. 3 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC No.5715, preservation date are on January 16th, 2012.
In addition, it is a kind of by described hybridoma cell strain excretory monoclonal antibody that the present invention also provides, and the monoclonal antibody called after BTV-4H7 that this hybridoma cell strain is secreted.
The IiT detected result shows that BTV-4H7 can specific reaction take place 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 the application of described hybridoma cell strain in preparation diagnosis or detection blue tongue virus (BTV) infection animal reagent again.And
Described monoclonal antibody is in the preparation diagnosis or detect the application in blue tongue virus (BTV) the infection animal reagent.
In sum, the present invention prepares and has identified a specific specificity to the proteic monoclonal antibody of BTV group specific antigen VP7.For the serological diagnostic method of setting up BT is laid a good foundation.
Description of drawings
Fig. 1 is a BTV-VP7 gene RT-PCR amplification;
1:DL5000 DNA Marker; 2,3:VP7 gene RT-PCR amplification.
Fig. 2 cuts evaluation for the enzyme of recombinant plasmid pMAL-c4X-VP7;
1:DL1500 DNA Marker; 2:EcoR I and Sal I enzyme are cut pMAL-c4X result; 3,4:EcoRI and Sal I enzyme are cut pMAL-c4X-VP7 result.
Fig. 3 is the SDS-PAGE analytical results of recombinant expressed BTV-VP7;
1: recon pMAL-c4X-VP7 is induced product not; 2: recon pMAL-c4X-VP7 induces 5 hours expression products; 3: recon pMAL-c4X-VP7 induces 10 hours expression products; 4: recon pMAL-c4X-VP7 induces 24 hours interior expression products of thalline; 5: recon pMAL-c4X-VP7 induces expression product in 24 hours bacterium liquid; 6: protein molecule standard (14.4-116KD); 7: empty carrier pMAL-c4X induces 24 hours expression products.
Fig. 4 is a recombinant protein pMAL-c4X-VP7 purification result.
1: purification of recombinant proteins not; 2: the recombinant protein behind the purifying; 3: protein molecular weight standard (10-200KD).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Main experiment material and source
1. albumen, cell and virus
Procaryotic cell expression and purifying BTV12-VP7 albumen, TB1 competent cell, BHK-21 mouse source kidney cell, SP2/0 myeloma cell, BTV1～24 C-type virus C strains, IBAV, CV and AKAV preserve by the 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 available from GIBCO company; Freund's adjuvant, 50%PEG, 50X HAT, 50X HT, 8-azaguanine (8-AG) and the anti-mouse IgG of fluorescein isothiocyanate (FITC) labelled goat are available from Sigma company; The anti-mouse IgG of horseradish peroxidase (HRP) labelled goat is the import packing of Bioisystech Co., Ltd of Golden Bridge; SBA ClonotypingTM System/HRP antibody subgroup identification test kit is purchased the company in Southern Biotechnology; PMAL
TMProtein Fusion and Purification System expressing fusion protein and purification kit are available from New ENGLAND Biolabs (NEB) company; Restriction enzyme EcoRI and SalI ThermoScript II, T4 ligase enzyme and LA Taq polysaccharase are available from precious biotechnology (Dalian) ltd.Glue reclaims test kit available from Shanghai Hua Shun company.
3. laboratory animal
6 the week age BALB/c mouse provide by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
Proteic prokaryotic expression of embodiment 1BTV-VP7 and purifying
1. design of primers
According to the BTV-12 S7 gene order of landing on the GenBank (sequence number is: AY263377) design pcr amplification primer, sequence is following:
BTVP7-EcoR I-18F:5'-
GACGAATTCATGGACACTATCGC-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 the Trizol method from the BHK-21 cell that infects BTV-12, to extract virus genome RNA, as the reverse transcription primer, carry out the synthetic viral cDNA of reverse transcription with BTVP7-Sal I-1067R primer as template.
The Trizol method is extracted the RNA step: results infect BHK-21 cell 1-5 * 10 of BTV-12
7Individual, add 1mL Trizol mixing, room temperature leaves standstill 5min, adds the 0.2mL chloroform, firmly jolting 15s; Incubated at room 2-3min, 12000g, 4 ℃ of centrifugal 15min, centrifugal back liquid divides three layers, carefully takes out the upper strata colourless liquid; The Virahol that adds the equal-volume precooling, incubated at room 10min behind the mixing, 12000g, 4 ℃ of centrifugal 10min abandon supernatant; Deposition adds the ethanol (preparation of DEPC water) of 1mL 75%, the 15s that gently shakes, 7500g, 4 ℃ of centrifugal 5min; Carefully abandon most supernatant, the air-dry 3-5min of deposition room temperature adds 20-30 μ L DEPC water dissolution ,-20 ℃ of preservations.
Carry out the synthetic cDNA of reverse transcription with the virus total RNA of extracting, system is following:
In the reaction process, earlier virus total RNA and primer are hatched 10min in 95 ℃, ice bath cooling 5min, with all the other reagent addings, mixing is hatched 60min for 42 ℃, hatches the 15min deactivation for 70 ℃ then.
3.BTV-12VP7 gene amplification and purifying
The cDNA that obtains with reverse transcription is a template, and as downstream primer, BTVP7-EcoR I-18F that utilization is designed increases BTV-12 C-type virus C VP7 gene fragment as upstream primer, BTVP7-Sal I-1067R.
PCR reaction system (50 μ L) is as follows:
Reaction conditions is 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, cyclic amplification 30 times; 72 ℃ are extended 10min.
Then PCR product glue is reclaimed; Whole pcr amplification products are carried out agarose gel electrophoresis; And under uv lamp, cut the blob of viscose that contains goal gene, as shown in Figure 1 according to the VP7 gene BTV-VP7 gene RT-PCR amplification that Shanghai China Shun biotechnology ltd glue recovery test kit specification sheets recovery pcr amplification goes out afterwards.The nucleotide sequence of VP7 gene is shown in SEQ ID NO.2.
4.BTV-VP7 gene is connected with expression vector
Glue is reclaimed the VP7 gene obtain carry out enzyme and cut, it is following that enzyme is cut system:
Simultaneously, prokaryotic expression carrier pMAL-c4X is carried out enzyme cut, it is following that enzyme is cut system:
Above-mentioned endonuclease reaction condition is 37 ℃ of water-baths, and be 4h action time, afterwards whole enzymes is cut product and carries out agarose gel electrophoresis and glue recovery.
The VP7 gene and the pMAL-c4X prokaryotic vector of double digestion and glue recovery purifying are connected, and linked system is following:
Condition of contact is to connect in the appearance 16 ℃ at DNA, spends the night.
5. transform and choose bacterium
The connection product that obtains in 4 is all changed in the E.coli DH5 α competent cell, and ice-water bath is placed 30min, and 42 ℃ of water-bath thermal stimulus 1.5min are placed on rapidly in the ice-water bath more afterwards, leave standstill 2min.Xiang Guanzhong adds 250 μ L LB liquid nutrient mediums, and wave and culture 1h in 37 ℃ of shaking tables coats 100 μ L bacterium liquid on the LB flat board that contains penbritin (100 μ g/mL) 37 ℃ of overnight cultures.The single bacterium colony of picking at random from the flat board is inoculated into 37 ℃ of shaking culture in 5mL LB (Amp+, the 100 μ g/mL) liquid nutrient medium respectively.
6. the enzyme of recombinant plasmid is cut and is identified and order-checking
1) plasmid that utilizes AXYGEN company is the extraction agent box in a small amount, extracts plasmid in the bacterium liquid of from 5, cultivating according to the operation of test kit specification sheets, and the plasmid that is extracted is carried out agarose gel electrophoresis with the pMAL-c4X vector plasmid, selects doubtful recombinant plasmid.
2) doubtful recombinant plasmid is carried out enzyme and cut evaluation, it is following that enzyme is cut system:
The enzyme tangent condition is 37 ℃ of water-baths, effect 2h.
3) above-mentioned enzyme is cut product and carry out agarose gel electrophoresis, according to electrophoresis result preliminary judgement positive plasmid.
4) will pass through and conclude that tentatively the bacterium liquid that contains positive plasmid delivers Nanjing Genscript Biotechnology Co., Ltd. and carry out sequencing, will identify correct recombinant plasmid called after pMAL-c4X-VP7 through sequencing.It is as shown in Figure 2 that the enzyme of recombinant plasmid pMAL-c4X-VP7 is cut qualification result.
7.BTV-12VP7 Prokaryotic Expression and purifying
According to 5 said method for transformation recombinant plasmid pMAL-c4X-VP7 is transformed into prokaryotic expression and uses the TB1 competent cell; Taking out 100 μ L bacterium liquid then coats on the LB flat board that contains penbritin (100 μ g/mL); 37 ℃ of overnight cultures; White on the picking LB plate culture medium isolates bacterium colony, is inoculated in 5mL and contains 37 ℃ of overnight cultures in the LB liquid nutrient medium of penbritin (100 μ g/mL).The condition that the induced expression condition adopts pMAL expressing fusion protein and purification kit specification sheets to recommend; Concrete operations are following: get in the LB substratum that 1mL reorganization bacterium bacterium liquid joins 100mL 37 ℃ of concussions and be cultured to OD600nm and be about at 0.5 o'clock; Add IPTG to final concentration be 0.5mmol/L, induce 24h for 16 ℃.
The expressed reorganization VP7 albumen of this test carries maltose binding protein (MBP) label, utilizes the amylose resin post that provides in pMAL expressing fusion protein and the purification kit, can carry out purifying to the VP7 albumen of amalgamation and expression.Purification step reference reagent box specification sheets carries out, and is specific as follows: will pass through inductive 500mL bacterium liquid 4000g, the centrifugal 10min of room temperature collects bacterial precipitation.With the resuspended bacterial precipitation of 25mL Column Buffer (pH7.0), and resuspended bacterium carried out ultrasonication.With the bacterium liquid 9000g after the ultrasonication, 4 ℃ of centrifugal 20min.Supernatant after centrifugal crossed post (2mL) 5 times in the amylose resin post after; ColumnBuffer (pH7.0) with 12 times of column volumes cleans resin column; Column Buffer (pH7.0) the wash-out fusion rotein that contains 10mM SANMALT-S at last with 5mL; Be the BTV VP7 albumen of expression and purification, the SDS-PAGE analytical results of recombinant expressed BTV-VP7 is as shown in Figure 3, and the SDS-PAGE analytical results is as shown in Figure 4 behind the recombinant protein pMAL-c4X-VP7 purifying.The expressed proteic aminoacid sequence of BTV VP7 is shown in SEQ ID NO.1.
1. mouse immune
With the reorganization of the prokaryotic expression behind purifying VP7 protein immunization female BALB/c mouse in age in 56 weeks; Immunity is 3 times altogether; One exempts from reorganization VP7 albumen and isopyknic Freund's complete adjuvant mixing and emulsifying; Two exempt from and three exempt from reorganization VP7 albumen and isopyknic Freund's incomplete adjuvant mixing and emulsifying, immunizing dose be 50 μ g/ only, immunization route is a peritoneal immunity.Exempt from and three exempt from one week of back to the mouse blood sampling of docking two respectively, (4 ℃ centrifugal, and 3000g 10min), detects antibody horizontal with indirect ELISA for separation of serum.In cytogamy preceding 3 days, the BALB/c mouse of good immune effect is carried out booster immunization again, every mouse peritoneal is injected 50 μ g immunizing antigens (not adding adjuvant).
2. cytogamy
Merge and prepare feeder layer cells previous day, get the BALB/c mouse peritoneal macrophage according to ordinary method and be laid in the 96 porocyte culture plates for use.Disconnected neck is put to death the mouse of waiting to get spleen, and aseptic spleen and 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 the fusion is laid on the accurate good feeder layer cells.
3. the screening of positive hybridoma cell strain and clone
Utilize the prokaryotic expression BTV12-VP7 albumen behind the purifying to set up the strain of indirect ELISA detection method screening positive hybridoma cell; Hybridoma enlarged culturing to reacting positive; Carry out the clone of hybridoma simultaneously with limiting dilution assay; Clone 3 takes turns, and the positive hybridoma cell that the clone is good is in time frozen.And finally obtain the hybridoma that a strain can the anti-BTV-VP7 protein monoclonal antibody of stably excreting; Called after BTV-4H7; Its called after of classifying: the hybridoma cell strain of secretion BTV-4H7 monoclonal antibody, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is institute of microbiology of the Chinese Academy of Sciences in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Its culture presevation is numbered: CGMCC No.5715, preservation date are 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 ages in week, 0.5mL/, 1 all pneumoretroperitoneum injections 1 * 10
6Individual hybridoma extracts ascites when the mouse web portion extreme expansion behind 7d～10d, take out once at a distance from 2d, with the centrifugal 10min of ascites 10000g/min that extracts, removes upper strata grease and deposition, and the supernatant packing is stored in-20 ℃.
The evaluation of embodiment 3 monoclonal antibodies
1. the subgroup identification of monoclonal antibody
SBA ClonotypingTM System/HRP antibody subgroup identification test kit carries out subgroup identification to embodiment 1 prepared monoclonal antibody, and concrete grammar carries out with reference to process specifications.
The result shows that the heavy chain of monoclonal antibody BTV-4H7 of the present invention is IgG
1, light chain is the κ chain.
2.IFA test
The BHK-21 cell that grows to 70%～80% is met poison 1～24 type BTV.Infected after 48 hours 4 ° of C of cold ethanol fixing 30 minutes, the BTV-VP7 ascites sense that 1:100 doubly dilutes is done 1 hour, and sheep anti mouse two anti-37 ° of C senses of the FITC mark that doubly dilutes of 1:128 are done 1 hour then.Last fluorescence microscope result.
In order to confirm the specificity of prepared monoclonal antibody of the embodiment of the invention 1 and BTV reaction, this experiment has also been set IBAV, AKAV, CV, BVDV, IBRV, BRV, BEV, RV and FMDV and has been contrasted.
Test-results confirms that the prepared monoclonal antibody BTV-4H7 of the present invention can not react (table 1) with IBAV, AKAV, CV, BVDV, IBRV, BRV, BEV, RV and FMDV with 1～24 type BTV reaction.
Table 1IFA identifies 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 that specifying, all reagent are analytical pure.
The sheep anti-mouse igg of HRP mark is available from company of middle shirt 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, being dissolved in the 950mL deionized water, adjustment pH value to 7.4 is settled to 1000mL, 121 ℃ of autoclaving 20min, room temperature preservation.
PBST:1000mL PBS damping fluid adds 500 μ L tweens (Tween) 20, and is existing with join at present.
Confining liquid: PBS damping fluid dissolved final concentration is 5% skimming milk, and is existing with join at present.
Stop buffer: with the 2M H of deionized water dilution
2SO
4Solution.
Encapsulate damping fluid: yellow soda ash (Na
2CO
3) 1.59g, sodium hydrogencarbonate (NaHCO
3) 2.93g, being dissolved in the 950mL deionized water, adjustment pH value to 9.6 is settled to 1000mL, 121 ℃ of autoclaving 20min, room temperature preservation.
Substrate buffer solution: Hydrocerol A 2.1g, Na
2HPO
4.12H
2O 71.6g is dissolved in the 950mL deionized water, and adjustment pH value to 5.0 is settled to 1000mL, 121 ℃ of autoclaving 20min, room temperature preservation.
Substrate colour developing liquid: 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
Reorganization VP7 albumen with being diluted to 10 μ g/mL encapsulates elisa plate, 1000ng/100 μ l/ hole, and 4 ℃ encapsulate and spend the night.Discard coating buffer then, add confining liquid, 100 μ L/ holes, 37 ℃ were sealed 2 hours, PBST washing 3 times, each 3 minutes, thieving paper was clapped dried elisa plate.MAb ascites is done 1000 times of dilutions, and MAb ascites and former times of serum (comprising yin and yang attribute serum and serum to be checked) that dilution is good add in the elisa plate simultaneously, each 50 μ L of every hole, and 37 ℃ of effects are after 2 hours, and PBST washes plate three times, each 3 minutes.The sheep anti-mouse igg of HRP mark is 1:4000 doubly dilutes, 100 μ L/ holes add elisa plate, and 37 ℃ act on 2 hours, and after PBST washed 3 times, thieving paper was clapped dried elisa plate.Every hole adds 50 μ L substrates colour developing liquid, and 37 ℃ of lucifuges developed the color 2M H 10 minutes
2SO
4After the termination reaction, detect the 492nm OD of place value with ELIASA.In the OD at 492nm place value, calculate the 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, confirm sample criterion.
1.2.2 specificity test
Utilize the C-ELISA detection method of setting up, detect IBAV, AKAV, BRV, FMDV and BTV-12 C-type virus C positive serum respectively, 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 the different operating person.
1.2.4ELISA main composition preservation period test
The elisa plate that purified recombinant VP7 albumen encapsulates, vacuum-packed 4 ℃ of preservations; MAb, 4 ℃ of preservations; The sheep anti-mouse igg of HRP mark ,-20 ℃ of preservations.After after the preservation the 3rd, 6,9 month, utilize the C-ELISA detection method of setting up that its stability is verified respectively.
1.2.5 known sample coincidence rate experiment
Utilize the C-ELISA detection method of instance 1 prepared MAb foundation 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, calculate both coincidence rates then.
1.2.6 terrain sample detection test
The C-ELISA detection method of utilizing instance 1 prepared MAb 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 statistical study.
2 test-results
2.1 confirming of criterion
Through detection to 50 parts of known background lowlenthal serums; Confirming to utilize 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 specificity test
The C-ELISA detection method of utilizing the embodiment of the invention 1 prepared MAb to set up detects IBAV positive serum, AKAV positive serum, BRV positive serum and FMDV positive serum, and the result shows that above each serum blocking-up rate all less than 30%, is judged to be feminine gender.And to the blocking-up rate of BTV-12 type positive serum greater than 40%, be judged to be the positive.The C-ELISA detection method that proof utilizes the embodiment of the invention 1 prepared MAb to set up has specificity (table 2) preferably.
Table 2 specificity test-results
2.3 replica test
3 bit manipulation persons detect 50 parts of known background lowlenthal serums, and the detected result basically identical proves that the C-ELISA detection method of utilizing the embodiment of the invention 1 prepared MAb to set up has satisfactory stability property and repeatability.
2.4 main composition is preserved test
Vacuum-packed ELISA Sptting plate and BTV-4H7MAb are after preserving 9 months, and through the C-ELISA checking, reactive nothing changes.
2.5 known sample coincidence rate test
Utilize the C-ELISA detection method of the embodiment of the invention 1 prepared MAb foundation and the test kit that IDEXX department produces; BTV standard positive serum and 50 parts of known background lowlenthal serums of detecting 15 kinds of anti-different serotypes simultaneously compare test; The result shows that 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 test
Utilize the C-ELISA detection method of the embodiment of the invention 1 prepared MAb foundation 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 picking up from the different areas, Guangxi simultaneously, the result shows that the test kit detected result coincidence rate of C-ELISA detection method that this research is set up and the production of IDEXX company is up to 98% (table 4).
Table 4 terrain sample detection test-results
The The above results explanation, the C-ELISA detection method of utilizing the embodiment of the invention 1 prepared MAb to set up can detect the antibody in the BTV infection animal serum fast and accurately, is fit to the 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 common micro-organisms center, and its culture presevation is numbered: CGMCC No.5715.
2. one kind by the described hybridoma cell strain excretory of claim 1 monoclonal antibody.
3. the application of the described hybridoma cell strain of claim 1 in preparation diagnosis or detection blue tongue virus infection animal reagent.
4. the application of the described monoclonal antibody of claim 2 in preparation diagnosis or detection blue tongue virus infection animal reagent.
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| CN103305473A (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 (BTV1-4B6), B-cell epitope polypeptide identified by BTV1-4B6 and application of BTV1-4B6 |
| 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 |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
| CN108384889A (en) * | 2018-03-23 | 2018-08-10 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-2 types, 3 types, 4 types, 7 types, 12 type genotypings differentiate |
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| CN115057925A (en) * | 2022-06-22 | 2022-09-16 | 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 |
| CN116836270A (en) * | 2023-08-03 | 2023-10-03 | 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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| 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 |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
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| 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 |
| CN115057925A (en) * | 2022-06-22 | 2022-09-16 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
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