CN103305471B

CN103305471B - Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application

Info

Publication number: CN103305471B
Application number: CN201310241664.2A
Authority: CN
Inventors: 吴东来; 徐青元; 刘霓红; 杨涛
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2013-06-18
Filing date: 2013-06-18
Publication date: 2014-11-05
Anticipated expiration: 2033-06-18
Also published as: CN103305471A

Abstract

The invention discloses a monoclonal antibody BTV8-VP2-4D9 resistant to bluetongue virus 8 type (BTV8) VP2 protein, B-cell epitope peptide identified thereby and application, and belongs to the field of BTV8 control. A selected hybridoma cell strain capable of stably secreting a BTV8-VP2 protein resistant monoclonal antibody is stored with a microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) No.7003. The experiment results show that the monoclonal antibody BTV8-VP2-4D9 secreted by the hybridoma cell strain can perform idiosyncratic reaction with the BTV8-VP2 protein but not reacts with the VP2 proteins of other serum types. The monoclonal antibody BTV8-VP2-4D9 and the BTV8-VP2 protein virus specific conserved B cell epitope peptide identified by the a monoclonal antibody can be prepared into an agent used for diagnosing BTV8 infection, thus laying a good foundation for creating serology differential diagnosis methods for BTV8 and other types of serum.

Description

The anti-blue tongue virus serum 8 monoclonal antibody BTV8-VP2-4D9 of type VP2 albumen and B cell epitope polypeptide and the application of identification

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-BTV8-VP2 protein monoclonal antibody and secreted monoclonal antibody thereof; The invention still further relates to a B cell epitope polypeptide, relate in particular to the BTV8-VP2 protein B cell epitope polypeptide of being identified by said monoclonal antibody; The invention still further relates to the application in preparation diagnosis, detection or prevention BTV8 medicine of above-mentioned hybridoma cell strain, monoclonal antibody and B cell epitope polypeptide, belong to the prevention and control field of BTV8.

Background technology

Bluetongue (Bluetongue, BT) is the ruminating animal arthropod borne infection being caused by the blue tongue virus of Reoviridae Orbivirus (Bluetongue virus, BTV).BTV can infect most of domestic and wild ruminating animals, because the susceptibility differing appearance of animal goes out different clinical symptom.The differential mortality of different Prevalent district susceptible animal is very large, generally all can reach 30%, even higher in certain areas.Due to the impact of BT on world economy, OIE (OIE) classifies BT as legal circular disease, and China is a class animal epidemic by its delimitation.The loss of related economic that BT causes is on the one hand by directly reducing the throughput of animal and increasing the mortality ratio of animal, the more important thing is owing to controlling flowing, control the outlet of ox seminal fluid and implementing the required expense of these measure of control and the animal trade loss that indirectly causes of animal.

BT is early than the sheep that is found in South Africa for 1876, and 1905 is " bluetongue " by definite designation.20 beginnings of the century, because causing BT, the introduction of hypersusceptible non-native sheep variety starts to spread in Africa.BT is considered to a kind of endemicity disease, and America, Africa, Asia, Australia of being positioned at 40 ° of S of latitude and 53 ° of N are hotspots, only within 1996, causes financial loss in global range up to 3,000,000,000 U.S.s.Within 1979, China finds bluetongue at Sheep from Yunnan first, all detects the positive domestic animal of BT subsequently, as Shanxi (1991), Gansu (1990), Sichuan (1988), Anhui (1985), Hubei (1983) etc. in 29 provinces.BT mainly propagates by insect vector storehouse midges bite.Since 2006, along with climate warming, BT especially BTV8 in Europe, many countries, as the outburst such as Belgian, Dutch, French, German, have caused serious financial loss, also expanded traditional distribution range of BTV simultaneously.Qin Shaomin etc. (2011) have carried out serosurvey to the regional goat BT in 11, Guangxi, and result shows that positive rate is 6.3%-45.1%, and average positive rate is 20.5%.This shows that BT has potential menace to the ruminating animal of China.

BTV serotype is numerous, has found at present 24 serotypes, and along with the new serotype of climate change constantly occurs, as the BTV25(Switzerland being in succession separated to again recently, 2008 years) and BTV26(Kuwait, 2011 years).But because cross immunity protectiveness between each serotype is poor, make that vaccine immunity is intricate can not play a very good protection, up to the present not yet there is effective vaccine.Early stage Accurate Diagnosis, early prevention, is the most effectual way that prevents the outburst of this disease.So, must strengthen the especially research of the related work of BTV8 of China BT, carry out necessary tachnical storage.

BTV belongs to Reoviridae (Reoviridae) Orbivirus (Orbivirus), and its genome is segmented linear double-stranded RNA, comprises 10 sections, and size is about 19kb, the 11 kinds of albumen of encoding altogether.BTV virus particle is made up of three layers of capsid protein, is one of main component of BTV coat protein by the VP2 of L2 genes encoding.VP2 is serotype specificity antigen, can stimulate neutralizing antibody to produce, and is also viral hemagglutinin antigen.Long 961 amino acid of BTV8-VP2, VP2 sequence difference maximum between different B TV serotype.The L2 gene order of 24 serotype type strains of BTV is carried out to phylogenetic tree comparison, between the variation of the L2 gene order of discovery coding VP2 and BTV serotype, have close ties.This shows that VP2, playing an important role aspect decision BTV serotype, is the desirable antigen of setting up the differential diagnosis of BTV serotype.So, filter out a strain and can secrete the hybridoma cell strain of anti-BTV8-VP2 protein specific antibody, and identify the BTV8-VP2 protein-specific B cell epitope that its secreted monoclonal antibody identifies BTV8 diagnosis, prevention or treatment are had to vital role, and lay a good foundation for the development of BTV8 subunit vaccine.

Summary of the invention

One of the object of the invention is to provide the hybridoma cell strain of a strain secretion BTV8-VP2 protein monoclonal antibody;

Two of the object of the invention is to provide a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain (Mab), this monoclonal antibody can with BTV8-VP2 albumen generation specific reaction, and do not react with other serotype VP2 albumen;

Three of the object of the invention is specific b cells epitope polypeptides of a BTV8-VP2 albumen of qualification.

Four of object of the present invention is to provide the application in preparation diagnosis or prevention BTV8 virus infective medicament of above-mentioned hybridoma cell strain, monoclonal antibody and B cell epitope polypeptide.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention adopts TRIZOL method to extract the total RNA of BTV8 C-type virus C, and then utilize reverse transcription technology to clone VP2 gene cDNA, this cDNA is inserted to prokaryotic expression carrier pMAL-c4X, utilize pMAL-c4X prokaryotic expression carrier to carry out prokaryotic expression to BTV8-VP2 gene, using expressed go out the BTV8-VP2 albumen of inclusion body form cut after glue purification as immunogen, immunity BALB/c mouse, gets its splenocyte and SP2/0 myeloma cell and merges.In addition, the present invention also utilizes baculovirus expression system to carry out eukaryotic expression to BTV8-VP2 gene, the BTV8-VP2 albumen of expressed inclusion body form is carried out after inclusion body purification as detection antigen, setting up indirect ELISA detection method screens positive hybridoma cell, the final hybridoma cell strain that obtains the anti-BTV8-VP2 protein monoclonal antibody of a strain stably excreting, called after BTV8-VP2-4D9, its microbial preservation number is: CGMCC No.7003; Classification And Nomenclature is: the hybridoma cell strain of secreting anti-BTV8-VP2-4D9 monoclonal antibody; The preservation time: on December 11st, 2012; Depositary institution is: Chinese common micro-organisms culture presevation administrative center; Preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.

It is a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain that the present invention also provides, called after BTV8-VP2-4D9, Western blot detected result show monoclonal antibody BTV8-VP2-4D9 can with BTV8-VP2 albumen generation specific reaction, and with contrast BHK-21 cell protein and do not react; IFA detected result shows that Mab BTV8-VP2-4D9 only with BTV8, specific reaction occurs, and TV does not react with other serotypes B.

The present invention utilizes the B cell epitope of the BTV8-VP2 albumen that pepscan identifies Mab BTV8-VP2-4D9 to identify, epi-position is accurately orientated as the most at last: ⁶²²aQYVFEKTCLYVLE ⁶³⁵(shown in SEQ ID NO.1.）。Meanwhile, sequence alignment result shows that this epi-position is by the peculiar and conservative B cell epitope of BTV8.

Therefore, the present invention proposes the application of described hybridoma cell strain in preparation diagnosis or prevention blue tongue virus serum 8 type infection medicines.And

The application of described monoclonal antibody in preparation diagnosis or prevention blue tongue virus serum 8 type infection medicines.And

The linear B cell epitope polypeptide of described BTV8-VP2 albumen ⁶²²aQYVFEKTCLYVLE ⁶³⁵(shown in SEQ ID NO.1.) diagnose the application in blue tongue virus serum 8 type infection medicines in preparation.

In sum, the present invention prepares and has identified a species specificity for the monoclonal antibody of BTV8-VP2 albumen and the B cell epitope polypeptide of identifying thereof, the conservative B cell epitope polypeptide of BTV8-VP2 prion specificity that monoclonal antibody of the present invention and this monoclonal antibody are identified can be used for being prepared into the reagent of diagnosis or prevention BTV8, thereby is that the serological diagnostic method of setting up BTV8 and other serotypes B TV is laid a good foundation.

Brief description of the drawings

Fig. 1 is BTV8-VP2 gene RT-PCR amplification;

1:DL5000DNA Marker; 2:BTV8-VP2 gene RT-PCR amplified production;

Fig. 2 is the double digestion qualification of recombinant plasmid pMAL-c4X-VP2;

1:DL5000DNA Marker; 2:BamH I and Hind III double digestion pMAL-c4X-VP2 result;

Fig. 3 is the SDS-PAGE analytical results of restructuring BTV8-VP2 albumen;

M: standard protein Marker; Product before 1:pMAL-c4X-VP2 induction; 4 hours expression products of 2:pMAL-c4X-VP237 ° of C induction; 3: the ultrasonic rear supernatant of restructuring BTV8-VP2 albumen; 4: the ultrasonic postprecipitation of restructuring BTV8-VP2 albumen; 5: product before empty carrier pMAL-c4X induction; 7: 4 hours expression products of empty carrier pMAL-c4X37 ° of C induction;

Fig. 4 is the reactive result that application Western blot test detects monoclonal antibody BTV8-VP2-4D9 and BTV8-VP2 albumen and BHK-21 cell;

1:BTV8-VP2-4D9 reacts with BHK-21 lysis precipitation; Reacting of the BHK-21 lysis precipitation that 2:BTV8-VP2-4D9 infects with BTV8; M: standard protein Marker;

Fig. 5 is that the SDS-PAGE of 24 small peptides of prokaryotic expression analyzes;

1-24:24 bar small peptide; M: standard protein Marker;

Fig. 6 is 24 small peptides and the reactive indirect ELISA analysis of BTV8-VP2-4D9 of prokaryotic expression;

Fig. 7 is synthesized small peptide and the reactive indirect ELISA analysis of BTV8-VP2-4D9 in table 2;

Fig. 8 is synthesized small peptide and the reactive indirect ELISA analysis of BTV8-VP2-4D9 in table 3.

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, virus

BTV8-VP2 albumen, SP2/0 cell, Sf9 cell, BHK-21 cell and the BTV1-24 type strain of BTV8-VP2 albumen, eukaryotic expression the purifying of prokaryotic expression purifying preserved by this laboratory.

2. main agents and medicine

Foetal calf serum, DMEM substratum are purchased from GIBCO company; 50%PEG, 50 × HAT, 50 × HT, FITC mark sheep anti-mouse igg antibody are purchased from Sigma company; The goat dynamics of diaminobenzidine (DAB) colouring reagents box, horseradish peroxidase (HRP) mark is purchased from Beijing company of Zhong Shan Golden Bridge; O-Phenylene Diamine (OPD) is purchased from Harbin Bo Rui Bioisystech Co., Ltd; Dye in advance albumen Marker purchased from Fermentas company; SBA ClonotypingTM System/HRP Subclass of antibody identification kit is purchased from Southern Biotechnology company; Restriction enzyme BamH I and Hind III, ThermoScript II, Ex Taq archaeal dna polymerase, T4DNA ligase enzyme 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 1BTV8-VP2 albumen

1. design of primers

According to listed BTV8VP2 gene order (accession number: AJ585129) in Genbank, design pcr amplification primer, sequence is as follows:

BTV8VP2-BamHI-atg-1-18:5’- CGGGATCCATGGAGGAGCTAGCAATTC-3’

BTV8VP2-HindⅢ-2903-2884R:5’- GTCAAGCTTCTATACATTGAGCAGRTTAG-3’

Underscore part is BamH I, the Hind III restriction enzyme site of introducing, and estimates that amplified production length is 2886bp.

The extraction of 2.BTV8 viral RNA and reverse transcription

Utilize Trizol method to extract virus genome RNA as template from the BHK-21 cell of infection BTV8, carry out reverse transcription with BTV8VP2-Hind III-2903-2884R primer and synthesize viral cDNA.

Trizol method is extracted RNA step: results infect BHK-21 cell 1-5 × 10 of BTV8 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 1mL75%, 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 template ribonucleic acid solution and downstream primer are hatched to 10min in 95 DEG C, the cooling 5min of ice bath, then adds all the other reagent, mixes, and room temperature is placed 10min, hatches 60min for 42 DEG C, cooling 2min in ice.

3.BTV8VP2 gene amplification and purifying

The cDNA obtaining taking reverse transcription, as template, utilizes designed pcr amplification primer, amplification BTV8VP2 gene (Fig. 1).

(50 μ L) is as follows for PCR reaction system:

Reaction conditions is 95 ° of C denaturation 5min; 95 ° of C sex change 30s, 54 ° of C annealing 30s, 72 ° of C extend 3min, circulate 30 cycles; 72 ° of C extend 10min.

Then PCR product glue is reclaimed; whole pcr amplification products are carried out to agarose gel electrophoresis; and under ultraviolet lamp, cut the blob of viscose that contains goal gene, reclaim test kit specification sheets according to Shanghai Hua Shun biotechnology company limited glue afterwards and reclaim the VP2 gene that pcr amplification goes out.

4.BTV8-VP2 the structure of albumen pronucleus expression recombinant vectors

Glue is reclaimed to the VP2 gene and the prokaryotic expression carrier pMAL-c4X that obtain and carry out respectively double digestion, it is as follows that enzyme is cut system:

Meanwhile, prokaryotic expression carrier pMAL-c4X is carried out to double digestion, it is as follows that enzyme is cut system:

After endonuclease reaction system is mixed, put standing 2h in 37 ° of C water-baths.System after L2 gene double digestion is carried out to purifying with the Shanghai PCR of Hua Shun biotechnology company limited product purification test kit, and operation steps is undertaken by test kit specification sheets.System after prokaryotic expression carrier pMAL-c4X double digestion is carried out to agarose gel electrophoresis, then carry out glue recovery.

L2 gene after double digestion is connected with prokaryotic expression carrier pMAL-c4X, and linked system is: 10 × T4DNA Ligase Buffer1 μ L, and enzyme is cut rear L2 gene 4 μ L, and enzyme is cut rear pMAL-c4X1.5 μ L, T4DNA ligase enzyme 1 μ L, ddH ₂o2.5 μ L.16 ° of C connections are spent the night.

5. transform and choose bacterium

The connection product obtaining in 4 is all proceeded in Ecoli DH5 α competent cell to ice bath 30min, 42 DEG C of water-bath thermal stimulus 90s afterwards, rapider ice bath 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 (Fig. 2).

4) by through tentatively concluding that the bacterium liquid that contains positive plasmid delivers the handsome Bioisystech Co., Ltd in Shanghai and carry out sequencing, correct recombinant plasmid called after pMAL-c4X-VP2 will be identified by sequencing.

The prokaryotic expression of 7.BTV8VP2 gene and purifying

According to method for transformation described in 5, recombinant plasmid pMAL-c4X-VP2 is transformed into BL21 for prokaryotic expression (DE3) 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).Induced expression concrete operations are as follows: get in the LB substratum that 1mL recombinant bacterium bacterium liquid joins 100mL 37 DEG C of concussions when being cultured to OD600nm and being about 0.5-1, adding IPTG is 1mmol/L to final concentration, 37 DEG C of induction 4-5h(Fig. 3).Expression product is carried out to SDS-PAGE electrophoresis, carry out purifying by cutting gluing method.Add appropriate PBS to be ground into thin broken particle the blob of viscose cutting, this micelle needn't add adjuvant and can be used for animal immune, can slowly release target protein.

The preparation of embodiment 2 monoclonal antibodies

1. mouse immune

To cut 5 of the prokaryotic expression recombinant VP 2 protein immunizations female BALB/c mouse in 6 week age of glue purification, immunity 3 times altogether, two weeks, each immune interval, immunizing dose be 50 μ g/ only, 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, 10000rpm, 20min), 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.

2. cytogamy

Merge and prepare feeder layer cells in first 1 day, 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 splenocyte and SP2/0 myeloma cell's ratio of 4: 1 with PEG, and the cell after fusion is laid on ready feeder cell.

3. the screening of positive hybridoma cell strain and clone

Utilize the eukaryotic expression BTV8-VP2 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 subclone of positive hybridoma cell with limiting dilution assay simultaneously, at least subclone 3 times, by frozen in time positive hybridoma cell good subclone.The final hybridoma cell strain that obtains a strain can the anti-BTV8-VP2 protein monoclonal antibody of stably excreting, its microbial preservation number is: CGMCC No.7003; And by the monoclonal antibody called after BTV8-VP2-4D9 of its secretion, hereinafter to be referred as 4D9.

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 7～10d in the time of mouse web portion extreme expansion, takes out once every 2d, by the centrifugal 10min of ascites 10000r/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

Monoclonal antibody embodiment 2 being obtained according to SBA ClonotypingTM System/HRP Subclass of antibody identification kit process specifications is carried out subgroup identification.

Result shows that the heavy chain of monoclonal antibody 4D9 of the present invention is IgG ₁, light chain is κ chain.

2.Western blot test

After cell precipitation after centrifugal results BTV8 virus supernatant and BHK-21 cell precipitation are processed, carry out SDS-PAGE electrophoresis, then through electric transfer printing by protein delivery to nitrocellulose filter, it is 18V voltage 30min that electricity turns condition; 4 ° of C of nitrocellulose filter after transfer printing sealing is spent the night with 5% skim-milk confining liquid; Add monoclonal antibody supernatant incubated at room 1h, PBS damping fluid with PBST(containing the pH7.4 of 0.5mL/L tween 20) wash three times, again with the goat dynamics incubated at room 1h of horseradish peroxidase (HRP) mark of 4000 times of dilutions, after PBST washing 3 times, with the colour developing of DAB colouring reagents box, sweep record.

Test-results confirms, the prepared monoclonal antibody 4D9 of the present invention can with BTV8-VP2 albumen generation specific reaction, and do not react (Fig. 4) with contrasting BHK-21 cell, infer that according to this experimental result the BTV8-VP2 protein B cell epitope that 4D9 identifies should be a linear epitope simultaneously.

3.IFA test

The BHK-21 cell that grows to 70%～80% is met to poison 1～24 type BTV.Infect 4 DEG C of fixing 30min of cold ethanol after 48 hours, the 4D9 ascites that 1:10 doubly dilutes is hatched 1 hour, and the sheep anti mouse two of the FITC mark that then 1:128 doubly dilutes resists 37 DEG C and hatches 1 hour.Last fluorescence microscope is taken pictures.

Test-results confirmation, only can there is specific reaction and not react (table 1) with other 23 BTV serotypes with BTV8 in the prepared monoclonal antibody 4D9 of the present invention.

Table 1IFA qualification monoclonal antibody 4D9 specificity

The qualification of embodiment 4B cell epitope polypeptide

1.BTV8-VP2 the segmentation shorten expression of albumen

Taking the gene order of recombinant plasmid pMAL-c4X-VP2 as template, design 24 pairs of primers, introduce respectively BamH I, Hind III restriction enzyme site at 5 ' end of every pair of upstream and downstream primer.After pcr amplification, by the method for embodiment 1, they are connected respectively to the upper construction recombination plasmid of prokaryotic expression carrier pMAL-c4X, called after pMAL-VP2-1 ﹣ pMAL-VP2-24 respectively, then it is converted into respectively to BL21(DE3) express in competent cell and carry out abduction delivering, every small peptide is about 50-53aa, called after VP2-1-VP2-24 respectively, an overlapping 10-13 aa between adjacent two peptides.It is carried out to SDS-PAGE electrophoretic analysis, determine small peptide successful expression (Fig. 5).

2. the preliminary evaluation of monoclonal antibody 4D9 epitope

By 24 small peptides of expressing after ultrasonication respectively with the coated ELISA Sptting plate in 100ng/ hole, hatch 1h taking 3E11 cells and supernatant as 37 ° of C of primary antibodie, then the mountain sheep anti-mouse igg of HRP mark is that two anti-37 ° of C are hatched 1h and carried out indirect ELISA experiment.Result shows that monoclonal antibody 4D9 only with VP2-17, specific reaction occurs, and does not all react (Fig. 6) with other small peptides, so the BTV8-VP2 protein B cell epitope Primary Location that monoclonal antibody 4D9 is identified in ⁶²²aQYVFEKTCLYVLELLSKHTMPSEDSEVTFEHPTVDPNIDIETWKIIDVSQLII ⁶⁷⁵.

3. the accurate location of monoclonal antibody 4D9 epitope

After the epitope of 4D9 is tentatively determined, by further VP2-17 brachymemma, utilize PepScan technology to synthesize following small peptide (table 2), overlapping 6 aa between adjacent two small peptides, carry out indirect ELISA experiment again and detect, package amount is 100ng/ hole, further determines epitope.Result shows that 4D9-1 and 4D9 are positive, other be all negative and react (Fig. 7) with 4D9, thus further 4D9 epitope is positioned at ⁶²²aQYVFEKTCLYVLELL ⁶³⁷.

Further synthesize following small peptide (table 3) according to above-mentioned indirect ELISA detected result, finally complete 4D9 and identify the accurate location of BTV8-VP2 protein B cell epitope.

Small peptide in his-and-hers watches 3 carries out indirect ELISA detection display monoclonal antibody 4D9 and only reacts with 4D9-1-5,4D9-1-6, when ⁶²²after A disappearance, all do not react with corresponding small peptide, infer ⁶²²a is key amino acid, when ⁶³⁵after E disappearance, do not react with corresponding small peptide, infer ⁶³⁵e is key amino acid, so 4D9 epitope is accurately navigated to ⁶²²aQYVFEKTCLYVLE ⁶³⁵(shown in SEQ ID NO.1.) (Fig. 8).

Table 2 is for the synthetic small peptide of monoclonal antibody 4D9

Table 3 is for the synthetic small peptide of monoclonal antibody 4D9

The conservative property of 4.B cell epitope polypeptide and virus-specific analysis

Utilize European Bioinformatics Institute(EBI) the aminoacid sequence Blast program that provides of database, identified BTV8-VP2 protein B cell epitope is carried out to conservative Analysis, whether this epitope sequences and other BTV serotype VP2 albumen respective section are compared, analyzing this epi-position is BTV8-VP2 protein-specific epi-position simultaneously.

The BTV8-VP2 protein B cell epitope that comparison result shows identifies is (table 4) guarded completely in the each strain of BTV8, and very low with the relatively demonstration homology of other BTV serotype VP2 albumen respective section, show that this epi-position is BTV8 peculiar (table 4).

Table 44D9 epitope conservative Analysis

Claims

1. anti-blue tongue virus serum 8 types (Bluetongue virus serotype8 is secreted in a strain, BTV8) hybridoma cell strain of VP2 protein monoclonal antibody, called after BTV8-VP2-4D9, it is characterized in that, the microbial preservation of described hybridoma cell strain number is: CGMCC No.7003.

2. the 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 prevention blue tongue virus serum 8 type infection medicines.

4. the application of monoclonal antibody claimed in claim 2 in preparation diagnosis or prevention blue tongue virus serum 8 type infection medicines.