CN103305471A - Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application - Google Patents

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

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 that is 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 animals is very large, generally all can reach 30%, in certain areas even higher.Because BT is on the impact of world economy, OIE (OIE) classifies BT as legal circular disease, and China is a class animal epidemic with its delimitation.The loss of related economic that BT causes is on the one hand by the throughput that directly reduces animal and the mortality ratio that increases animal, the more important thing is because the flowing of control animal, the seminal fluid outlet of control ox and implement the required expense of these measure of control and the animal trade loss that indirectly causes.

BT is early than the sheep that was found in South Africa in 1876, and 1905 is " bluetongue " by definite designation.20 beginnings of the century are because the introduction of hypersusceptible non-native sheep variety causes BT to begin 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 the hotspots, only causes the interior financial loss of global range up to 3,000,000,000 U.S.s in 1996.China found bluetongue at Sheep from Yunnan first in 1979, all detected the positive domestic animal of BT in 29 provinces subsequently, such as Shanxi (1991), Gansu (1990), Sichuan (1988), Anhui (1985), Hubei (1983) etc.BT mainly propagates by insect vector storehouse midges bite.Since 2006, along with climate warming, BT especially BTV8 many countries have caused serious financial loss such as the outburst such as Belgian, Dutch, French, German in Europe, also enlarged traditional distribution range of BTV simultaneously.Qin Shaomin etc. (2011) have carried out serosurvey to the goat BT in 11 areas, Guangxi, and the 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, such as the BTV25(Switzerland that in succession is separated to again recently, 2008 years) and BTV26(Kuwait, 2011 years).Yet because the cross immunity protectiveness is poor between each serotype, can not play a very good protection so that vaccine immunity is intricate, up to the present effective vaccine is not yet arranged.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.The BTV virus particle is comprised 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 also is viral hemagglutinin antigen.Long 961 amino acid of BTV8-VP2, the VP2 sequence difference is maximum between the different B TV serotype.The L2 gene order of 24 serotype type strains of BTV is carried out phylogenetic tree relatively, and finding has close ties between the variation of L2 gene order of coding VP2 and the BTV serotype.This shows that VP2 playing an important role aspect the 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 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 provides the hybridoma cell strain of strain secretion BTV8-VP2 protein monoclonal antibody;

Two of the object of the invention provides 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 to identify the specific b cells epitope polypeptide of a BTV8-VP2 albumen.

Four of purpose of the present invention provides 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 the TRIZOL method to extract the total RNA of BTV8 C-type virus C, and then utilize the reverse transcription technology to clone the VP2 gene cDNA, this cDNA is inserted prokaryotic expression carrier pMAL-c4X, utilize the pMAL-c4X prokaryotic expression carrier that the BTV8-VP2 gene is carried out prokaryotic expression, the BTV8-VP2 albumen of the expressed inclusion body form that goes out is cut behind the glue purification as immunogen, the immunity BALB/c mouse is got its splenocyte and SP2/0 myeloma cell and is merged.In addition, the present invention also utilizes baculovirus expression system that the BTV8-VP2 gene is carried out eukaryotic expression, BTV8-VP2 albumen to expressed inclusion body form carries out using antigen as detecting behind the inclusion body purification, 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; The 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 do not react with contrast BHK-21 cell protein; The 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 the SEQ ID NO.1.）。Simultaneously, the sequence alignment result shows that this epi-position is 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 the SEQ ID NO.1.) preparing the application of diagnosing in the blue tongue virus serum 8 type infection medicines.

In sum, the present invention's preparation has also 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 the 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.

Description of drawings

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

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

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

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; 1:pMAL-c4X-VP2 induces front product; 2:pMAL-c4X-VP237 ° of C induces 4 hours expression products; 3: the ultrasonic rear supernatant of restructuring BTV8-VP2 albumen; 4: the ultrasonic postprecipitation of restructuring BTV8-VP2 albumen; 5: empty carrier pMAL-c4X induces front product; 7: an empty carrier pMAL-c4X37 ° C induces 4 hours expression products;

Fig. 4 detects the reactive result of monoclonal antibody BTV8-VP2-4D9 and BTV8-VP2 albumen and BHK-21 cell for using Western blot test;

The reaction of 1:BTV8-VP2-4D9 and BHK-21 lysis precipitation; The reaction of the BHK-21 lysis precipitation that 2:BTV8-VP2-4D9 and BTV8 infect; 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 the reactive indirect ELISA analysis of synthetic small peptide and BTV8-VP2-4D9 in the table 2;

Fig. 8 is the reactive indirect ELISA analysis of synthetic small peptide and BTV8-VP2-4D9 in the 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 only are exemplary, scope of the present invention are not consisted of 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 make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.

Main experiment material and source

1. albumen, cell, virus

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

2. main agents and medicine

Foetal calf serum, DMEM substratum are available from GIBCO company; 50%PEG, 50 * HAT, 50 * HT, FITC mark sheep anti-mouse igg antibody are available from Sigma company; The goat dynamics of diaminobenzidine (DAB) colouring reagents box, horseradish peroxidase (HRP) mark is available from company of China fir Golden Bridge in Beijing; O-Phenylene Diamine (OPD) is available from Harbin Bo Rui Bioisystech Co., Ltd; Dye in advance albumen Marker available 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 available from precious biotechnology (Dalian) company limited; Glue reclaims test kit available from Shanghai Hua Shun company.

3. laboratory animal

6 the week age BALB/c mouse 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 among the Genbank (accession number: AJ585129), design pcr amplification primer, sequence is as follows:

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

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

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

2.BTV8 the extraction of viral RNA and reverse transcription

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

The Trizol method is extracted the RNA step: the BHK-21 cell 1-5 of results infection BTV8 * 10 ⁷Individual, add 1mL Trizol mixing, room temperature leaves standstill 5min, add the 0.2mL chloroform, the jolting 15s that exerts oneself, incubated at room 2-3min, 12000g, 4 ℃ of centrifugal 15min, centrifugal rear liquid divides three layers, the careful upper strata colourless liquid that takes out, the Virahol that adds the equal-volume precooling, incubated at room 10min behind the mixing, 12000g, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation adds the ethanol (preparation of DEPC water) of 1mL75%, and 15s gently shakes, 7500g, 4 ℃ of centrifugal 5min carefully abandon most supernatant, and settling chamber's warm air is done 3-5min, add 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 as follows:

In the reaction process, first template ribonucleic acid solution and downstream primer are hatched 10min in 95 ℃, then ice bath cooling 5min adds all the other reagent, mixing, and room temperature is placed 10min, hatches 60min for 42 ℃, cools off 2min in the ice.

3.BTV8VP2 gene amplification and purifying

The cDNA that obtains take reverse transcription utilizes designed pcr amplification primer, amplification BTV8VP2 gene (Fig. 1) as template.

(50 μ L) is as follows for the 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 agarose gel electrophoresis; and under ultraviolet lamp, cut the blob of viscose that contains goal gene, reclaim the test kit specification sheets according to Shanghai China 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 VP2 gene and the prokaryotic expression carrier pMAL-c4X obtain carry out respectively double digestion, it is as follows that enzyme is cut system:

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

Behind endonuclease reaction system mixing, put in 37 ° of C water-baths and leave standstill 2h.System behind the L2 gene double digestion is carried out purifying with Shanghai China Shun biotechnology PCR of company limited product purification test kit, and operation steps is undertaken by the test kit specification sheets.System behind the prokaryotic expression carrier pMAL-c4X double digestion is carried out agarose gel electrophoresis, then carry out glue and reclaim.

L2 gene behind the double digestion is connected with prokaryotic expression carrier pMAL-c4X, and linked system is: 10 * T4DNA Ligase Buffer1 μ L, enzyme 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 that obtains in 4 is all changed in the Ecoli DH5 α competent cell over to ice bath 30min, 42 ℃ of water-bath thermal stimulus 90s afterwards, more rapid ice bath 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 incubation.The single bacterium colony of random picking from the flat board is inoculated into respectively 37 ℃ of shaking culture in 5mL LB (Amp+, the 100 μ g/mL) liquid nutrient medium.

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 cultivating from 5 according to the operation of test kit specification sheets, and the plasmid that extracts 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 as follows 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 (Fig. 2).

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

7.BTV8VP2 the prokaryotic expression of gene and purifying

According to 5 described method for transformation recombinant plasmid pMAL-c4X-VP2 is transformed into prokaryotic expression BL21 (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 ℃ of overnight incubation, white on the picking LB plate culture medium isolates bacterium colony, is inoculated in 37 ℃ of overnight incubation in the LB liquid nutrient medium that 5mL contains penbritin (100 μ g/mL).The induced expression concrete operations are as follows: get in the LB substratum that 1mL recombinant bacterium bacterium liquid joins 100mL 37 ℃ of concussions when being cultured to OD600nm and being about 0.5-1, add IPTG to final concentration be 1mmol/L, induce 4-5h(Fig. 3 for 37 ℃).Expression product is carried out the SDS-PAGE electrophoresis, carry out purifying by cutting gluing method.The blob of viscose that downcuts is added an amount of PBS be ground into thin broken particle, this micelle needn't add adjuvant and namely can be used for animal immune, can slowly release target protein.

The preparation of embodiment 2 monoclonal antibodies

1. mouse immune

With the prokaryotic expression recombinant VP 2 protein immunization female BALB/c mouse in age in 56 weeks of cutting glue purification, immunity is 3 times altogether, and in each two weeks of immune interval, immunizing dose is 50 μ g/, and immunization route is peritoneal immunity.

Respectively two exempt to exempt from three after a week to the mouse blood sampling of docking, separation of serum (4 ℃, 10000rpm, 20min) detects antibody horizontal with indirect ELISA.In cytogamy front 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.

2. cytogamy

Merge and prepared feeder layer cells in front 1 day, get the BALB/c mouse peritoneal macrophage according to ordinary method and be laid in the 96 porocyte culture plates stand-by.Disconnected neck is put to death the mouse of spleen to be got, and aseptic spleen and the separating Morr. cell got carries out cytogamy in splenocyte and 4: 1 ratio of SP2/0 myeloma cell with PEG, and the cell after the fusion is laid on the ready feeder cell.

3. the screening of positive hybridoma cell strain and clone

Utilize the eukaryotic expression BTV8-VP2 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 simultaneously the subclone of positive hybridoma cell with limiting dilution assay, at least subclone is 3 times, and the positive hybridoma cell that subclone is good is in time frozen.The final hybridoma cell strain that obtains a strain can the anti-BTV8-VP2 protein monoclonal antibody of stably excreting, its microbial preservation number are: CGMCC No.7003; And with 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 ages in week, 0.5mL/, 1 all pneumoretroperitoneum injections 1 * 10 ⁶Individual hybridoma extracts ascites when the mouse web portion extreme expansion behind 7～10d, take out once every 2d, with the centrifugal 10min of ascites 10000r/min that extracts, removes upper strata grease and precipitation, and the supernatant packing is stored in-20 ℃.

The evaluation of embodiment 3 monoclonal antibodies

1. the subgroup identification of monoclonal antibody

According to SBA ClonotypingTM System/HRP Subclass of antibody identification kit process specifications embodiment 2 resulting monoclonal antibodies are carried out subgroup identification.

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

2.Western blot test

Will be centrifugal cell precipitation behind the results BTV8 virus supernatant and BHK-21 cell precipitation carry out the SDS-PAGE electrophoresis after processing, then through electric transfer printing with protein delivery to nitrocellulose filter, it is 18V voltage 30min that electricity turns condition; Spend the night with 4 ° of C sealings of the nitrocellulose filter of 5% skim-milk confining liquid after with transfer printing; Add monoclonal antibody supernatant incubated at room 1h, the PBS damping fluid that contains the pH7.4 of 0.5mL/L tween 20 with PBST() washing is three times, again with the goat dynamics incubated at room 1h of horseradish peroxidase (HRP) mark of 4000 times of dilutions, after the 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 contrast 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 poison 1～24 type BTV.Infect after 48 hours fixedly 30min of 4 ℃ of cold ethanol, the 4D9 ascites that 1:10 doubly dilutes was hatched 1 hour, then the FITC mark that doubly dilutes of 1:128 sheep anti mouse two anti-37 ℃ hatched 1 hour.Last fluorescence microscope is taken pictures.

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

Table 1IFA identifies monoclonal antibody 4D9 specificity

The evaluation of embodiment 4B cell epitope polypeptide

1.BTV8-VP2 the segmentation shorten expression of albumen

Take 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.Behind pcr amplification, method by embodiment 1 is connected respectively to the upper construction recombination plasmid of prokaryotic expression carrier pMAL-c4X with them, difference called after pMAL-VP2-1 ﹣ pMAL-VP2-24, then it is converted into respectively BL21(DE3) express in the competent cell and carry out abduction delivering, every small peptide is about 50-53aa, difference called after VP2-1-VP2-24, an overlapping 10-13 aa between adjacent two peptides.It is carried out the SDS-PAGE electrophoretic analysis, determine small peptide successful expression (Fig. 5).

2. the preliminary evaluation of monoclonal antibody 4D9 epitope

With 24 small peptides of expressing after ultrasonication respectively with the coated ELISA Sptting plate in 100ng/ hole, 37 ° of C are hatched 1h take the 3E11 cells and supernatant as 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 the indirect ELISA experiment.The 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 definite, with the further brachymemma of VP2-17, utilize the PepScan technology to synthesize following small peptide (table 2), overlapping 6 aa between adjacent two small peptides, carry out the indirect ELISA experiment again and detect, package amount is the 100ng/ hole, further the defined antigen epi-position.The result shows that 4D9-1 and 4D9 are positive, other with the 4D9 reaction (Fig. 7) that all is negative, thereby further the 4D9 epitope is positioned at ⁶²²AQYVFEKTCLYVLELL ⁶³⁷

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

Small peptide in the 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 ⁶²²All do not react with corresponding small peptide after the A disappearance, infer ⁶²²A is key amino acid, when ⁶³⁵Do not react with corresponding small peptide after the E disappearance, infer ⁶³⁵E is key amino acid, so the 4D9 epitope is accurately navigated to ⁶²²AQYVFEKTCLYVLE ⁶³⁵(shown in the 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

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

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

The BTV8-VP2 protein B cell epitope that comparison result shows identifies is (table 4) guarded fully in 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 (6)

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 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.BTV8-VP2 the linear B cell epitope polypeptide of albumen is characterized in that the aminoacid sequence of described B cell epitope polypeptide is that described epitope polypeptide is identified by monoclonal antibody claimed in claim 2 shown in the SEQ ID NO.1.

4. the application of hybridoma cell strain claimed in claim 1 in preparation diagnosis or prevention blue tongue virus serum 8 type infection medicines.

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

6. the application of the linear B cell epitope polypeptide of BTV8-VP2 albumen claimed in claim 3 in preparation diagnosis blue tongue virus serum 8 type infection medicines.

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