CN103740650A

CN103740650A - Monoclonal antibody BTV16-3G10 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope polypeptide identified by monoclonal antibody BTV16-3G10, and applications of monoclonal antibody BTV16-3G10

Info

Publication number: CN103740650A
Application number: CN201310652908.6A
Authority: CN
Inventors: 吴东来; 徐青元; 杨涛
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2013-12-06
Filing date: 2013-12-06
Publication date: 2014-04-23

Abstract

The invention discloses monoclonal antibody BTV16-3G10 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope polypeptide identified by the monoclonal antibody BTV16-3G10, and applications of the monoclonal antibody BTV16-3G10, and belongs to the field of bluetongue prevention. The invention also relates to a hybridoma cell strain which is capable of secreting the monoclonal antibody BTV16-3G10, and monoclonal antibody secreted by the hybridoma cell strain. It is shown by indirect immunofluorescence assay that the specific reaction is observed between the monoclonal antibody and BTV16, and no cross reaction is observed between the monoclonal antibody and other serum types of bluetongue virus, Ibaraki virus or Chuzan disease virus. Further, BTV16-VP2 protein B-cell epitope polypeptide which can be identified by the monoclonal antibody is determined via cutting expression antigen short peptide and peptide scanning technology. The monoclonal antibody, and the BTV16-VP2 protein B-cell epitope polypeptide identified by the monoclonal antibody can be used for preparation of BTV16 specific differential diagnosis reagents, and foundation is provided for development of BTV epitope labeled vaccines.

Description

B cell epitope polypeptide and the application of anti-blue tongue virus serum 16 type VP2 protein monoclonal antibody BTV16-3G10 and 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-BTV16 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 BTV16 VP2 protein B cell epitope polypeptide of being identified by said monoclonal antibody; The invention still further relates to the application in preparation diagnosis or prevention BTV16 infection medicine of above-mentioned hybridoma cell strain, monoclonal antibody and B cell epitope polypeptide, belong to the prevention and control field of bluetongue.

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, early than the sheep that is found in South Africa for 1876, is named as " anti-malarial sheep catarrhal fever " for 1902, 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 dollars.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.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%.

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 research of the related work 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.Wherein VP2 albumen is positioned at viral outermost, is the outstanding virus surface of spike-shaped, participates in absorption and the intrusion of virus to mammalian cell, has hemagglutination activity, and neutralization is active, is the major antigen that determines serological specificity.Therefore, filter out the hybridoma cell strain that a strain can secretor type specific antibody, and identify the foundation to BTV16 type specificity diagnostic method of BTV16-VP2 protein-specific B cell epitope that its secreted monoclonal antibody identifies, and the prevention of disease or treatment have vital role, and lay a good foundation for the development of BTV Epitope tag vaccine.

Summary of the invention

One of the object of the invention is to provide a strain secretes the hybridoma cell strain of anti-blue tongue virus serum 16 types (Bluetongue virus 16, BTV16) 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, can there is specific reaction with BTV16 in this monoclonal antibody, and not with other serotypes of blue tongue virus (BTV1-15,17-24), Ibaraki virus (Ibaraki virus, IBAV), middle mountain virus (Chuzan virus, CV) produces cross reaction;

Three of the object of the invention is to identify the BTV16 type specificity B cell epitope of a BTV16-VP2 albumen.

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

The BTV16 VP2 recombinant protein that the present invention expresses by Bac-to-Bac baculovirus expression system is immunogen immune Balb/c mouse, gets mouse spleen lymphocyte and SP2/0 cytogamy after immunity.In addition, the present invention also utilizes eucaryon recombinant VP 2 albumen as envelope antigen, through indirect ELISA method screening, to obtain the hybridoma cell strain of the anti-BTV16 VP2 of strain stably excreting protein monoclonal antibody, called after BTV16-3G10, Classification And Nomenclature is: the hybridoma cell strain of secreting the monoclonal antibody of anti-BTV16 VP2 protein monoclonal antibody 3G10; Its microbial preservation number is: CGMCC No.8153, preservation time: on August 28th, 2013; 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, the aminoacid sequence of the linear B cell epitope of the BTV16 VP2 albumen of its specific recognition of described monoclonal antibody BTV16-3G10 be shown in SEQ ID NO.1 ( ₃₄eWSGHDVTEIPNRRMF ₄₉).

It is a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain that the present invention also provides, its called after BTV16-3G10, Western blot detected result show BTV16-3G10 can with BTV16-VP2 albumen generation specific reaction, and do not react with BHK-21 cell, indirect immunofluorescence assay (IFA) shows that this monoclonal antibody only presents specific reaction with BTV16, and not with other serotypes of blue tongue virus (BTV1-15,17-24), Ibaraki virus (IBAV), the sick virus in middle mountain (CV) produces cross reaction.

The present invention utilizes the B cell epitope of the BTV16-VP2 albumen that prokaryotic expression small peptide technology and pepscan identify BTV16-3G10 to identify, this epi-position is accurately orientated as the most at last: ₃₄eWSGHDVTEIPNRRMF ₄₉(shown in SEQ ID NO.1).Meanwhile, sequence alignment result shows ₃₄eWSGHDVTEIPNRRMF ₄₉in the strain isolated of BTV16 type different areas, it is high conservative, and other serotypes of blue tongue virus and and blue tongue virus be in Reoviridae Orbivirus together other represent between virus almost there is no conservative property, illustrate that this epi-position is the specificity epitope of BTV16 type.

In addition, the invention allows for the application of described hybridoma cell strain in preparation diagnosis BTV16 virus infective medicament.And

The application of described monoclonal antibody in system diagnosis or prevention BTV16 virus infective medicament.And

The linear B cell epitope polypeptide of described BTV16-VP2 albumen ₃₄eWSGHDVTEIPNRRMF ₄₉(shown in SEQ ID NO.1) application in preparation diagnosis BTV16 infection medicine.

In sum, the present invention prepares and has identified the monoclonal antibody of a species specificity for BTV16-VP2 albumen, and the BTV16-VP2 prion specific b cells epitope polypeptide that monoclonal antibody of the present invention and this monoclonal antibody are identified is that the functional study of setting up BTV16 type specificity immunological detection method and VP2 albumen is laid a good foundation.

Accompanying drawing explanation

Fig. 1 is eukaryotic expression and the purifying that SDS-PAGE identifies recombinant VP 2 albumen;

M: albumen Marker; 1: wild-type baculovirus infects Sf9 cell pyrolysis liquid; 2: the Sf9 cell pyrolysis liquid that does not infect baculovirus; 3: the Sf9 cell pyrolysis liquid that infects recombinant baculovirus; 4: the BTV-16VP2 albumen of purifying; 5: the washing lotion (not containing BTV-16 VP2 albumen, but containing other foreign proteins) of purifying BTV-16 VP2 albumen;

Fig. 2 is the reactive result that application Western blot test detects monoclonal antibody BTV16-3G10 and BTV16-VP2 albumen;

1: inoculation has the insect cell lysate of wild-type baculovirus; 2: by the BTV16VP2 recombinant protein of the expression of Bac-to-Bac baculovirus expression system, purifying; The BHK-21 lysis precipitation that 3:BTV16 infects; 4:BHK-21 lysis precipitation; M: standard protein Marker

Fig. 3 is the reactive result that application IFA test detects monoclonal antibody BTV16-3G10 and BTV16;

A:MAb BTV16-3G10; The contrast of b:BTV16 type mouse positive serum; C: mouse negative serum contrast

Fig. 4 is prokaryotic expression restructuring MBP small peptide and the reactive indirect ELISA analysis of monoclonal antibody BTV16-3G10;

Fig. 5 synthesizes small peptide and the reactive indirect ELISA analysis of monoclonal antibody BTV16-3G10 by pepscan;

Fig. 6 by conservative property and the specificity analyses of evaluation B cell epitope;

The conservative Analysis of the B cell that A: monoclonal antibody BTV16-3G10 identifies between BTV1-26; The B cell that B: monoclonal antibody BTV16-3G10 identifies represents the conservative Analysis between virus in Reoviridae Orbivirus;

Fig. 7 is the not quite identical small peptide of the B cell epitope respective section identified with BTV16-3G10 on the VP2 albumen of BTV16 type different areas isolated strain and the indirect ELISA analysis of BTV16-3G10.

Embodiment

Below in conjunction with specific embodiment, further describe the present invention, 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 modifications and replacement all fall within the scope of protection of the present invention.

Main experiment material and source

1. cell and virus

BHK-21 cell, SP2/0 myeloma cell and BTV1-24 type strain, the sick virus in Ibaraki, the sick virus in middle mountain are preserved by this research department.

2. main agents

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.

3. laboratory animal

6-8 BALB/c mouse in age in week is provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.

Embodiment 1BTV-16VP2 albumen eukaryotic expression and purifying

1, design of primers

Eukaryotic expression adopts Bac-to-Bac baculovirus expression system, according to L2 gene order (JN671907) the design pcr amplification primer of the BTV16 logining in GenBank, BTV16L2-22F:5 '-ACGAATTCATGGAGGAGCTAGTTATACC-3 ' (EcoR I); BTV16L2-2901R:5 '-CCGTCGACTTAAATATTTAGAAGCTTCGTG-3 ' (Sal I).

2, eukaryotic expression and the purifying of the construction of eukaryotic expression vector of BTV-16VP2 albumen and VP2 albumen

First utilize primer BTV16L2-22F and BTV16L2-2901R amplification BTV-16 VP2 gene (amplified fragments size is 2896bp), and carry out glue recovery.Then utilize restriction enzyme EcoR I and Sal I to reclaiming the VP2 gene of acquisition and the shuttle vectors pFastBac of baculovirus ^tMhT A carries out respectively double digestion.Utilize the quick ligase enzyme of T4DNA by enzyme is cut VP2 gene and pFastBac ^tMhT A connects, and at DNA, connects 25 ℃ of connection 5min in instrument.By above-mentioned connection product Transformed E .coli DH5 α competent cell, 37 ℃ of constant temperature are inverted incubated overnight.The random single bacterium colony of picking, is inoculated into respectively 5mL LB liquid nutrient medium (Amp ⁺100 μ g/mL) in, 37 ℃ of incubated overnight.Extract recombinant plasmid and carry out preliminary screening, empty carrier pFastBac ^tMhT A is as negative control.Doubtful recombinant plasmid is carried out respectively to double digestion and identify the evaluation with PCR.Whole above-mentioned evaluation correct bacterium liquid that contains positive recombinant plasmid is checked order, the recombinant plasmid called after pFast-VP2 that checks order correct.

By recombinant plasmid pFast-VP2 transformed competence colibacillus cell E.coli DH10Bac ^tM, first by the competent cell DH10Bac of-80 ℃ of preservations ^tM(100 μ L) is placed on ice and melts; Recombinant plasmid pFast-VP2 1 μ L is joined to the DH10Bac of thawing ^tMin mix, rapid 42 ℃ of heat shock 60s after ice bath 30min, then ice bath 5min.In super clean bench, to the SOC liquid nutrient medium 900 μ L that add the antibiotic-free of room temperature in mixture, 4h are cultivated in 37 ℃ of concussions.Above-mentioned bacterium liquid is done respectively to 10 times, 100 times dilutions, by on each the bacterium liquid of former times, 10 times and 100 times dilution 100 μ L difference coated plates (LA-Bac flat board contains the LB solid medium of 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, 100 μ g/mL X-gal and 40 μ g/mL IPTG), 37 ℃ of constant temperature are inverted and are cultivated 48h, then carry out blue hickie screening.Streak culture being inoculated on new LA-Bac flat board of the single white colony of random choose done further culture identification, picking white colony is seeded to respectively in the LB liquid nutrient medium of 5mL containing 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, picking blue colonies, as negative control, is extracted respectively restructuring rod granule and empty rod granule after 37 ℃ of cultivation 24h simultaneously.PCR identifies correct restructuring rod granule, and called after BAC-VP2 obtains recombinant baculovirus for transfection.

Utilize baculovirus transfection reagent Cellfectin Reagent, the rod granule BAC-VP2 transfection Sf 9 insect cell of recombinating, acquisition is with the recombinant baculovirus BACV-VP2 of VP2 gene, sets up respectively the transfection of wild-type rod granule and independent transfection reagent as negative control.Concrete operations are as follows:

(1) use Sf9 cell bed board.In six porocyte culture plates, 4.5 × 105/mL of every hole Sf9 cell (cell viability is more than 97%) at least cultivates 1h in 27 ℃ of constant incubators, makes cell completely adherent.

(2) (every hole consumption) prepared in transfection:

A:A liquid is used Sf9 basic culture solution (1 bottle, Grace powder (1 × 1L), yeast extract 3.3g, lactoalbumin hydrolysate 3.3g, sodium bicarbonate (NaHCO3) 0.35g) that 1 μ g restructuring rod granule BAC-VP2 is diluted in 100 μ L;

B:B liquid is used Sf9 basic culture solution that 6 μ L Cellfectin Reagent transfection reagents are diluted to 100 μ L;

After c:A liquid and B liquid mix, incubated at room 15-45min.

Control group 1: 1 μ g wild-type rod granule is joined in 100 μ L Sf9 basic culture solutions and mixed with B liquid;

Control group 2: only add B liquid.

(3) during transfection, first discard the original Sf9 complete culture solution in Tissue Culture Plate, then clean cell with Grace basic culture solution, 2mL/ time, clean altogether 2 times.

(4) 800 μ L Grace basic culture solutions are joined in the mixture of the A liquid of having hatched and B liquid, then mix and lipid-DNA mixture of the approximately 1mL mixing is joined in Sf9 insect cell culture plate.

(5) 27 ℃ of constant-temperature incubation 5h, discard transfection liquid, add new Sf9 complete culture solution (0.1%1000 × dual anti-, 10% foetal calf serum (FBS), 89%Sf9 insect cell basic culture solution), and 3.0mL/ hole is hatched 72h at 27 ℃ or occurs to pathology.Harvested cell culture supernatant is generation recombinant baculovirus.Then get generation recombinant baculovirus and continue inoculation Sf9 insect cell, find out optimal Sf9 insect cell stand density, best recombinant baculovirus generation and optimum receipts poison time.

By the Sf9 cell precipitation of recombinate shape virus infection, through ultrasonic treatment and carry out SDS-PAGE electrophoresis, result shows that restructuring BTV16 VP2 albumen obtains expression in Sf9 cell, and molecular mass is about 110ku, and expects that size conforms to.Use Ni2+NTA resin to carry out purifying to VP2 albumen, result shows the VP2 albumen (shown in Fig. 1) that acquisition is purer.

The preparation of embodiment 2 monoclonal antibodies

1. mouse immune

Expressing by Bac-to-Bac baculovirus expression system, 4 of the BTV16 VP2 recombinant protein of purifying immunity female BALB/c mouse in 6 week age, immunity 3 times altogether, two weeks, each immune interval, one exempts from recombinant VP 2 albumen and isopyknic Freund's complete adjuvant mixing and emulsifying, two exempt to exempt from recombinant VP 2 albumen and isopyknic Freund's incomplete adjuvant mixing and emulsifying with three, immunizing dose is 50 μ g/, and immunization route is peritoneal immunity.

Respectively two exempt to exempt from three after one week to the mouse blood sampling of docking, separation of serum (4 ℃, 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 (not adding adjuvant).

2. cytogamy

Merge and prepare feeder cell in first 1 day, according to ordinary method, get BALB/c mouse peritoneal macrophage 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

Utilization is set up the strain of indirect ELISA detection method screening positive hybridoma cell by the BTV16 VP2 recombinant protein of the expression of Bac-to-Bac baculovirus expression system, purifying, to the hybridoma enlarged culturing of reacting positive, with limiting dilution assay, carry out the subclone of positive hybridoma cell 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-BTV16-VP2 protein monoclonal antibody of stably excreting, its microbial preservation number is: CGMCC No.8153; And by the monoclonal antibody called after BTV16-3G10 of its secretion.

4. a large amount of preparations of monoclonal antibody

Give the healthy BALB/c mouse abdominal injection paraffin oil about 10 week age, only, within 1 week, pneumoretroperitoneum injects 1 × 10 to 0.5mL/ ⁶individual hybridoma extracts ascites after 7～10d when mouse web portion extreme expansion, every 2d, takes out once, by the centrifugal 10min of ascites 10000r/min extracting, removes upper strata grease and precipitation, and supernatant packing is stored in-20 ℃.

The evaluation of embodiment 3 monoclonal antibodies

1. the subgroup identification of monoclonal antibody

Monoclonal antibody embodiment 1 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 BTV16-3G10 of the present invention is IgG ₁, light chain is κ chain.

2.Western blot test

Inoculation is had to the insect cell lysate of wild-type baculovirus, by the BTV16VP2 recombinant protein of the expression of Bac-to-Bac baculovirus expression system, purifying, cell precipitation after centrifugal results BTV16 virus supernatant and BHK-21 cell precipitation carry out SDS-PAGE electrophoresis after processing, then through electric transfer printing by protein delivery to nitrocellulose filter, it is 18V voltage 30min that electricity turns condition; 4 ℃ of sealings of nitrocellulose filter after transfer printing are 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 BTV16-3G10 of the present invention can with by Bac-to-Bac baculovirus expression system, express, BTV16 VP2 recombinant protein and the natural B TV16-VP2 albumen generation specific reaction of purifying, and do not have the insect cell lysate of wild-type baculovirus and BHK-21 cell to react (Fig. 2) with control group inoculation, according to this experimental result, infer that the BTV16VP2 protein B cell epitope that BTV16-3G10 identifies should be a linear epitope simultaneously.

3.IFA test

The reactive result of application IFA test detection monoclonal antibody BTV16-3G10 and BTV16 as shown in Figure 3.

The evaluation of embodiment 4 B cell epitopes

1.MBP merge the expression of small peptide and the preliminary evaluation of B cell epitope polypeptide

For the B cell linear epitope of identifying that BTV16-3G10 identifies, we are according to the synthetic 87 pairs of complementary primers of the nucleotide sequence of BTV16 VP2 albumen (Gene Bank accession no.JN671907) design, 16 amino acid of every pair of primer coding, the aminoacid sequence of adjacent two pairs of primers coding has 5 amino acid overlaps.87 pairs of coded aminoacid sequences of primer cover BTV16 VP2 albumen total length, and 87 MBP that express are merged to small peptide called after MBP-VP2-1 respectively, MBP-VP2-2 etc.The primer of synthesized is connected respectively to pMAL by EcoRI and SalI restriction enzyme site after annealing ^tMon-C4x plasmid, successfully build 87 recombinant plasmids.After order-checking is identified correctly, recombinant plasmid is transformed into respectively to (DE3 in E.coli BL21 competent cell; Novagen, USA), after evaluation transforms successfully, the intestinal bacteria that carry recombinant plasmid are cultured to OD value for 0.5-0.7 in LB/Amp substratum, adding final concentration is the IPTG of 0.5mM, 37 ℃ are continued to cultivate 6 hours, so that albumen gives full expression to.After bacterium liquid 9000g is centrifugal, (10min, 4 ℃) carry out ultrasonication, by SDS-PAGE and Western blot test, determine that 87 MBP merge the successful expression of small peptide.

87 MBP that express are merged to small peptide as the coated elisa plate of envelope antigen, MBP label protein is contrasted as envelope antigen simultaneously, package amount is 100ng/ hole, by indirect ELISA Preliminary Experiment, identifies the epitope regions that BTV16-3G10 identifies.

Result show BTV16-3G10 can with MBP-VP2-4 ( ₃₄eWSGHDVTEIPNRRMF ₄₉) generation specific reaction (Fig. 4).And then the BTV16-VP2 protein B cell epitope Primary Location that BTV16-3G10 is identified in ₃₄eWSGHDVTEIPNRRMF ₄₉, shown in SEQ ID NO.1.

The accurate location of 3.B cell epitope

Utilize pepscan to synthesize following small peptide (table 1), and it is carried out to indirect ELISA detection, package amount is 100ng/ hole, and then accurately locates B cell epitope.

Table 1 is for the small peptide of BTV16-3G10 synthesized

Work as deletion ₃₄eWSGHDVTEIPNRRMF ₄₉on ³⁴e(Glu) time, the reacting value of small peptide VP2-4-1 and the monoclonal antibody BTV16-3G10 (OD that decreases ₄₉₂value is reduced to 1.254 by 3.499).Similarly, when ⁴⁹when F(Phe) deleted, also there is reduction (OD in the reacting value of VP2-4-2 and monoclonal antibody BTV16-3G10 ₄₉₂value is reduced to 0.974 by 3.499).But, when ³⁴e and ⁴⁹when F deletes simultaneously, the specific binding power of VP2-4-2 and BTV16-3G10 completely loses.

The above results explanation ₃₄eWSGHDVTEIPNRRMF ₄₉(shown in SEQ ID NO.1) is the accurate epitope regions that monoclonal antibody BTV16-3G10 identifies, ³⁴e and ⁴⁹the epi-position that F identifies BTV16-3G10 is two key amino acids, and their disappearance can completely lose the bonding force of BTV16-3G10 epi-position corresponding with it.In sum, the BTV16-VP2 protein B cell epitope that the present invention identifies BTV16-3G10 is accurately orientated as ₃₄eWSGHDVTEIPNRRMF ₄₉(shown in SEQ ID NO.1), synthesizes small peptide and the reactive indirect ELISA analysis of monoclonal antibody BTV16-3G10 as shown in Figure 4 by pepscan.

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

The aminoacid sequence Blast program of utilizing UniProt database to provide, identified BTV16-VP2 protein B cell epitope is carried out to conservative Analysis, whether this epitope sequences and BTV1-26 and other Orbivirus virus VP 2 albumen respective section are compared, analyzing this epi-position is BTV16 specificity epitope simultaneously.

Blast result shows that the BTV16-VP2 protein B cell epitope identifying is (table 2) of high conservative in the isolated strain of BTV16 serotype different areas, and with other viral VP2 albumen respective section of other serotypes of BTV and Orbivirus without homology (Fig. 6).

The conservative type analysis of the BTV16-VP2 protein B cell epitope that table 2 monoclonal antibody BTV16-3G10 identifies in the isolated strain of BTV16 serotype different areas

According to sequence alignment result, utilize pepscan to synthesize the not quite identical small peptide of B cell epitope respective section of identifying with BTV16-3G10 on the VP2 albumen of BTV16 type different areas isolated strain, by indirect ELISA detection method, determine whether specific reaction to occur with monoclonal antibody BTV16-3G10.

Result shows: monoclonal antibody BTV16-3G10 can with the B cell epitope respective section generation specific reaction (Fig. 7) identified with BTV16-3G10 on the VP2 albumen of BTV16 type different areas isolated strain, there is good type specificity.

Claims

1. anti-blue tongue virus serum 16 types (Bluetongue virus 16 is secreted in a strain, BTV16) hybridoma cell strain of VP2 protein monoclonal antibody, called after BTV16-3G10, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its microbial preservation number is: CGMCC No.8153, the aminoacid sequence of the linear B cell epitope of BTV16 VP2 albumen of its specific recognition is shown in SEQ ID NO.1.

2. the monoclonal antibody of being secreted by hybridoma cell strain claimed in claim 1.

The linear B cell epitope polypeptide of 3.BTV16 VP2 albumen, is characterized by: its aminoacid sequence is that shown in SEQ ID NO.1, described epitope polypeptide is identified by monoclonal antibody claimed in claim 2.

4. the application of hybridoma cell strain in preparation diagnosis or prevention blue tongue virus serum 16 type infection medicines described in claim 1.

5. the application of monoclonal antibody in preparation diagnosis or prevention blue tongue virus serum 16 type infection medicines described in claim 2.

6. the application of the linear B cell epitope polypeptide of BTV16 VP2 albumen in preparation diagnosis blue tongue virus serum 16 type infection medicines described in claim 3.