CN104046594B - A kind of Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof - Google Patents
A kind of Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof Download PDFInfo
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Abstract
The invention discloses a kind of Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain ' [2G8D10 and monoclonal antibody thereof.The deposit number of this hybridoma cell strain 2G8D10 is CCTCC NO:C201471.And this monoclonal antibody is produced by the hybridoma cell strain 2G8D10 secretion that deposit number is CCTCC NO:C201471.This monoclonal antibody can occur highly sensitive specific antigen-antibody to react with Orf virus protein ORFV086, it is possible to is effective to Orf virus protein ORFV086 is included the biological diagnosis of ELISA, western-blot, immunofluorescence and Immunohistochemical detection.
Description
Technical field
The present invention relates to cell engineering field, concrete Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof.
Background technology
Sheep infective pustule (Orf) is scorching also known as sheep infective pustular, is commonly called as sore mouth virus, is that the one of goat and the sheep caused by blue tongue virus (ORFV) is acute, contagious disease.Its characteristics of lesion is to suffer from sheep lip etc. skin and mucosa formation erythema, pimple, abscess, ulcer and excipuliform incrustation.Lamb is very easily felt, and mass-sends more.Primary disease all has generation all over the world, and main sheep raising district of China also more typically, is one of principal disease endangering flock of sheep.This viral resistance is strong, and flock of sheep are once infected, and not easy-clear, sustainable harm flock of sheep for many years, cause heavy economic losses to animal husbandry.
ORFV Tobamovirus Poxviridae, parapoxvirus belongs to.Virion is brick shape or ellipse, and its surface is rope sample criss-cross arrangement structure.This virus can grow on cattle, sheep, the nephrocyte of goat and the testicular cell of calf and lamb, and produces cytopathy.ORFV causes the Histopathological Features of focus to include the change of Keratinocytic cavity, swelling, intercellular substance hyaline degeneration, epidermal hyperplasia is notable, micro-swelling in epidermis, and subcutaneous tissue neutrophilic granulocyte, dendritic cell (DCs), T cell and B cell are assembled.
Sore mouth virus is widely distributed, and infectiousness is strong, once outburst can cause serious economic loss.Virus is at diseased region sustainable existence, and the easy repeated infection of infected animal, at present without effective vaccine, prevention and control are difficult.Therefore, the early stage of sheep of virus, accurately detection have great importance for control and the prevention of disease.On veterinary clinic, the diagnosis of sore mouth mainly diagnoses according to its classical symptom, but cannot distinguish with foot and mouth disease (foot-and-mouthdisease, FMD) and sheep pox (Capripox).Currently used laboratory diagnostic method is divided into, (1) immunological method (ELIS, Westernblotting)；(2) histopathology；(3) PCR and restriction fragment length polymorphism (RFLP) analysis etc..Wherein mostly important with amynologic diagnostic method, because it has sensitive, feature fast and accurately, and specific antibody especially monoclonal antibody is most important instrument in immunology diagnosis.In recent years, quick-fried in the flock of sheep in Jilin Province of China and Gansu Province send out sore mouth virus epidemic situation, owing to the shortage of specific antibody, Disease epizootic and further research work cannot effectively be carried out.
ORFV full-length genome is about 138kb, G+C rich content (63%-64%), containing 132 genes.By the genome analysis of sheep of virus not homophyletic (being) is shown, ORFV086 gene code is a virion core protein, the about 100.05KD of this albumen.This protein localization, in mature virion, is important structural protein, similar to other homology poxvirus protein structures of vaccinia virus precursor protein P4a.In the middle of the research of vaccinia virus, it is fully hydrolyzed the abundantest major structural protein p4a of content available 62,23 and tri-fragments of 9kDa are ripe infective requisite architecture basics of VV progeny virus.Therefore, essential core albumen p4a and vaccinia virus 39-kDa albumen (VVA4L gene code) form a stable complex, VVA4L gene and ORFV080 genetic homology are higher. and therefore prepare the monoclonal antibody for core texture proteantigen and be significant, can be used to study ORFV086 Proteolytic enzyme, how viral integrase, sheep infective pustule (Orf) mechanism of causing a disease and research control the diffusion of disease.
At home and abroad there is no the antibody of commercialization ORFV086 at present, and its monoclonal antibody hypotype is IgG2b type, ELISA, western-blot, immunofluorescence and Immunohistochemical detection can be used for, thus being that this albumen is verified as clinical target and solid foundation has been established in the research of function.
Summary of the invention
Technical problem solved by the invention is in that to overcome above-mentioned the deficiencies in the prior art, there is provided a kind of Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof, it is possible to for Orf virus protein ORFV086 being included the biological diagnosis of ELISA, western-blot, immunofluorescence and Immunohistochemical detection.
The invention provides Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10, this cell strain on April 23rd, 2014 China typical culture collection center (address: China, Wuhan, Wuhan University) preservation, deposit number is CCTCCNO:C201471.It addition, the invention provides Orf virus protein ORFV086 monoclonal antibody, this monoclonal antibody is produced by the hybridoma cell strain 2G8D10 secretion that deposit number is CCTCCNO:C201471.The Orf virus protein ORFV086 monoclonal antibody of the present invention is that the ORFV086 albumen expressed with RT-PCR obtains as antigen immune BALB/C mice, and the hybridoma cell strain 2G8D10 producing this antibody is obtained with cell-fusion techniques, the antibody of hybridoma secretion is IgG2b, and light chain is κ type.
The present invention obtains above-mentioned Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof by the following method:
1) prepared by restructuring ORFV086 antigen
In the present invention, the aminoacid sequence of Orf virus protein ORFV086 is AFX81702.1, as shown in SEQIDNO:1.In the present invention, the gene order of Orf virus protein ORFV086 is JQ729675.1, as shown in SEQIDNO:2.In the present invention, the full-length gene of ORFV086 is to be template by sheep of virus genome, pcr amplification obtain.The primer sequence of amplification ORFV086 gene is: forward primer: 5 '-TAGGATCCGATGACGGCCCCAAACGTGCA-3 ' (SEQIDNO:3), downstream primer: 5'-GCAAGCTTCTCACTGTCAAAAGAAAACGGC-3 ' (SEQIDNO:4).Upstream introduces BamHI restriction enzyme site, and downstream introduces HindIII restriction enzyme site.Prokaryotic expression carrier selection pET-33b (+).
With sheep of virus genome for template, total length ORFV086 gene is obtained through pcr amplification, build RT-PCR expression vector PET33b (+)/ORFV086 expresses in escherichia coli EscherichiacoliBL21, carbamide is utilized to open inclusion body, electrophoresis carries out rubber tapping purification after running glue, it is thus achieved that purity reaches the restructuring ORFV086 albumen of more than 90%.
2) immune mouse
Recombinant antigen immunity 6-8 week old female BAl BIc/c mice with purification.
First time immunity: after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L；
Second time immunity: after 2 weeks, after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L；
Third time immunity: again after 2 weeks, after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount lumbar injection BALB/c mouse of every 50 μ g/500 μ L；After immune 10 days for the third time, mouse tail vein takes blood, is coated with recombinant antigen, and ELISA detects serum titer, takes titer more than 1:105Mouse boosting cell and myeloma cell merge.Wherein, Bentonite adjuvant commodity in use product, market purchase obtain.
3) immune serum titration
Indirect elisa method is adopted to measure immune serum titer.Taking 50 μ g and recombinate ORFV086 protein dissolution in 10ml0.05MpH9.6 carbonate buffer solution, be coated micro-96 orifice plates of polystyrene, 100 μ l/ holes, 4 DEG C overnight.PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, with 10mMPBS containing 1%BSA confining liquid 100 μ l/ hole, close 2h for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, mice in third time immunity latter 10 days mouse tail veins blood sampling, Mus immune serum with containing 1%BSA10mMPBS with 10-2～10-8Dilution again, add 96 orifice plates, after 100 37 DEG C of μ l/ hole 1h, PBS (containing 0.05% (V/V) Tween-20) wash plate three times, add 1:10000 times and dilute horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.), 100 37 DEG C of μ l/ hole 30min, after ibid washing plate, TMB develops the color, 100 μ l/ holes, room temperature lucifuge 10min, add 50 μ l/ hole 2MH2SO2Terminating reaction, survey 450nm absorption value, before immunity, mice serum is as negative control, must compare >=2.1 titers judging immune serum for the positive with measured value with control value.
4) preparation of hybridoma
Take serum titer more than 1:105Mice, merge first 3 days, after taking recombinant antigen and isopyknic PBS mixing, carry out booster immunization with the amount lumbar injection BALB/c mice to be fused of every 50 μ g/500 μ L.Aseptic take mouse spleen, make splenocyte suspension to mix in the ratio of 1:1 with the murine myeloma cell strain SP2/0 of exponential phase, 1000 × g room temperature is centrifuged 5min, abandon supernatant, flick bottom centrifuge tube with finger, make precipitation loose, centrifuge tube is placed in 37 DEG C of water-baths, by the 50% Polyethylene Glycol (PEG at 37 DEG C of water bath heat preservations, MW4000, Sigma) with in the one after another drop of addition centrifuge tube of dropper, drip while shake centrifuge tube, drip off in 1min, 2min is stood after dripping off, the serum-free 1640 culture medium 1ml of 37 DEG C of preheatings is added every 1 minute, 2ml, 3ml, 4ml, 5ml and 10ml terminates the effect of Polyethylene Glycol, cell mixture 1000 × g room temperature is centrifuged 5min, abandon supernatant, add HAT culture fluid (hypoxanthine (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma)) re-suspended cell gently, cell is divided to 96 orifice plates, every hole 200 μ l.After cultivating three days, observation of cell merges situation, changes half HAT culture fluid, the continuous a few days, until there being Clone formation, merging latter seven days and changing HT culture fluid (hypoxanthine (H) and thymidine (T) (HT, Sigma)) cultivation.
5) hybridoma of anti-ORFV086 monoclonal antibody is secreted in screening
Indirect elisa method screening cells and supernatant, the higher positive colony hybridoma of titer is selected to carry out subcloning, and with limiting dilution assay continuous cloning 2-3 time, until to 100% cell positive rate, finally obtain the anti-ORFV086 cell strain of monoclonal antibody of stably excreting, be labeled as 2G8D10.Positive rate after cloning is reached 100% cell amplification cultivate after liquid nitrogen cryopreservation.
6) preparation and purification of ascites
By 2G8D10 hybridoma cell strain with 1 × 106/ amount only injects the BALB/c female mice abdominal cavity of the 8-10 week old of liquid paraffin pretreatment, breeding observing after 10-14 days mouse web portion extract ascites when expanding.Adopting affinity chromatography ProteinGSepharoseFastFlow monoclonal antibody purification, measure the purity of monoclonal antibody with SDS-PAGE, purity reaches more than 90%.
Compared with prior art, characteristic of the present invention is as follows:
The present invention is coated elisa plate with ORFV086 albumen of recombinating, and is detected the positive of antibody purification by ELISA method, and with this antibody purification, the Orf virus protein of purification is carried out Western-blot and prove that this antibody may identify which ORFV086 albumen.
This antibody purification is used for carrying out the immunohistochemical staining of the immunofluorescence dyeing of sheep of virus and sore mouth virus tissue by the present invention proves that it can recognise that natural ORFV086 albumen.The ORFV086 function that monoclonal antibody is further research of this strain of hybridoma secretion and the diagnostic reagent of exploitation ORFV086 are laid a good foundation, and provide strong instrument for it as the checking of sore mouth virus clinical diagnosis and therapeutic targets.
Its immunogen of the monoclonal antibody of the present invention is that RT-PCR expresses ORFV086 full-length proteins, and its aminoacid sequence is AFX81702.1,905 aminoacid of total length.The monoclonal antibody of the present invention can also be applied to cellular immunofluorescence except can apply to ELISA and Western and detecting degeneration ORFV086 albumen and clinical sore mouth virus tissue specimen immunohistochemical study detects unmodified natural ORFV086 albumen, reduces application cost simultaneously.There is no both at home and abroad for sheep of virus specific antibody at present, and the antibody of the present invention can not only be combined with Denatured protein, moreover it is possible to be combined with the native protein with higher structure, it is thus possible to be applied to immunofluorescence and immunohistochemical experiment.The present invention is directed to the monoclonal antibody that ORFV086 full-length proteins prepares, to detecting this albumen, there is higher specificity and sensitivity, the application of this antibody will for ORFV086 functional study and its provide as the clinical samples checking work of sore mouth virus mark and to support.
Accompanying drawing explanation
Fig. 1 is ORFV086 Protein reconstitution expression identification result of the present invention.
Fig. 2 is the monoclonal antibody 2G8D10 of the present invention result to sheep of virus immunoblotting, and it is about 100kDa in conjunction with the molecular weight of band, and each fragment of hydrolysis can be detected.
Fig. 3 is the monoclonal antibody 2G8D10 of the present invention result to sheep of virus immunofluorescence dyeing；
Fig. 4 is monoclonal antibody 2G8D10 of the present invention to the result of sore mouth virus immunohistochemical staining and negative control result (microscope magnification 100 times).
Detailed description of the invention
For making the present invention easier to understand, below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.
The preparation of embodiment 1 monoclonal antibody of the present invention and qualification
1, the preparation of monoclonal antibody 2G8D10
1) prepared by restructuring ORFV086 antigen
With sheep of virus genome for template, total length ORFV086 gene is obtained through pcr amplification, build RT-PCR expression vector PET33b (+)/ORFV086 expresses in escherichia coli EscherichiacoliBL21, carbamide is utilized to open inclusion body, electrophoresis carries out rubber tapping purification after running glue, it is thus achieved that purity reaches the restructuring ORFV086 albumen of more than 90%.
2) immune mouse
Recombinant antigen immunity 6-8 week old female BAl BIc/c mice with purification.
First time immunity: after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L；
Second time immunity: after 2 weeks, after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L；
Third time immunity: again after 2 weeks, after taking recombinant antigen and the mixing of isopyknic Bentonite adjuvant, with the amount lumbar injection BALB/c mouse of every 50 μ g/500 μ L；After immune 10 days for the third time, mouse tail vein takes blood, is coated with recombinant antigen, and ELISA detects serum titer, takes titer more than 1:105Mouse boosting cell and myeloma cell merge；
Bentonite adjuvant commodity in use product.
3) immune serum titration
Indirect elisa method is adopted to measure immune serum titer.Taking 50 μ g and recombinate ORFV086 protein dissolution in 10ml0.05MpH9.6 carbonate buffer solution, be coated micro-96 orifice plates of polystyrene, 100 μ l/ holes, 4 DEG C overnight.PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, with 10mMPBS containing 1%BSA confining liquid 100 μ l/ hole, close 2h for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, mice in third time immunity latter 10 days mouse tail veins blood sampling, Mus immune serum with containing 1%BSA10mMPBS with 10-2～10-8Dilution again, add 96 orifice plates, after 100 37 DEG C of μ l/ hole 1h, PBS (containing 0.05% (V/V) Tween-20) wash plate three times, add 1:10000 times and dilute horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.), 100 37 DEG C of μ l/ hole 30min, after ibid washing plate, TMB develops the color, 100 μ l/ holes, room temperature lucifuge 10min, add 50 μ l/ hole 2MH2SO2Terminating reaction, survey 450nm absorption value, before immunity, mice serum is as negative control, must compare >=2.1 titers judging immune serum for the positive with measured value with control value.
4) preparation of hybridoma
Take serum titer more than 1:105Mice, merge first 3 days, after taking recombinant antigen and isopyknic PBS mixing, carry out booster immunization with the amount lumbar injection BALB/c mice to be fused of every 50 μ g/500 μ L.Aseptic take mouse spleen, make splenocyte suspension to mix in the ratio of 1:1 with the murine myeloma cell strain SP2/0 of exponential phase, 1000 × g room temperature is centrifuged 5min, abandon supernatant, flick bottom centrifuge tube with finger, make precipitation loose, centrifuge tube is placed in 37 DEG C of water-baths, by the 50% Polyethylene Glycol (PEG at 37 DEG C of water bath heat preservations, MW4000, Sigma) with in the one after another drop of addition centrifuge tube of dropper, drip while shake centrifuge tube, drip off in 1min, 2min is stood after dripping off, the serum-free 1640 culture medium 1ml of 37 DEG C of preheatings is added every 1 minute, 2ml, 3ml, 4ml, 5ml and 10ml terminates the effect of Polyethylene Glycol, cell mixture 1000 × g room temperature is centrifuged 5min, abandon supernatant, add HAT culture fluid (hypoxanthine (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma)) re-suspended cell gently, cell is divided to 96 orifice plates, every hole 200 μ l.After cultivating three days, observation of cell merges situation, changes half HAT culture fluid, the continuous a few days, until there being Clone formation, merging latter seven days and changing HT culture fluid (hypoxanthine (H) and thymidine (T) (HT, Sigma)) cultivation.
5) hybridoma of anti-ORFV086 monoclonal antibody is secreted in screening
Indirect elisa method screening cells and supernatant, the higher positive colony hybridoma of titer is selected to carry out subcloning, and with limiting dilution assay continuous cloning 2-3 time, until to 100% cell positive rate, finally obtain the anti-ORFV086 cell strain of monoclonal antibody of stably excreting, be labeled as 2G8D10.Positive rate after cloning is reached 100% cell amplification cultivate after liquid nitrogen cryopreservation.
6) preparation and purification of ascites
By 2G8D10 hybridoma cell strain with 1 × 106/ amount only injects the BALB/c female mice abdominal cavity of the 8-10 week old of liquid paraffin pretreatment, breeding observing after 10-14 days mouse web portion extract ascites when expanding.Adopting affinity chromatography ProteinGSepharoseFastFlow monoclonal antibody purification, measure the purity of monoclonal antibody with SDS-PAGE, purity reaches more than 90%.
Embodiment 2, monoclonal antibody CHARACTERISTICS IDENTIFICATION
1) mensuration of antibody concentration: obtain ORFV086 monoclonal antibody 2G8D10 after the hybridoma CCTCCNO:C201471 ascites prepared is purified, the SmartSpecplus nucleic acid-protein analyzer that BIO-RAD company produces is used to measure respectively, its concentration respectively 0.51mg/ml.
2) antibody subtype is identified: adopting the Mus monoclonal antibody hypotype identification kit of Roche company to identify the hypotype of hybridoma cell strain, the hypotype of 2G8D10 secretory antibody is IgG2b type, and light chain is κ chain, as shown in Figure 1.
3) titer of antibody purification is identified: 50 μ g restructuring ORFV086 antigens are dissolved in the 0.05M carbonate of 10mlpH9.6 and are coated in buffer, add the 96 every hole 100 μ L of orifice plate, and 4 DEG C overnight.PBS (containing 0.05% (V/V) Tween-20) washes plate three times, with 10mMPBS containing 1%BSA confining liquid 150 μ l/ hole, close 2h for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, every hole adds 100 μ l antibody purifications, hatch 1h for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) washes plate three times, add horseradish peroxidase-labeled sheep anti-mouse igg polyclonal antibody be two resist, hatch 30min for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) washes plate three times, every hole adds 100 μ lTMB colour developings, 37 DEG C hatch 15min after, add 2MH2SO4Solution terminates reaction, and microplate reader detects at absorbance 450nm place.
4) Westernblot of antibody identifies: the Orf virus protein of purification loading after the cracking of 2 × SDS lysis buffer, with Bio-Rad electrotransfer device by protein delivery to pvdf membrane after 12%SDS-PAGE, 5% defatted milk powder closes 1h, the Tris-HCl buffer (containing 0.1% (V/V) Tween-20) of pH7.4 washes film 3 times, each 5min, 1:1000 adds the purified hybridoma CCTCCNO:C201471 anti-ORFV086 monoclonal antibody 2G8D10 prepared, 4 DEG C of overnight incubation, the Tris-HCl buffer (containing 0.1% (V/V) Tween-20) of pH7.4 washes film 3 times, each 5min, add 1:10000 dilution sheep anti-mouse igg polyclonal antibody (Sigma) be two resist, incubated at room 2h, TBST washes film 3 times, unnecessary solution is sucked with filter paper, it is laid on clean tin foil, add 1.4mlPierce-ThermoScientificECL series Western chemical luminous substrate reactant liquor (A:B=1:1), make film complete wetting in reactant liquor, take out rapidly, surplus liquid is sucked with filter paper, it is laid on another tin foil, with tin foil, film is wrapped, put into X-ray magazine, dark place develops.Anti-ORFV086 monoclonal antibody 2G8D10 prepared by hybridoma CCTCCNO:C201471 all appears for the specific band of each fragment of ORFV086, the carrier 4A-086 expressing ORFV086 gene does transient expression, western-blotting checking is done with monoclonal antibody 2G8D10, result shows ORFV086 total length and the GGS-905 of the recognizable eukaryotic expression of this monoclonal antibody, and result is as shown in Figure 2.
5) identified by immunofluorescence of antibody: collect primary sheep fetus turbinates (OFTu) cell and be laid on sheet glass and grow overnight, sheep of virus (ORFV) infects (MOI=5) 24h, PBS washed cell, use the fixing cell of paraformaldehyde of pre-cooling, it is separately added into the hybridoma CCTCCNO:C201471 anti-ORFV086 monoclonal antibody 2G8D10 (1:1000 dilution) prepared, incubated at room 1h, with PBS for negative control.After PBS develops a film, 1:1000 adds fluorescent labeling sheep anti mouse two anti-(Sigma), incubated at room 1h, and DAPI dyes 10min, after PBS develops a film, fluorescence microscopy Microscopic observation, all observes red fluorescence through the anti-ORFV086 monoclonal antibody 2G8D10 cell dyeed, as shown in Figure 3.Result proves that the hybridoma CCTCCNO:C201471 anti-ORFV086 monoclonal antibody 2G8D10 prepared can recognize that natural ORFV086 albumen.
6) SABC of antibody is identified: collects and suffers from sheep pathological tissues, the anti-ORFV086 monoclonal antibody 2G8D10 (1:1000 dilution) prepared of the hybridoma CCTCCNO:C201471 after purification is utilized to carry out immunohistochemical staining, Fujian is used to step the immunohistochemical kit of new company, colouring method is with reference to stepping novel agent box description, DAB develops the color, negative control group replaces primary antibodie with PBS, found that all find granule in Cytoplasm on the tissue specimen of anti-ORFV086 monoclonal antibody 2G8D10 dyeing, as shown in Figure 4, by ORFV-Jilin virus, ((MOI2.5) infects the OFTu cell 0 cultivated on the cover slip respectively, 3, 5, 8, 10, 12 and 24 hours (post-infection (p.i.), and fix with the paraformaldehyde of 4% in different time points, use 0.25%TritonX-100 incubated at room 10 minutes subsequently, after fixing with the PBS containing 1%BSA again, by monoclonal antibody 2G8D10 incubated at room one hour, wash off after the unnecessary antibody being not bound with by IgGAlexaFluor488 (Green) incubated at room one hour of anti-mouse, finally with 4 ', 6 '-diamidino-2-phenylindole (DAPI) hatch 10 minutes.From part B it will be seen that viral infection after 8 hours gene start express, after 24 hours albumen start express.The immunohistochemical staining result of pathological tissues proves that the hybridoma CCTCCNO:C201471 anti-ORFV086 monoclonal antibody 2G8D10 prepared can recognize that natural ORFV086 albumen.
Finally be should be noted that; above example is only in order to illustrate technical scheme but not limiting the scope of the invention; although the present invention being explained in detail with reference to preferred embodiment; it will be understood by those within the art that; technical scheme can be modified or equivalent replacement, without deviating from the spirit and scope of technical solution of the present invention.
Claims (2)
1. an Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10, its deposit number is CCTCCNO:C201471.
2. an Orf virus protein ORFV086 monoclonal antibody, it is characterised in that: this monoclonal antibody is produced by the hybridoma cell strain 2G8D10 secretion that deposit number is CCTCCNO:C201471.
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| CN102533666A (en) * | 2012-01-19 | 2012-07-04 | 南方医科大学 | Orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody |
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| CN102533666A (en) * | 2012-01-19 | 2012-07-04 | 南方医科大学 | Orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody |
| CN102559605A (en) * | 2012-01-19 | 2012-07-11 | 南方医科大学 | Orf virus protein ORFV059 monoclonal antibody hybridoma A3 and monoclonal antibody |
| CN103602636A (en) * | 2013-10-22 | 2014-02-26 | 中国农业科学院兰州兽医研究所 | Anti-Orf virus B2L protein monoclonal antibody and application thereof |
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