CN102533666A - Orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody - Google Patents
Orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody Download PDFInfo
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Abstract
The invention discloses an orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and a monoclonal antibody. The ORFV059 monoclonal antibody hybridoma G3 was collected in China General Microbiological Culture Collection Center (CGMCC) on November 4, 2011, wherein the collection number is CGMCC No.5425. The recombinant ORFV059 protein coats an enzyme-linked immuno sorbent assay (ELISA) plate; activity of a purified antibody is detected by an ELISA method; and the purified ORFV059 protein is subjected to Western-blot by the purified antibody, which proves that the antibody can identify the ORFV059 protein. The purified antibody is used for performing immunofluorescent staining on the ORFV059 and immunohistochemical staining on the orf tissue, which proves that the purified antibody can identify the natural ORFV059 protein. The monoclonal antibody secreted by the hybridoma cell lays basis for further development of ORFV059 function and a diagnosis reagent for developing the ORFV059 and provides a powerful tool for clinical diagnosis on the orf and confirmation of a therapeutic target.
Description
Technical field
The present invention relates to the cell engineering field, be specifically related to sheep of virus structural protein ORFV059 monoclonal antibody hybridoma cell strain G3 and monoclonal antibody.
Background technology
Sheep infective pustule (Orf) claims that again the sheep infective pustular is scorching, is commonly called as the sheep aphtha, is the goat that caused by blue tongue virus (ORFV) and a kind of acute, the contagious disease of sheep.Its characteristics of lesion is to suffer from sheep lip etc. to locate skin and form erythema, papule, running sore, ulcer and excipuliform with mucous membrane and form a scab.Lamb is very easily felt, and multigroup is sent out.This disease all has generation all over the world, China main sheep raising district is also more common, is one of principal disease of harm flock of sheep.This viral resistance is strong, the easy-clear not in case flock of sheep are infected, and sustainable harm flock of sheep are caused heavy economic losses to livestock industry for many years.
ORFV Tobamovirus Poxviridae, parapoxvirus belongs to.Virus particle is brick shape or ellipse, and its surface is rope appearance criss-cross arrangement structure.This virus can be grown on the testicular cell of the kidney cell of ox, sheep, goat and calf and lamb, and produces cytopathy.The histopathology characteristic that ORFV causes focus comprises that Keratinocytic cavity changes, swelling, the intercellular substance hyaline degeneration, and epidermal hyperplasia is remarkable, little swelling in the epidermis, subcutis neutrophil leucocyte, BMDC (DCs), T cell and B cell aggregation.
The sheep aphtha distributes extensively, and infectivity is strong, in case outburst can cause serious economy loss.Virus continues to exist at diseased region, and infected animal is prone to superinfection, does not have effective vaccine at present, the prevention and control difficulty.Therefore, the control and the prevention that in early days, accurately detect for disease of sheep of virus have great importance.The diagnosis of sore mouth is mainly diagnosed according to its classical symptom on the veterinary clinic, but can't with foot and mouth disease (foot-and-mouth disease, FMD) and sheep pox (Capripox) distinguish.The laboratory diagnostic method that is adopted at present is divided into, (1) immunological method, and like ELISA, Western blotting, immunohistochemistry (IHC) etc.; (2) histopathology; (3) molecular biology method is like PCR and restriction fragment length polymorphism (RFLP) analysis etc.Wherein important with amynologic diagnostic method, because of it has sensitivity, characteristics fast and accurately, and specific antibody especially monoclonal antibody be most important instrument in the immunology diagnosis.In recent years, in the flock of sheep in China Jilin Province and Gansu Province, break out sheep aphtha epidemic situation several times, because the shortage of specific antibody, disease control can't effectively be carried out with further research work.
The about 138kb of ORFV genome total length, G+C content enriches (63%-64%), contains 132 genes.Through to sheep of virus not the genome analysis of homophyletic (being) show, the ORFV059 genes encoding be an immundominance albumen.This albumen is positioned the mature virion after birth; It is C end anchorin; Similar with vaccinia virus H3L immundominance protein structure, it has vital role in virus maturation, absorption, viral morbific molecular mechanism and processes such as medical diagnosis on disease and subunit vaccine research and development.Therefore; ORFV059 has the possibility as potential clinical diagnosis target; The monoclonal antibody of preparation ORFV059 has crucial meaning, can lay the foundation for the research of this protein function, for its feasibility as disease marker the experimental data support is provided simultaneously.
Because ORFV059 is positioned the mature virion after birth, expression amount is higher in tissue sample usually, has the value of clinical detection and checking.Thus, preparation can be used for the ORFV059 monoclonal antibody of clinical samples immunohistochemical methods detection and carry out the checking of tissue sample in enormous quantities, can be its checking as sheep aphtha clinical diagnosis target strong instrument is provided.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of and can be used for the anti-sore mouth toxalbumin ORFV059 monoclonal antibody that ELISA, western-blot, immunofluorescence and immunohistochemistry detect.
To achieve these goals, the present invention adopts following technical scheme:
The antibody of detection sore mouth toxalbumin ORFV059 of the present invention is to obtain as the antigen immune BALB/C mice with the recombinant expressed ORFV059 albumen of protokaryon; And to produce the hybridoma cell strain G3 of this antibody with cell-fusion techniques; Hybridoma excretory antibody is that IgG2b is positive, and light chain is the κ type.Said anti-sore mouth toxalbumin ORFV059 monoclonal antibody hybridoma G3 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 4th, 2011, and preserving number is CGMCC No.5425.
The aminoacid sequence of sore mouth toxalbumin ORFV059 is AAR98154 among the present invention, shown in SEQ ID NO:1.The gene order of sore mouth toxalbumin ORFV059 is AY386263 among the present invention, shown in SEQ ID NO:2.
The full-length gene of ORFV059 is to be template by the sheep of virus genome among the present invention, is obtained by pcr amplification.The primer sequence of amplification ORFV059 gene is: upstream primer: 5 '-CATTAAC CATGGATCCACCCGAAATCACGGC-3 ' (SEQ ID NO:3), downstream primer: 5'-AATCATCTCGAGCACGATGGCCGTGACCAGCAGC-3 ' (SEQ ID NO:4).The NcoI restriction enzyme site is introduced at the upper reaches, and the XhoI restriction enzyme site is introduced in downstream.Prokaryotic expression carrier is selected pET-28a (+).
Described preserving number is that the preparation method of the hybridoma cell strain G3 of CGMCC No.5425 is: get serum titer greater than 1:10
5BALB/c mouse splenocyte and SP2/0 myeloma cell merge with 50% PEG-4000 by ordinary method; HAT RPMI-1640 screening of medium fused cell with containing 20% calf serum encapsulates elisa plate with recombinant expressed ORFV059 albumen, carries out the ELISA screening; Through limiting dilution repeatedly, obtain the hybridoma cell strain of the anti-ORFV059 monoclonal antibody of stably excreting at last, be labeled as G3.Use this strain of hybridoma with 1 * 10
6/ amount is only injected the pretreated 8-10 of the paraffin BALB/c female mice abdominal cavity in age in week, extracts ascites when mouse web portion expands after breeding observing 10-14 days.Adopt affinity chromatography Protein G Sepharose Fast Flow monoclonal antibody purification, with the purity of SDS-PAGE evaluation monoclonal antibody, purity reaches more than 90%.
Compared with prior art, the present invention has following beneficial effect:
The present invention encapsulates elisa plate with reorganization ORFV059 albumen, detects the activity of antibody purification through the ELISA method, and with this antibody purification the sore mouth toxalbumin of purifying is carried out Western-blot and prove that this antibody can discern ORFV059 albumen.
The present invention this antibody purification is used to carry out sheep of virus immunofluorescence dyeing and sheep aphtha tissue the immunohistochemical staining proof its can discern natural ORFV059 albumen.This strain of hybridoma excretory monoclonal antibody is laid a good foundation with the diagnostic reagent of exploitation ORFV059 for the ORFV059 function of further research, for its checking as clinical diagnosis of sheep aphtha and treatment target provides strong instrument.
Its immunogen of monoclonal antibody of the present invention is the recombinant expressed ORFV059 full-length proteins of protokaryon, and its aminoacid sequence is AAR98154,338 amino acid of total length.Monoclonal antibody of the present invention except being applied to ELISA and Western detection sex change ORFV059 albumen, can also be applied to cellular immunofluorescence and clinical sheep aphtha tissue sample immunohistochemical study detects unmodified natural ORFV059 albumen, has reduced application cost simultaneously.The ORFV059 albumen of the multiple sheep of virus strain isolated of monoclonal anti physical efficiency specific recognition of the present invention is with other virus of Poxviridae, like no cross reactions such as capripox virus, bird pox virus, vaccinia viruss.Still needleless is to the sheep of virus specific antibody both at home and abroad at present, and antibody of the present invention can not only combine with metaprotein, can also combine with the native protein with higher structure, thereby can be applied to immunofluorescence and immunohistochemical experiment.The present invention is directed to the monoclonal antibody that the ORFV059 full-length proteins prepares and have higher specificity and sensitivity to detecting this albumen, the application of this antibody will provide support for ORFV059 functional study and its clinical samples checking work as sheep aphtha mark.The antibody that commercialization ORFV059 is not arranged at present both at home and abroad as yet; And its monoclonal antibody hypotype is the IgG2b type; Can be used for ELISA, western-blot, immunofluorescence and immunohistochemistry and detect, thereby be that this albumen has been established solid basis as the research of clinical target checking and function thereof.
Description of drawings
Fig. 1 is the result of monoclonal antibody G3 of the present invention to the sheep of virus immunoblotting, and it combines the molecular weight of band to be about 39kDa.1.10 μ g OFTu lysis albumen; 2.2 μ g purified virus albumen; 3. 2 μ g purification of Recombinant ORFV059 albumen.
Fig. 2 is the result of monoclonal antibody G3 of the present invention to the sheep of virus immunofluorescence dyeing; 5 MOI sheep of virus infect the OFTu cell; Respectively at 10, the 12 and 24 hours collecting cells in inoculation back, 4% Paraformaldehyde 96 is fixed, and successively resists with the sheep anti mouse two of monoclonal antibody G3 and green fluorescence mark and hatches; DAPI dyeing, observations under the fluorescent microscope.
Fig. 3 is the result of monoclonal antibody G3 of the present invention to the histochemical stain of sore mouth change histogenic immunity, the anti-ORFV059 monoclonal antibody of A. G3, B. normal mouse serum (negative control), (400 times of microscope magnifications).
Embodiment
1, the preparation of monoclonal antibody G3
1) reorganization ORFV059 antigen prepd
With the sheep of virus genome is template, obtains total length ORFV059 gene through pcr amplification, makes up protokaryon recombinant expression vector pET28a (+)/ORFV059, intestinal bacteria
Escherichia coliExpress among the BL21, utilize Ni Sepharose affinitive layer purification post to carry out purifying, obtain purity and reach the reorganization ORFV059 albumen more than 90%.
2) immune mouse
With female BALB/c mouse in purified recombinant antigen immune 6-8 age in week.
Immunity for the first time: after getting recombinant antigen and isopyknic Bentonite adjuvant mixing, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L;
Immunity for the second time: after 2 weeks, get recombinant antigen and isopyknic Bentonite adjuvant mixing after, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L;
Immunity for the third time: again after 2 weeks, get recombinant antigen and isopyknic Bentonite adjuvant mixing after, with the amount abdominal injection BALB/c mouse of every 50 μ g/500 μ L; Mouse tail vein is got blood after immune 10 days for the third time, encapsulates with recombinant antigen, and ELISA detects serum titer, gets and tires greater than 1:10
5Mouse boosting cell and myeloma cell merge;
Bentonite adjuvant commodity in use product.
3) immune serum titration
Adopting indirect elisa method to measure immune serum tires.Get 50 μ g reorganization ORFV059 protein dissolution in 10ml 0.05M pH9.6 carbonate buffer solution, encapsulate little 96 orifice plates of PS, 100 μ l/ holes, 4 ℃ are spent the night.Use PBS (containing 0.05% (V/V) Tween-20) to wash plate three times; Contain 1%BSA confining liquid 100 μ l/ holes with 10mM PBS; 37 ℃ of sealing 2h; Use PBS (containing 0.05% (V/V) Tween-20) to wash plate three times, mouse tail vein blood sampling in immune for the third time back 10 days, the mouse immune serum carries out 10 with containing 1%BSA 10mM PBS
-2～10
-8Doubly dilution adds 96 orifice plates, the 37 ℃ of 1h in 100 μ l/ holes; After PBS (containing 0.05% (V/V) Tween-20) washes plate three times, add 1:10000 doubly dilute the horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.); The 37 ℃ of 30min in 100 μ l/ holes, the same wash plate after, TMB colour developing; 100 μ l/ holes, room temperature lucifuge 10min adds 50 μ l/ hole 2M H
2SO
2Termination reaction is surveyed the 450nm absorption value,, must compare>=2.1 with measured value and control value and positively judge tiring of immune serum as negative control with mice serum before the immunity.
4) preparation of hybridoma
Get serum titer greater than 1:10
5Mouse, merged preceding 3 days, get recombinant antigen and isopyknic PBS mixing after, carry out booster immunization with the amount abdominal injection BALB/c mouse to be merged of every 50 μ g/500 μ L.The aseptic mouse spleen of getting is processed the mixed of the murine myeloma cell strain SP2/0 of splenocyte suspension and logarithmic phase by 1:1, the centrifugal 5min of 1000 * g room temperature; Abandon supernatant, flick the centrifuge tube bottom, make deposition loose with finger; Centrifuge tube places 37 ℃ of water-baths, will be at 50% polyoxyethylene glycol (PEG, the MW4000 of 37 ℃ of water bath heat preservations; Sigma) with in the one after another drop of adding centrifuge tube of dropper, shake centrifuge tube, drip off in the 1min while dripping; Leave standstill 2min after dripping off, the serum-free 1640 substratum 1ml, 2ml, 3ml, 4ml, 5ml and the 10ml that whenever added 37 ℃ of preheatings at a distance from 1 minute stop the effect of polyoxyethylene glycol, the centrifugal 5min of cell mixture 1000 * g room temperature; Abandon supernatant, (xanthoglobulin (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma)) be re-suspended cell gently to add the HAT nutrient solution; Cell is divided to 96 orifice plates every hole 200 μ l.Cultivate after three days, observation of cell merges situation, changes half HAT nutrient solution, and continuously a few days, until there being the clone to form, (xanthoglobulin (H) and thymidine (T) (HT, Sigma)) are cultivated to merge back seven days replacing HT nutrient solutions.
5) hybridoma of the anti-ORFV059 monoclonal antibody of screening secretion
Indirect elisa method screening cells and supernatant; The selection high positive clone hybridization oncocyte of tiring carries out subcloning, and with the continuous cloning of limiting dilution assay 2-3 time, until to 100% cell positive rate; Obtain the anti-ORFV059 cell strain of monoclonal antibody of stably excreting at last, be labeled as G3.Positive rate after the cloning is reached 100% cell amplification cultivate the back liquid nitrogen cryopreservation.
6) preparation of ascites and purifying
With the G3 hybridoma cell strain with 1 * 10
6/ amount is only injected the pretreated 8-10 of the whiteruss BALB/c female mice abdominal cavity in age in week, extracts ascites when mouse web portion expands after breeding observing 10-14 days.Adopt affinity chromatography Protein G Sepharose Fast Flow monoclonal antibody purification, with the purity of SDS-PAGE mensuration monoclonal antibody, purity reaches more than 90%.
2, the CHARACTERISTICS IDENTIFICATION of monoclonal antibody of the present invention
1) mensuration of AC: after the ascites of hybridoma CGMCC No.5425 preparation is purified, obtain ORFV059 monoclonal antibody G3; The Smart Spec plus nucleic acid-protein determinator that uses BIO-RAD company to produce is measured, and its concentration is 0.51mg/ml.
2) antibody subtype is identified: adopt the hypotype of the mouse monoclonal antibody hypotype identification kit evaluation hybridoma cell strain of Roche company, the hypotype of G3 secretory antibody is the IgG2b type, and light chain is the κ chain.
3) evaluation of tiring of antibody purification: the 0.05M carbonate that 50 μ g reorganization ORFV059 antigen is dissolved in 10ml pH9.6 encapsulates in the damping fluid, adds 96 orifice plates, every hole 100 μ L, and 4 ℃ are spent the night.PBS (containing 0.05% (V/V) Tween-20) washes plate three times, contains 1%BSA confining liquid 150 μ l/ holes with 10mM PBS, 37 ℃ of sealing 2h; Use PBS (containing 0.05% (V/V) Tween-20) to wash plate three times, every hole adds 100 μ l antibody purifications, hatches 1h for 37 ℃; PBS (containing 0.05% (V/V) Tween-20) washes plate three times, and the sheep anti-mouse igg polyclonal antibody that adds horseradish peroxidase-labeled is two anti-, hatches 30min for 37 ℃; PBS (containing 0.05% (V/V) Tween-20) washes plate three times, and every hole adds 100 μ l, the TMB colour developing; 37 ℃ hatch 15min after, add 2M H
2SO
4The solution termination reaction, ELIASA detects at absorbance 450 nm places.
4) the Western blot of antibody identifies: the sore mouth toxalbumin of OFTu lysis albumen, purifying and reorganization ORFV059 albumen are gone up appearance after with the cracking of 2 * SDS lysis buffer, and through with Bio-Rad electrotransfer device albumen being transferred on the pvdf membrane behind the 12%SDS-PAGE, 5% skim-milk seals 1h; The Tris-HCl damping fluid of pH7.4 (containing 0.1% (V/V) Tween-20) is washed film 3 times, and each 5min, 1:1000 add the anti-ORFV059 monoclonal antibody G3 of purified hybridoma CGMCC No.5425 preparation; 4 ℃ of incubated overnight, the Tris-HCl damping fluid of pH7.4 (containing 0. 1% (V/V) Tween-20) is washed film 3 times, each 5min; The sheep anti-mouse igg polyclonal antibody (Sigma) that adds the 1:10000 dilution is two anti-, and incubated at room 2h, TBST wash film 3 times; Go excessive solution with the filter paper suction; Be tiled on the clean tin foil, add 1.4ml Pierce-Thermo Scientific ECL series Western chemical luminous substrate reaction solution (A:B=1:1), make film be infiltrated in the reaction solution fully; Take out rapidly; Remove unnecessary liquid with the filter paper suction, be laid on another tin foil, wrap film with tin foil; Put into X ray photography magazine, in dark place, develop.Single specific band appears in the anti-ORFV059 monoclonal antibody G3 of hybridoma CGMCC No.5425 preparation, and the result is as shown in Figure 1.
5) identified by immunofluorescence of antibody: collect former generation sheep fetus turbinal bone (OFTu) cell and be laid on grow overnight on the sheet glass; Sheep of virus (ORFV) infects (MOI=5), the collecting cell respectively at 10,12, behind the 24h, PBS washed cell; Use the Paraformaldehyde 96 fixed cell of precooling; Add the anti-ORFV059 monoclonal antibody G3 (1:1000 dilution) of hybridoma CGMCC No.5425 preparation, incubated at room 1h is with the negative contrast of PBS.1:1000 added the sheep anti mouse two anti-(Sigma) of Alex488 (green fluorescence) mark after PBS developed a film; Incubated at room 1h; DAPI (blueness) 10min that dyes, after PBS developed a film, fluorescent microscope was observed down; All observe green fluorescence through the painted cell of anti-ORFV059 monoclonal antibody G3, as shown in Figure 2.The result proves that the anti-ORFV059 monoclonal antibody G3 of hybridoma CGMCC No.5425 preparation can discern natural ORFV059 albumen.
6) immunohistochemical methods of antibody (IHC) is identified: collect and suffer from sheep disease change tissue; Utilize the anti-ORFV059 monoclonal antibody G3 (1:1000 dilution) of the hybridoma CGMCC No.5425 preparation behind the purifying to carry out immunohistochemical staining; Use Fujian to step the immunohistochemical methods test kit of new company; Dyeing process is with reference to stepping novel agent box specification sheets, and DAB develops the color, and negative control group replaces one to resist with normal mouse serum; The result finds that cell is dyed pale brown look on the painted tissue sample of anti-ORFV059 monoclonal antibody G3, and is as shown in Figure 3.The immunohistochemical staining result of pathological tissues proves that the anti-ORFV059 monoclonal antibody G3 of hybridoma CGMCC No.5425 preparation can discern natural ORFV059 albumen.
7) antibody detects the reactivity of different sheep of virus strain isolateds and other poxvirus: adopt ELISA and IHC method to detect the reactivity of anti-ORFV059 monoclonal antibody G3 to different sheep of virus strain isolateds and other kind poxvirus.The result is as shown in table 1; Anti-ORFV059 monoclonal antibody G3 can discern from isolating variant geographic ORFV strain isolated of China and USS ORFV strain isolated OV-IA82; And with the poxvirus of other kind, like vaccinia virus, capripox virus and bird pox virus no cross reaction.
Table 1. monoclonal antibody G3 of the present invention is to the reactivity of different sheep of virus strain isolateds and other poxvirus
SEQUENCE LISTING
< 110>Nanfang Medical Univ
< 120>a kind of sore mouth toxalbumin ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody
<130>
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<170> PatentIn version 3.5
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Pro Pro Ala Pro His Pro Lys Gly Asp His Val Leu Lys Ala Val Glu
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Ser Asp Asp Asp Ile Val Leu Val Val Asp Asn Asp Met Thr Leu Arg
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Thr Pro Leu Phe Ser Phe Met Gly Leu Phe Asp Val Asp Val Val Asp
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cccgcaccta agcctgctcc ctctcctgcg ccggcccccg caccggcccc cgcgccagca 180
cccaagccat ctcctcccgc gccgcacccc aagggcgacc acgtgctcaa ggcggtggaa 240
tggaaagacg ttgactccaa agactacccg cacttcttca cggacatgtg caagtccacg 300
tgtccgaagg agatgcagcg ccgcgcggca caccacctca acctctggga gagcatatcg 360
gccggcaccg tctccaccaa gtactccgac gatgacatcg tcctggtggt cgacaacgac 420
atgaccctcc gcaagcccga gatggtaaag ccgctcatcg aggcgatgag gacgaacggc 480
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ggcaccggcg accccgagct catggtctac cccggcgggt acgacgtctc gctagacgcc 600
tacatcatca acgtcggcgg catgaagaag ctctacgacg cgatcatcaa ggagggaggg 660
ctgcgcagcg gcctgctcac cgaggtgttc acgctggaga agcggctctc tctggcgcgc 720
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cggctaagcg gcgcgccctc cctgtggtcg ctgctcgcca cgtggctggc gcgcttctgg 840
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Claims (4)
1. an anti-sheep of virus ORFV059 monoclonal antibody hybridoma cell strain G3 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 4th, 2011, and preserving number is CGMCC No.5425.
2. an anti-sheep of virus ORFV059 monoclonal antibody G3 is characterized in that by the said hybridoma cell strain G3 secretion of claim 1 gained.
3. the preparation method of the said anti-sheep of virus ORFV059 monoclonal antibody hybridoma cell strain G3 of claim 1 is characterized in that: get serum titer greater than 1:10
5BALB/c mouse splenocyte and SP2/0 myeloma cell merge with 50%PEG-4000 by ordinary method; HAT RPMI-1640 screening of medium fused cell with containing 20% calf serum encapsulates elisa plate with recombinant expressed sheep of virus ORFV059 albumen, carries out the ELISA screening; Through limiting dilution repeatedly, obtain the hybridoma cell strain of the anti-sheep of virus ORFV059 of stably excreting monoclonal antibody at last, be labeled as G3.
4. an immunologic function test reagent that detects sheep of virus ORFV059 expression is characterized in that containing the said anti-sheep of virus ORFV059 monoclonal antibody G3 of claim 2.
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Cited By (3)
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| CN104046594A (en) * | 2014-06-16 | 2014-09-17 | 广州洪祥生物医药科技有限公司 | Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof |
| CN109112111A (en) * | 2017-09-12 | 2019-01-01 | 华中农业大学 | The preparation and application of pig δ coronavirus N protein monoclonal antibody |
| CN113265005A (en) * | 2021-04-02 | 2021-08-17 | 贵州大学 | Sore mouth disease virus antibody capture agent, kit, detection method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104046594A (en) * | 2014-06-16 | 2014-09-17 | 广州洪祥生物医药科技有限公司 | Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof |
| CN104046594B (en) * | 2014-06-16 | 2016-06-29 | 广州洪祥生物医药科技有限公司 | A kind of Orf virus protein ORFV086 monoclonal antibody hybridoma cell strain 2G8D10 and monoclonal antibody thereof |
| CN109112111A (en) * | 2017-09-12 | 2019-01-01 | 华中农业大学 | The preparation and application of pig δ coronavirus N protein monoclonal antibody |
| CN113265005A (en) * | 2021-04-02 | 2021-08-17 | 贵州大学 | Sore mouth disease virus antibody capture agent, kit, detection method and application thereof |
| CN113265005B (en) * | 2021-04-02 | 2022-09-30 | 贵州大学 | Sore mouth disease virus antibody capture agent, kit, detection method and application thereof |
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