CN110078820A - Bovine leukemia virus antibody and detection kit - Google Patents
Bovine leukemia virus antibody and detection kit Download PDFInfo
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- CN110078820A CN110078820A CN201811639057.0A CN201811639057A CN110078820A CN 110078820 A CN110078820 A CN 110078820A CN 201811639057 A CN201811639057 A CN 201811639057A CN 110078820 A CN110078820 A CN 110078820A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The present invention relates to bovine leukemia virus BLV P24 monoclonal antibody and its active fragments, target the main neutralizing epitope of P24 albumen of bovine leukemia virus.The invention further relates to use BLV P24 monoclonal antibody or its active fragment to be used to prevent and treat the method and composition of bovine leucosis caused by bovine leukemia virus, and the method and kit of determination, identification and/or quantitative sample or the bovine leukemia virus BLV in vaccine.
Description
Technical field
The present invention relates to a kind of antiviral antibody and virus detection kits, antibody and inspection specific to bovine leukemia virus
Test agent box.
Background technique
Bovine leukemia virus (bovine leukemia virus, BLV) is that one kind can infect the inverse of many ruminants
Retroviral.Its main host infected is milk cow and sheep, and can cause endemicity bovine leucosis (enzootic
Bovine leukosis, EBL), main clinic symptoms show as lymphocyte neoplasm, the output of milk of milk cow reduce and
The progressive emaciation etc. of beef cattle.The disease has been reported that in many countries such as Europe, the U.S. and Japan, produces to cattle-raising larger
Harm.China's 1970s find this after being ill in Shanghai for the first time, and many provinces in the whole nation have been reported that.Recently, Yang Y
Et al carries out bovine leucosis investigation to 15, China province, and in clinical infection than more serious, infection can drop discovery BLV
Low milk production of cow produces serious harm to China's cattle-raising and milk industry；Clinical inspection metropolitan to Heilongjiang Province 6 in 2015
Survey shows BLV in universal Infection trend, and some areas infection is serious, and infection rate is more than 50%.It is worth noting that, having in the recent period
The report of BLV proviral DNA is detected in human milk glandular tissue, it was demonstrated that human breast carcinoma case is related to BLV infection.Therefore, BLV is controlled
Infection it is not only significant to Animal husbandry production, it is also significant to public bio-safety.Currently, clinically there is no
The vaccine for BLV prevention and treatment of effect.The prevention and control of China and the U.S. and Japan and other countries BLV are isolated based on detecting and eliminate disease
Suffer from host, European Union member countries avoid the transmission of infection of the disease in a manner of detecting and slaughter.
The detection of BLV infection can carry out the antibody test of the virus in viral nucleic acid and antigen detection and host.
Viral nucleic acid detection method mainly detects virus in the intracorporal proviral DNA of host, can apply nested PCR method and fluorescence
Quantifying PCR method.Currently, there are many articles of the method for BLV provirus detection of nucleic acids to deliver.The problem is that testing
These detection methods of application are fine within the scope of room.And extensive pattern detection, these methods are special to clinical technique personnel's
Industry level requirement is higher, and once management control is not stringent, be easy to cause small range detection environment DNA pollution, gives testing result
Accuracy brings very big influence.
In market scope, molding detection kit is to apply the antibody level of the virus in detection host,
It there is no the kit for directly detecting the viral antigen, partly cause is that the virus is Low-level Replication, amount of antigen in host
It is very low, it is high to the sensitivity requirement of kit research and development.For BLV GP51 envelope protein and P24 capsid protein in host
Antibody level highest, is easy to clinical detection.Currently, BLV GP51 antibody assay kit has been commercialized, mainly external one
A little Reagent Company's R&D and production are completed, and detection effect is more satisfactory, but testing cost is expensive, for connecting for a long time for population
It is necessary to have certain economic strengths for continuous property detection.Also there is the kit of simple detection BLV GP51 antibody in the country, for kind
The preliminary screening detection of group has better effects.However still there is a distance for the target for needing to reach eradication.It is overall and
Speech, the country, China lack the antibody assay kit of effective detection BLV infection.It should be pointed out that there is no both at home and abroad effectively
, commercialization the detection kit for BLV P24 albumen.Although external import GP51 detection kit detection essence
Accuracy is ideal, but if can be used cooperatively with efficient P24 detection kit, may be more advantageous to and reach BLV infection kind
The purpose of group's purification.
Summary of the invention
Based on the above circumstances, the present inventor is quasi- develops the efficient antibody assay kit for being directed to BLV P24 albumen, specifically
It is prepared for P24 albumen using escherichia coli prokaryotic expression system, after external affinity tag purifies immune mouse, successfully screening is made
Good base is provided for the monoclonal antibody of two plants of anti-BLV P24 albumen, the research and development for the subsequent BLV antibody assay kit
Plinth.
The present invention relates to bovine leukemia virus BLV P24 monoclonal antibody and its active fragments, target bovine leucosis disease
The main neutralizing epitope of P24 albumen of poison.Further the invention further relates to use BLV P24 monoclonal antibody or its active fragment to use
In the method and composition for preventing and treating bovine leucosis caused by bovine leukemia virus.And further to determination, identification
And/or the method and kit of quantitative sample or the bovine leukemia virus BLV in vaccine.
Based on this, on the one hand, the present invention provides a kind of major confonnational epitope for being specific to bovine leukemia virus BLV P24
Monoclonal antibody and its active fragment, that is, antigen-binding fragment (also referred to as antibody fragment).The monoclonal antibody or its segment are special
The opposite sex combines the comformational epitope of bovine leukemia virus BLV P24.The comformational epitope first is that the full length amino acid of P24 albumen
In the 304th to the 315th, sequence is shown in QKLQACAHW, and the corresponding monoclonal antibody is by mouse hybridoma
The P24-44-10 that 293T cell generates.It is further P24- by the monoclonal antibody that mouse hybridoma 293T cell generates
115-5, identification P24 protein epitope is the 216th to the 225th in full length amino acid, and sequence is GDLRSQYQN institute
Show, the corresponding monoclonal antibody is another plant of P24-115-5 generated by murine hybridoma 293T cell.Due to described two plants
Monoclonal antibody identifies the different zones of BLV P24 albumen, and because identification epitope is different, two strain antibodies may be in combination with the same BLV
P24 protein molecular, it is also possible in conjunction with different BLV P24 molecules.
Further, the present invention provides the nucleic acid for encoding the monoclonal antibody or its antigen-binding fragment, the nucleic acid
Encode P24-44-10 or P24-115-5 or its antigen-binding fragment.Further, the present invention provides include the nucleic acid
Carrier and include and the cell of expressing the carrier.
In another aspect, the present invention provides use P24-44-10 or P24-115-5 or its segment to prevent and treat the white blood of ox
The method and composition of disease.The further present invention provides a kind of pharmaceutical composition, and it includes the monoclonal antibodies that the present invention describes
And pharmaceutically acceptable diluent or carrier, the monoclonal antibody are P24-44-10 or P24-115-5.Further, the medicine
Compositions include antigen-binding fragment of the present invention and pharmaceutically acceptable diluent or carrier, the antigen binding
Segment is the antigen-binding fragment of P24-44-10 or P24-115-5.Further, described pharmaceutical composition includes described in coding
The nucleic acid molecules and pharmaceutically acceptable diluent or carrier of antibody or antibody fragment.Further, described pharmaceutical composition includes
Carrier and pharmaceutically acceptable diluent or carrier with the nucleic acid.Further, described pharmaceutical composition includes expression institute
State the cell and pharmaceutically acceptable diluent or carrier of carrier.
Further, the present invention provides a kind of reduction object and infects or reduce bovine leucosis in object by bovine leukemia virus
The method of risk of virus infection.Wherein the method includes the monoclonal antibodies of the present invention to affected animal application therapeutically effective amount
Or its antigen-binding fragment, polynucleotides comprising encoding said antibody or antibody fragment nucleic acid molecules, include the multicore
The carrier of thuja acid or the cell of the expression carrier.The monoclonal antibody is P24-44-10 or P24-115-5.
The third aspect, the present invention are provided using the monoclonal antibody or the identification of its segment, characterization and/or quantitative P24 table
The method and composition reached.In the present invention, the monoclonal antibody is P24-44-10 or P24-115-5, the method includes
Detect the combination of bovine leukemia virus and monoclonal antibody of the present invention or its segment.The present invention is exempted from by protein-bonded
Epidemic disease fluoremetry (IFA), Immunohistochemistry and other methods, including ELISA, blood clotting inhibit in (HI) measurement and virus
(VN) measurement.
In the present invention, the method and composition of the identification and/or quantitative BLV P24 expression be for identifying and/or
Bovine leukemia virus immunogenic substance in quantitative vaccine.The immunogenic substance includes BLV P24 albumen or its antigen
Property part, or coding P24 albumen or its antigenic portions nucleic acid.Further, the antigenic portions include the table of P24
Position.In the present invention, the immunogenic substance is the virus comprising P24.Further, the virus is inactivation；More into one
Step, the virus is the virus of attenuation or the virus is virion (virosome) form.Further, the virus is
Derived from egg or derive from cell culture.In the present invention program, the immunogenic substance is the lytic virus comprising P24
(split virus) or lytic virus antigenic preparation.Further, the immunogenic substance is P24 or its antigenicity
Part.Further, the P24 or its antigenic portions are separated.In further embodiment of the present invention, the P24
Or its antigenic portions is generated by expression system, the expression system is any expression system, such as virus expression carrier.
Fourth aspect, the present invention provides be directed in bovine leukemia virus in detection biological sample or detection biological sample
The kit and method of the antibody of bovine leukemia virus BLV P24.One of bovine leukemia virus BLV in detection biological sample
In scheme, the method includes contacting sample with first antibody, the first antibody is monoclonal antibody of the present invention
P24-44-10 or P24-115-5 or its antibody fragment (being referred to as capture antibody).Further, the method further includes will
Sample is contacted with the secondary antibody of specific binding bovine leukemia virus BLV P24 as described in the present invention or its antibody fragment,
Wherein secondary antibody contains or is conjugated with detectable element and (is referred to as detection antibody).Further, the method includes will
The biological sample is contacted with third antibody, and the third antibody refers to other immunogenes with BLV virus in addition to P24 albumen
Property substance combine antibody such as GP51.
In a concrete scheme of the invention, the kit is comprising first antibody and carries out detection bovine leukemia virus
Measurement specification, the first antibody is the monoclonal antibody that describes of the present invention or its antibody fragment.The monoclonal is anti-
Body is P24-44-10 or P24-115-5.Further, the kit further includes specific binding bovine leukemia virus BLV
The secondary antibody of P24 epitope, wherein secondary antibody contains or is conjugated with detectable element.The secondary antibody is P24-44-
10 or P24-115-5, the secondary antibody contain radioactive atom, are conjugated with fluorescent molecule, or are conjugated with enzyme.More into one
Step, the kit contain the antibody such as GP51 of specific binding other antigenicity substances of bovine leukemia virus BLV.
The hybridoma cell strain P44 of the present invention for generating monoclonal antibody P24-44-10 existed on October 12nd, 2018
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are as follows: CGMCCNO.16682 is generated single
The hybridoma cell strain P115 of clonal antibody P24-115-5 entrusts on October 12nd, 2018 in Chinese microorganism strain preservation management
Member's meeting common micro-organisms center preservation, deposit number are as follows: CGMCC NO.16683.
Detailed description of the invention
The expression and purification of Fig. 1: BLV P24, wherein the expression and purification of A.P24-GST, B.P24-6 × His recombinate egg
White expression and purification, M, protein marker.
Fig. 2: the serum antibody titer after mouse immune P24 albumen, it is effective serum titer that wherein OD value, which is greater than 0.2,.
Fig. 3: more rounds screening of the hybridoma positive strain of anti-BLV P24 albumen.
Fig. 4: different hybridoma positive strains and the identification of BLV Gag albumen and the cellular level of binding ability are verified.
Fig. 5: hybridoma secrete monoclonal antibody hypotype detection.
Fig. 6: the monoclonal screening of hybridoma cell strain.
The Epitope Identification of Fig. 7: two plants of monoclonal antibodies.The Epitope Identification of A:45-10 monoclonal antibody, No. B:115-5
The Epitope Identification of monoclonal antibody.
The specific detection of Fig. 8: two plants of monoclonal antibodies.The indirect immunofluorescence of A:44-10/115-5 and BLV P24 is examined
It surveys, the indirect immunofluorescene assay of B:44-10/115-5 and BVDV, the ELISA of C:44-10/115-5 and FMDV are detected.
Specific embodiment
Term used herein " antibody " is art recognized term, refers to molecule or molecule in conjunction with known antigens
The immunoactive portions of active fragment, especially immunoglobulin molecules and immunoglobulin molecules, i.e., containing specific binding
The molecule of the binding site of antigen.Immunoglobulin is comprising substantially by immunoglobulin κ and λ, α, γ, δ, ε and μ constant region
The albumen of one or more polypeptides of gene and numerous immune globulin variable region genes coding.Light chain is classified as κ or λ.Heavy chain
It is classified as γ, μ, α, δ or ε, and then defines immunoglobulin class IgG, IgM, IgA, IgD and IgE respectively.The subclass of heavy chain
It is also known that.
" specific binding " about antibody of the invention used refer to antibody combine the affinity of its target antigen be higher than its with
The binding affinity of the different antigen of structure.
Known typical immunoglobulin structure unit includes the tetramer.Each tetramer is by two pairs of identical polypeptide chain groups
At each pair of that there is one " light chain " (about 25kD) and one " heavy chain " (about 50-70kD).The N-terminal of every chain defines one big
The variable region of about 100-110 or more amino acid, is mainly responsible for antigen recognizing.Term variable light (VL) and variable heavy chain
(VH) refer respectively to these light chains and heavy chain.
Antibody with the complete antibody of overall length or with it is many sufficiently identify by various peptases or compound digestion produce
Raw segment and exist." antibody " of the present invention includes chimeric monoclonal antibody and its active fragment.In conjunction with known anti-
The active fragment of former molecule is for example including separated light chain and heavy chain, Fab, Fab/c, Fv, Fab' and F (ab')2Segment, packet
Include the product in Fab immunoglobulin expression library and the epitope binding fragments of above-mentioned any antibody and segment.
Term " monoclonal antibody " is also sufficiently approved in this field, refers to the product of the antibody producing cells of monoclonal.It is single
Clonal antibody typically by the antibody-producing B cells and fast-growth cell such as cancer cell for merging normal short survival (sometimes referred to as
Make " immortality cell ") and generate.Resulting hybrid cell or hybridoma quickly double, and obtain the clone for generating antibody.
Term " segment " refers to a part of antibody or antibody chain, and it includes than complete or complete antibody or antibody chain
Less amino acid residue.Segment can or complete antibody complete by chemistry or enzymatic treatment or antibody chain obtain.Segment
It can be obtained by recombination form.The segment of citing includes Fab, Fab', F (ab')2, Fabc and/or Fv segment.Term " antigen
Binding fragment " refers to the combination antigen of immunoglobulin or antibody or (specifically binds) with complete antibody competitive binding antigen
Polypeptide fragment.Binding fragment is to be generated by recombinant DNA technology, or pass through enzyme or chemical cracking intact immunoglobulins
It generates.Binding fragment includes Fab, Fab', F (ab')2, Fabc, Fv, single-stranded and single-chain antibody.
Refer to the biomolecule without its natural adjoint at least some component about " separation " used in of the invention.
The present invention is described by following examples, and the embodiment is for the illustration present invention.
Embodiment 1: plasmid construction
After the synthesis of BLV P24 capsid protein gene, directed cloning to prokaryotic expression p-coldIII-GST carrier and pet-
32a-6 × His carrier.BLV Gag (expands and saves sample in laboratory) full-length gene directed cloning to carrier for expression of eukaryon
PcDNA3.1 (+) (end C- HA label).After P24 truncated gene segment is connect with pCAGGS- carrier (end C- FLag label),
Series P 24 Gene truncation Series P 24-1, P24-2, P24-3, P24-4,1A, 1B, 1C, 3A, 3B, 3C, 3D are constructed, and is prepared into
Corresponding recombinant vector pcDNA3.1-HA, the Epitope Identification for monoclonal antibody.Vector construction application Vazemy recombinant clone
Technology is completed.Wherein p24-1 gene order (namely overall length P24 gene) and protein sequence are respectively such as sequence table SEQ ID NO:
Shown in 1 and 2, p24-2 gene orders and protein sequence be respectively as shown in sequence table SEQ ID NO:3 and 4, p24-3 gene order
With protein sequence respectively as shown in sequence table SEQ ID NO:5 and 6, p24-4 gene order and protein sequence be respectively such as sequence table
Shown in SEQ ID NO:7 and 8, p24-5 gene order and protein sequence be respectively as shown in sequence table SEQ ID NO:9 and 10,24-
1A gene order and protein sequence are respectively as shown in sequence table SEQ ID NO:11 and 12, p24-1B gene order and protein sequence
Respectively as shown in sequence table SEQ ID NO:13 and 14, p24-1C gene order and protein sequence be respectively such as sequence table SEQ ID
Shown in NO:15 and 16, p24-3A gene order and protein sequence be respectively as shown in sequence table SEQ ID NO:17 and 18, p24-3B
Gene order and protein sequence are respectively as shown in sequence table SEQ ID NO:19 and 20, p24-3C gene order and protein sequence point
Not as shown in sequence table SEQ ID NO:21 and 22, p24-3D gene order and protein sequence be respectively such as sequence table SEQ ID NO:
Shown in 23 and 24.Wherein it should be noted that P24 albumen is smaller, therefore the part NC of GAG albumen is merged at its end N-, this
Sample can increase the molecular weight of albumen, be easy to vitro detection, and have no effect on identification of the monoclonal antibody to its sequence.
Embodiment 2:P24 protein expression and detection and label protein purifying and detection
24-p-coldIII-GST carrier is converted to BL21 (DE3) competent escherichia coli cell, overnight 18 DEG C of low temperature trainings
It supports, 1mM IPTG induction collected thallus after 18 hours.P24-pet-32a-6 × His carrier is converted to BL21 (DE3) large intestine bar
Bacterium competence cell, 30 DEG C are incubated overnight, and collect thallus after 18 hours.The above coli somatic is after excusing from death cracking, respectively
After collecting supernatant sample and precipitating sample, SDS-PAGE electrophoresis detection is carried out.Gag-pcDNA3.1-HA transfection 293T cell, 48
Sample is collected after hour and carries out SDS-PAGE, and WB detects the specificity of Gag gene expression and verifying antibody identification.P24 gene
Truncated series recombinant vector transfects 293T cell, and sample is collected after 48 hours and carries out SDS-PAGE, WB detects antibody epitope identification.
Gag-pcDNA3.1-HA transfects 293T cell, and after 24 hours after 0.1%Triton punching, P24 hybridoma ascites are incubated for, and carries out
Simple indirect immunofluorescence experiment (IFA) verifies monoclonal antibody specificity.
P24-p-coldIII-GST recombinant plasmid transformed is broken through ultrasound after IPTG induction to BL21 (DE3) Escherichia coli
Broken, low-speed centrifugal abandons precipitating and takes supernatant.The magnetic bead low temperature of supernatant and coupling GST tag antibody is incubated overnight, then with elution
ELISA after liquid washing for hybridoma screens (SolarBio company).Similarly, P24-pet-32a-6 × His is passed through
Ni- column purification system obtains recombinant protein (SolarBio company), and mouse is immunized to prepare monoclonal antibody.Gag-
After pcDNA3.1-HA recombinant plasmid sequence verification, anti-HA- label H RP- labelled antibody recombinant protein 293T intracellular expression.
The result is shown in Figure 1: the wherein expression and purification 1 of A.P24-GST, supernatant before inducing；2, precipitating before inducing；3, induction 14
Hour supernatant；4, induce precipitate within 14 hours；5, albumen precipitation urea Purification result；M, protein marker.B.P24-6×
The expression and purification 1 of His recombinant protein, supernatant before inducing；2, precipitating before inducing；3, induce 1 hour supernatant；4, it induces 1 hour
Precipitating；5, induce 2 hours supernatants；6, induce precipitate within 2 hours；7, induce 4 hours supernatants；8, induce precipitate within 4 hours；9, albumen is heavy
Shallow lake urea Purification result；M, protein marker.This result shows that we construct P24 protein gene and GST- label and
The expression vector of His- label coupling obtains the higher P24 albumen obtained of purity after GST- affine resin and Ni column purification.
Embodiment 3: immune and hybridoma the preparation and screening of recombinant protein
Mouse (intraperitoneal injection) is immunized after quantitative in the recombination P24-His albumen of Ni particle purifying, and every mouse is immunized every time
100 μ g recombinate P24 albumen, and 5 mouse are immunized altogether.After first immunisation, secondary immunity after 4 weeks is immunized three times after 6 weeks, exempts from three times
P24 antibody titer in serum detection Mice Body is taken after epidemic disease, is chosen and is generated the high mouse of antibody titer, is taking spleen and marrow
The first three days of oncocyte Sp2/0 fusion, booster immunization are primary.
After immune, choose serum titer and reach the mouse that fusion requires, win immune mouse spleen and Sp2/0 marrow
Oncocyte carries out cell fusion under fusion agent (PEG3350) effect, and cell is dispensed 96 porocyte culture plates after fusion, uses
HAT culture medium carries out selective culture, is cloned with ELISA method detection, is detected through excessive round, obtains anti-BLV P24 albumen
Hybridoma positive clone strain.The screening of hybridoma is coated with 96 orifice plates with the P24-GST recombinant protein prepared, with EILSA method
Hybridoma supernatant is detected, selects clone strain according to positive colony standard screen.Positive cell strain is screened, its supernatant is taken to be resisted
Original identification specific detection.After 293T cell transfecting Gag-pcDNA3.1-HA (Gag gene includes complete BLV P24 gene),
It, which can be detected, with the hybridoma supernatant of acquisition identify the BLV P24 recombinant protein of eukaryotic cell expression.
As a result it after seeing that Fig. 2 and Fig. 3, Fig. 2 show 5 mouse of recombination P24 protein immunization of His label purifying, is marked with GST-
The recombination P24 albumen of label purifying is coated with 96 orifice plates, and ELISA method detects the intracorporal antibody titer of every mouse.After No. 1 mouse is immune
Serum antibody titer highest, antibody titer still with higher after ten thousand times of 1:20 dilutions.Therefore, No. 1 mouse is further used
Splenocyte merges with Sp2/0 myeloma cell and prepares hybridoma.After splenocyte is merged with Sp2/0, plantation in 96 orifice plates,
The pressurization screening of HAT culture medium.Hybridoma Cell Culture 10 is screened for the first time later.Antibody titer is higher in screening each time
Culture hole continues next round screening, amounts to and carries out 3 wheel screenings.As shown in figure 3, obtaining amount to 14 altogether by multi-turns screen
Strain hybridoma positive colony.
Embodiment 4: prepared by monoclonal screening and the ascites of hybridoma positive strain
The hybridoma positive hole that will be screened above obtains monoclonal cell strain using Endpoint Dilution Method, is used in combination
The antibody titer for the monoclonal cell strain that ELISA method verifying obtains.Under the positive monoclonal hybridoma strain of verifying continues
It is prepared by the ascites of trip.Mouse immune system is activated in preparation one Zhou Qianyong incomplete Freund's adjuvant of ascites, is injected intraperitoneally every 500
μL.Intraperitoneal injection of mice abdominal cavity hybridoma, every mouse inject 10 after a week for activation6Hybridoma.After injection 10 days,
Observation mouse peritoneal daily can take ascites to carry out antibody titer detection after its enlargement.
Embodiment 5: the activity of monoclonal antibody is analyzed with specificity
The supernatant of the P24 Monoclonal hybridomas positive strain of acquisition identifies that it is anti-with antibody isotypes detection kit (Sigma)
Body type, the i.e. type detection of IgM, IgG and IgA.Antibody continues the epitope identification work of antibody after subtype identification
Make.The above P24 serial genes constructed of WB method detection truncate recombinant protein, determine the epitope of monoclonal antibody identification.
Specially in Gag gene of the 293T cell inner expression containing overall length P24 gene, hybridization obtained is verified with it
Can tumor positive strain identify the BLV P24 albumen expressed in mammalian cell.First 10 plants of hybridomas tentatively higher to OD value
The supernatant of cell is detected, as shown in figure 4, supernatant detected can be reacted with BLV GAG, shows these these hybridization
The antibody of tumor cell strain secretion may identify the wild type BLV virus under native state.The antibody that different hybridomas generate is to BLV
The recognition capability of P24 is different, and such as No. 115, No. 173, No. 180, No. 55 and No. 358 responds are stronger；And No. 36, No. 248,57
Number, No. 44 and No. 178 responds it is weaker.
Embodiment 6: the subtype identification of monoclonal antibody
Preliminary subtype identification is carried out to the hybridoma positive strain screened above, as shown in figure 5, different hybridoma sun
Property strain secretion monoclonal antibody type it is different.Wherein, the monoclonal antibody that No. 44, No. 115, No. 178 and No. 358 clones generate
Type is relatively simple, shows that the hybridoma in hole is relatively simple；No. 36, No. 57, No. 173, No. 180 and No. 248, antibody class
Type disunity shows that hole inner cell is the mixture of different hybridomas, and is to be mixed with the cell hole containing IgM antibody；55
Number be also hybridoma mixture, the monoclonal antibody of two kinds of hypotypes of IgG secretion 1 and IgG3.No. 44 and No. 115 reaction bondeds
Ability is stronger, selects this two strain of hybridoma further progress Colony Culture.As shown in fig. 6, No. 44-10 and No. 115-5
Antibody titer is higher, this 3 plants of cells is selected to carry out ascites preparation, to be used for subsequent detection work,
Embodiment 7: ascites preparation is identified with epitope
1, the antibody epitope identification of monoclonal antibody P44-10
Further it is prepared for the ascites of 44-10 and 115-5.As shown in fig. 7, constructing a series of BLV P24 Gene truncations
Expression plasmid, because P24 albumen is smaller, has merged the part NC of GAG albumen at its end N- in building, in this way can be with
The molecular weight for increasing albumen, is easy to vitro detection, and have no effect on identification of the monoclonal antibody to its sequence.Fused egg
The white fusion protein for NC-P24.5 albumen altogether is constructed altogether, is named as P24-1, P24-2, P24-3, P24-4 and P24-
5, and this five molecular weight of albumen are sequentially reduced, each albumen 40 amino acid residue left sides fewer than a upper PROTEIN C-end
Right (molecular size range sequence is P24-1 > P24-2 > P24-3 > P24-4 > P24-5).P24-1, P24-2, P24-3 will be encoded,
The plasmid (i.e. p24-1, p24-2, p24-3, p24-4 and p24-5 are shown in embodiment 1) of five genes of P24-4 and P24-5 transfects
293T cell collects cell sample after transfection 48 hours, carry out protein immunoblotting experiment (western blotting,
WB).Protein binding that monoclonal antibody can only be identified with it simultaneously generates signal.Monoclonal antibody P44-10 detects these albumen samples
This.The result shows that P44-10 can be reacted with p24-1 albumen.Therefore, primarily determine monoclonal antibody P44-10 identification be
The end C- of BLV P24 albumen.On this basis, further p24-1 albumen truncate again and be expressed, it is pressed from the end C-
Every 10 amino acid sequences gradually truncate, and are named as p24-1A, p24-1B and p24-1C.These genes are transfected into 239T cell
Afterwards, cell sample is collected after 48 hours, continues to carry out WB detection with monoclonal antibody P44-10.The result shows that monoclonal antibody
P44-10 but therefore can cannot primarily determine its identification with P24-1C protein binding in conjunction with albumen P24-1A and P24-1B
Epitopic regions be P24-1B C- end sequence, i.e. epitope GQKLQACAHW.
2, the antibody epitope identification of monoclonal antibody P115-5
It is anti-to monoclonal with same means and method referring to the antibody epitope identification work of monoclonal antibody P44-10
The antibody epitope of body P115-5 identification is identified.Plasmid p24-1, p24-2, p24-3, p24-4 and p24-5 are transfected into 293T
Cell 48 as a child, collects cell sample and carries out WB detection.The result shows that monoclonal antibody P115-5 can be with albumen P24-3
In conjunction with but can not be in conjunction with albumen P24-4.Therefore, the region identified should be in the end C- of albumen P24-3.To albumen
The albumen that the end C- of P24-3 has carried out 10 amino acid or so length sequentially truncates, and is respectively designated as P24-3A, P24-3B,
P24-3C and P24-3D.The result shows that monoclonal antibody P115-5 can identify albumen P24-3C, but it cannot identify albumen P24-
3D, therefore, the epitope identified are determined as the protein sequence GDLRSQYQN of the end C- of P24-3C.
The specific detection of 8: two plants of monoclonal antibodies of embodiment
To detect whether monoclonal antibody obtained has antigen-recognition specificity, bovine viral diarrhea virus is used
(Bovine viral diarrhea virus, BVDV) and foot and mouth disease virus (Foot and mouth disease virus,
FMDV the specificity of two plants of monoclonal antibodies of 44-10 and 115-5) is detected.As shown in figure 8, two plants of monoclonal antibodies can identify
BLV P24 albumen, but can not identify intracellular BVDV virus.ELISA detection shows two plants of monoclonals of 44-10 and 115-5
Antibody can not identify Asia I type, A type and O-shaped foot and mouth disease virus, it was demonstrated that two plants of monoclonal antibody specificities of acquisition are good.
As shown in figure 8, after BVDV is proliferated in the cell, BVDV specific antibody can identify intracellular virus, and of the invention two
Strain monoclonal antibody can not identify the virus, i.e., in the cell without green florescent signal.Similarly, it is detected with FMDV antigen
The detection monoclonal antibody of kit.It was found that two plants of monoclonal antibodies of the invention are unable to identify O-shaped, A type and Asia I type
Foot-and-mouth disease virus antigen, and positive control antibodies can identify these antigens, show that antibody specificity of the invention is good,
There is no cross reaction with generally popular FMDV.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture is (in China Animal Health and Epidemiology Center Harbin point
The heart)
<120>bovine leukemia virus antibody and detection kit
<160>24
<170>PatentIn Version 2.1
<210>1
<211>996
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>1
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCATCGTCCAAGGCCCCGCCGAAAGCTATGTAGAGTTTGTCAACCGGTTA 780
CAAATTTCATTAGCTGACAACCTTCCCGACGGAGTCCCTAAGGAACCCATTATTGACTCC 840
CTTAGTTATGCAAATGCTAACAAAGAGTGCCAGCAAATTTTGCAGGGGCGAGGCCTAGTG 900
GCCGCCCCGGTGGGGCAAAAACTGCAGGCTTGCGCACATTGGGCCCCCAAGGTGAAACAG 960
CCTGCAGTTCTCGACTACAAAGACGATGACGACAAG 996
<210>2
<211>332
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>2
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Ile Val Gln Gly Pro Ala Glu Ser Tyr Val Glu Phe
245 250 255
Val Asn Arg Leu Gln Ile Ser Leu Ala Asp Asn Leu Pro Asp Gly Val
260 265 270
Pro Lys Glu Pro Ile Ile Asp Ser Leu Ser Tyr Ala Asn Ala Asn Lys
275 280 285
Glu Cys Gln Gln Ile Leu Gln Gly Arg Gly Leu Val Ala Ala Pro Val
290 295 300
Gly Gln Lys Leu Gln Ala Cys Ala His Trp Ala Pro Lys Val Lys Gln
305 310 315 320
Pro Ala Val Leu Asp Tyr Lys Asp Asp Asp Asp Lys
325 330
<210>3
<211>876
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>3
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCATCGTCCAAGGCCCCGCCGAAAGCTATGTAGAGTTTGTCAACCGGTTA 780
CAAATTTCATTAGCTGACAACCTTCCCGACGGAGTCCCTAAGGAACCCATTATTGACTCC 840
CTTAGTTATGCAGACTACAAAGACGATGACGACAAG 876
<210>4
<211>292
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>4
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Ile Val Gln Gly Pro Ala Glu Ser Tyr Val Glu Phe
245 250 255
Val Asn Arg Leu Gln Ile Ser Leu Ala Asp Asn Leu Pro Asp Gly Val
260 265 270
Pro Lys Glu Pro Ile Ile Asp Ser Leu Ser Tyr Ala Asp Tyr Lys Asp
275 280 285
Asp Asp Asp Lys
290
<210>5
<211>756
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>5
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCGACTACAAAGACGATGACGACAAG 756
<210>6
<211>252
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>6
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Asp Tyr Lys Asp Asp Asp Asp Lys
245 250
<210>7
<211>636
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>7
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGGACTACAAAGACGATGACGACAAG 636
<210>8
<211>212
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>8
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Asp Tyr Lys Asp
195 200 205
Asp Asp Asp Lys
210
<210>9
<211>516
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>9
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAAGACTACAAAGACGATGACGACAAG 516
<210>10
<211>172
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>10
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Asp Tyr Lys Asp Asp Asp Asp Lys
165 170
<210>11
<211>972
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>11
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCATCGTCCAAGGCCCCGCCGAAAGCTATGTAGAGTTTGTCAACCGGTTA 780
CAAATTTCATTAGCTGACAACCTTCCCGACGGAGTCCCTAAGGAACCCATTATTGACTCC 840
CTTAGTTATGCAAATGCTAACAAAGAGTGCCAGCAAATTTTGCAGGGGCGAGGCCTAGTG 900
GCCGCCCCGGTGGGGCAAAAACTGCAGGCTTGCGCACATTGGGCCCCCAAGGTGAAACAG 960
CCTGCAGTTCTC 972
<210>12
<211>324
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>12
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Ile Val Gln Gly Pro Ala Glu Ser Tyr Val Glu Phe
245 250 255
Val Asn Arg Leu Gln Ile Ser Leu Ala Asp Asn Leu Pro Asp Gly Val
260 265 270
Pro Lys Glu Pro Ile Ile Asp Ser Leu Ser Tyr Ala Asn Ala Asn Lys
275 280 285
Glu Cys Gln Gln Ile Leu Gln Gly Arg Gly Leu Val Ala Ala Pro Val
290 295 300
Gly Gln Lys Leu Gln Ala Cys Ala His Trp Ala Pro Lys Val Lys Gln
305 310 315 320
Pro Ala Val Leu
<210>13
<211>942
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>13
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCATCGTCCAAGGCCCCGCCGAAAGCTATGTAGAGTTTGTCAACCGGTTA 780
CAAATTTCATTAGCTGACAACCTTCCCGACGGAGTCCCTAAGGAACCCATTATTGACTCC 840
CTTAGTTATGCAAATGCTAACAAAGAGTGCCAGCAAATTTTGCAGGGGCGAGGCCTAGTG 900
GCCGCCCCGGTGGGGCAAAAACTGCAGGCTTGCGCACATTGG 942
<210>14
<211>314
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>14
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Ile Val Gln Gly Pro Ala Glu Ser Tyr Val Glu Phe
245 250 255
Val Asn Arg Leu Gln Ile Ser Leu Ala Asp Asn Leu Pro Asp Gly Val
260 265 270
Pro Lys Glu Pro Ile Ile Asp Ser Leu Ser Tyr Ala Asn Ala Asn Lys
275 280 285
Glu Cys Gln Gln Ile Leu Gln Gly Arg Gly Leu Val Ala Ala Pro Val
290 295 300
Gly Gln Lys Leu Gln Ala Cys Ala His Trp
305 310
<210>15
<211>912
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>15
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACCATCGTCCAAGGCCCCGCCGAAAGCTATGTAGAGTTTGTCAACCGGTTA 780
CAAATTTCATTAGCTGACAACCTTCCCGACGGAGTCCCTAAGGAACCCATTATTGACTCC 840
CTTAGTTATGCAAATGCTAACAAAGAGTGCCAGCAAATTTTGCAGGGGCGAGGCCTAGTG 900
GCCGCCCCGGTG 912
<210>16
<211>304
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>16
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr Ile Val Gln Gly Pro Ala Glu Ser Tyr Val Glu Phe
245 250 255
Val Asn Arg Leu Gln Ile Ser Leu Ala Asp Asn Leu Pro Asp Gly Val
260 265 270
Pro Lys Glu Pro Ile Ile Asp Ser Leu Ser Tyr Ala Asn Ala Asn Lys
275 280 285
Glu Cys Gln Gln Ile Leu Gln Gly Arg Gly Leu Val Ala Ala Pro Val
290 295 300
<210>17
<211>732
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>17
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCTACTCGTCCTTCAGTACAA 720
CCTTGGTCCACC 732
<210>18
<211>244
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>18
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro Thr Arg Pro Ser Val Gln
225 230 235 240
Pro Trp Ser Thr
<210>19
<211>702
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>19
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAACCTCTGGCTTCAGGCCTGGAAAAATCTCCCT 702
<210>20
<211>234
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>20
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
Leu Trp Leu Gln Ala Trp Lys Asn Leu Pro
225 230
<210>21
<211>672
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>21
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCCGGGGATCTTAGAAGT 660
CAATATCAAAAC 672
<210>22
<211>224
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>22
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala Gly Asp Leu Arg Ser Gln Tyr Gln Asn
210 215 220
<210>23
<211>645
<212>DNA
<213>bovine leukemia virus (bovine leukemia virus)
<400>23
ATGGGAAATTCCCCCTCCTATAACCCCCCCGCTGGTATCTCCCCCTCAGACTGGCTCAAC 60
CTTCTGCAAAGCGCGCAAAGGCTCAATCCGCGACCCTCTCCCAGCGATTTTACCGATTTA 120
AAGAATTACATCCATTGGTTTCATAAGACCCAGAAAAAACCATGGACTTTCACTTCTGGT 180
GGCCCCACCTCATGTCCACCCGGGAGATTCGGCCGGGTTCCCCTTGTCTTGGCCACCCTA 240
AACGAAGTGCTCTCAAACGATGGGGGCGCCCCGGGTGCATCGGCCCCAGAAGAACAACCC 300
CCCCCTTATGACCCCCCCGCCGTTTTGCCAATCATATCTGAAGGGAATCGCAACCGCCAT 360
CGTGCTTGGGCACTCCGAGAATTACAAGATATCAAAAAGGAAATTGAAAATAAGGCACCG 420
GGTTCGCAAGTATGGATACAAACACTACGACTTGCAATCCTGCAGGCCGACCCTACTCCG 480
GCTGACCTAGAACAACTTTGCCAATATATTGCTTCCCCGGTCGACCAAACGGCCCATATG 540
ACCAGCCTAACGGCAGCAATAGCCGCCGCTGAAGCGGCCAACACCCTCCAGGGTTTTAAC 600
CCCCAAAACGGGACCCTAACCCAACAATCAGCTCAGCCCAACGCC 645
<210>24
<211>215
<212>PRT
<213>bovine leukemia virus (bovine leukemia virus)
<400>24
Met Gly Asn Ser Pro Ser Tyr Asn Pro Pro Ala Gly Ile Ser Pro Ser
1 5 10 15
Asp Trp Leu Asn Leu Leu Gln Ser Ala Gln Arg Leu Asn Pro Arg Pro
20 25 30
Ser Pro Ser Asp Phe Thr Asp Leu Lys Asn Tyr Ile His Trp Phe His
35 40 45
Lys Thr Gln Lys Lys Pro Trp Thr Phe Thr Ser Gly Gly Pro Thr Ser
50 55 60
Cys Pro Pro Gly Arg Phe Gly Arg Val Pro Leu Val Leu Ala Thr Leu
65 70 75 80
Asn Glu Val Leu Ser Asn Asp Gly Gly Ala Pro Gly Ala Ser Ala Pro
85 90 95
Glu Glu Gln Pro Pro Pro Tyr Asp Pro Pro Ala Val Leu Pro Ile Ile
100 105 110
Ser Glu Gly Asn Arg Asn Arg His Arg Ala Trp Ala Leu Arg Glu Leu
115 120 125
Gln Asp Ile Lys Lys Glu Ile Glu Asn Lys Ala Pro Gly Ser Gln Val
130 135 140
Trp Ile Gln Thr Leu Arg Leu Ala Ile Leu Gln Ala Asp Pro Thr Pro
145 150 155 160
Ala Asp Leu Glu Gln Leu Cys Gln Tyr Ile Ala Ser Pro Val Asp Gln
165 170 175
Thr Ala His Met Thr Ser Leu Thr Ala Ala Ile Ala Ala Ala Glu Ala
180 185 190
Ala Asn Thr Leu Gln Gly Phe Asn Pro Gln Asn Gly Thr Leu Thr Gln
195 200 205
Gln Ser Ala Gln Pro Asn Ala
210 215
Claims (10)
1. the monoclonal antibody or its active fragment of the neutralization comformational epitope of bovine leukemia virus BLV P24 are specifically bound,
Described in neutralize comformational epitope: (a) comprising amino acid GQKLQACAHW, or (b) include amino acid GDLRSQYQN.
2. monoclonal antibody as described in claim 1 or its active fragment, wherein in the bovine leukemia virus BLV P24
And comformational epitope: (a) be mouse monoclonal antibody P24-44-10 specific binding epitope, or be (b) mouse monoclonal antibody
The epitope of P24-115-5 specific binding, the hybridoma cell strain P44 for generating the monoclonal antibody P24-44-10 are deposited in
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are as follows: CGMCC NO.16682 is generated
It is general that the hybridoma cell strain bacterial strain P115 of monoclonal antibody P24-115-5 is deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center preservation, deposit number are as follows: CGMCC NO. is 16683.
3. monoclonal antibody as described in claim 1 or its active fragment, wherein the monoclonal antibody is: (a) mouse Dan Ke
Grand antibody P24-44-10, or (b) mouse monoclonal antibody P24-115-5.
4. the monoclonal antibody P24-44-10 or P24-115-5 that are generated by hybridoma 293T cell, generate the monoclonal antibody
The hybridoma cell strain P44 of P24-44-10 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center guarantor
Hiding, deposit number are as follows: CGMCC NO.16682 generates the hybridoma cell strain bacterial strain P115 of the monoclonal antibody P24-115-5
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are as follows: CGMCC NO. is
16683。
5. a kind of method of bovine leukemia virus BLV in Testing in vitro biological sample, including sample is contacted with first antibody,
Described in first antibody be any one of claim 1-4 monoclonal antibody or its active fragment, and determine bovine leukemia virus
The existence or non-existence of BLV.
6. method for claim 5 further comprises by the P24 epitope of sample and specific binding bovine leukemia virus BLV
Secondary antibody contact, wherein the secondary antibody contains detectable element or is conjugated with detectable element, wherein it is described with
Secondary antibody contact carries out before the determination.
7. method for claim 6, wherein secondary antibody is the monoclonal antibody or its active tablet of any one of claim 1-4
Section, wherein first antibody is one of monoclonal antibody P24-44-10 or monoclonal antibody P24-115-5, and secondary antibody is
Another kind in the monoclonal antibody P24-44-10 or monoclonal antibody P24-115-5.
8. the kit of bovine leukemia virus BLV in a kind of detection biological sample, it includes first antibody, wherein first antibody is
The monoclonal antibody or antibody fragment of any one of claim 1-4.
9. the kit of claim 8 further comprises the second of the P24 epitope of specific binding bovine leukemia virus BLV
Antibody, wherein the secondary antibody contains detectable element or is conjugated with detectable element.
10. the kit of claim 9, wherein secondary antibody is the monoclonal antibody or its activity of any one of claim 1-4
Segment, and secondary antibody is the another kind in the monoclonal antibody P24-44-10 or monoclonal antibody P24-115-5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811639057.0A CN110078820A (en) | 2018-12-29 | 2018-12-29 | Bovine leukemia virus antibody and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811639057.0A CN110078820A (en) | 2018-12-29 | 2018-12-29 | Bovine leukemia virus antibody and detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110078820A true CN110078820A (en) | 2019-08-02 |
Family
ID=67412905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811639057.0A Pending CN110078820A (en) | 2018-12-29 | 2018-12-29 | Bovine leukemia virus antibody and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110078820A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117777283A (en) * | 2024-02-26 | 2024-03-29 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody of bovine leukemia virus p24 protein and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988001745A1 (en) * | 1986-08-28 | 1988-03-10 | Institut National De La Sante Et De La Recherche M | Method of immunological detection of bovine leucosis, means for implementing such method and prophylactic application thereof |
| DD255001A1 (en) * | 1986-11-24 | 1988-03-16 | Akad Wissenschaften Ddr | METHOD FOR OBTAINING A MONOCLONAL ANTIBODY AGAINST PROTEIN P24 OF THE BOVINE ELECTRIC VIRUS (BLV) |
| JPH032664A (en) * | 1989-05-31 | 1991-01-09 | Tonen Corp | Cattle-leukemia diagnostic medicine |
| CN101303351A (en) * | 2008-06-26 | 2008-11-12 | 中华人民共和国徐州出入境检验检疫局 | Test paper strip for testing bovine leukemia antibody with colloidal gold immune chromatography and preparation method thereof |
| CN108330214A (en) * | 2018-04-26 | 2018-07-27 | 石河子大学 | The quickly RPA primers and reagent and kit of detection bovine leucosis provirus |
-
2018
- 2018-12-29 CN CN201811639057.0A patent/CN110078820A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988001745A1 (en) * | 1986-08-28 | 1988-03-10 | Institut National De La Sante Et De La Recherche M | Method of immunological detection of bovine leucosis, means for implementing such method and prophylactic application thereof |
| DD255001A1 (en) * | 1986-11-24 | 1988-03-16 | Akad Wissenschaften Ddr | METHOD FOR OBTAINING A MONOCLONAL ANTIBODY AGAINST PROTEIN P24 OF THE BOVINE ELECTRIC VIRUS (BLV) |
| JPH032664A (en) * | 1989-05-31 | 1991-01-09 | Tonen Corp | Cattle-leukemia diagnostic medicine |
| CN101303351A (en) * | 2008-06-26 | 2008-11-12 | 中华人民共和国徐州出入境检验检疫局 | Test paper strip for testing bovine leukemia antibody with colloidal gold immune chromatography and preparation method thereof |
| CN108330214A (en) * | 2018-04-26 | 2018-07-27 | 石河子大学 | The quickly RPA primers and reagent and kit of detection bovine leucosis provirus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117777283A (en) * | 2024-02-26 | 2024-03-29 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody of bovine leukemia virus p24 protein and application thereof |
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