CN106381288B - A kind of anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody hybridoma 2D10 and monoclonal antibody - Google Patents
A kind of anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody hybridoma 2D10 and monoclonal antibody Download PDFInfo
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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
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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Abstract
The invention discloses a kind of anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody hybridoma 2D10 and monoclonal antibodies.The hybridoma cell strain was preserved in China typical culture collection center on August 12nd, 2016, and deposit number is CCTCC NO:C2016155.The hybridoma cell strain secretion yield that the present invention obtains is high, for the monoclonal antibody specificity with higher of H3N2 hypotype canine influenza virus NP preparation and sensitivity, and denaturation NP albumen can be combined in ELISA and Western-blot detection, and the natural NP albumen with higher structure is combined in Immunofluorescence test, scientific basis can be provided for the research of the structure function of NP albumen and the development of various hypotype external diagnosis reagents, and provide experimental data as the feasibility of disease marker for it and support.
Description
Technical field
The invention belongs to field of immunology, and in particular to a kind of anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody
Hybridoma 2D10 and monoclonal antibody.
Background technique
Influenza A virus is common influenza virus, and influenza A virus is easiest to morph, influenza A virus packet
Include multiple hypotypes.Have now been found that a variety of subtype influenza virus can infect dog, including H1, H3, H5 and H9 hypotype.Wherein H3N8 and
H3N2 subtype influenza virus can be popular in dog group, mainly causes respiratory symptom.Dog influenza occurs not only threatening the health of dog,
Also threat is brought to public health.Recently the study found that the HA gene of H3N2 hypotype canine influenza virus is possibly through reconfiguration
It is introduced into human influenza virus, and the introducing of new HA gene is likely to result in person-to-person flu outbreak.
A type canine influenza virus is sub-thread minus-stranded rna virus, is made of 8 independent segments RNA, encodes 10 genes
, wherein that the 5th segment RNA coding is nucleocapsid protein (NP).NP is a kind of polypeptide of monomer phosphorylation, includes 498 ammonia
Base acid, molecular weight is about 60KD, wherein being rich in arginine, glycine and serine, is the main component for constituting nucleocapsid, has
Type specificity, antigenic specificity are one of foundations of influenza virus parting.NP is also a kind of multifunctional protein, in influenza disease
In the transcription and replication of poison and determine that the host specificity etc. of virus plays a role.NP makes a variation frequently during virus evolution
Rate is low, and structure height is conservative in each different subtype, and has very strong immunogenicity, immune response can be caused to generate anti-
Body.Therefore, NP can be used as influenza A virus clinical diagnosis and therapeutic targets, and preparation is directed to each hypotype NP albumen conservative antigen
The monoclonal antibody in site has a very important significance, and can examine in vitro for the research of the structure function of the albumen and various hypotypes
The development of disconnected reagent provides scientific basis, and provides experimental data as the feasibility of disease marker for it and support.
Summary of the invention
The purpose of the present invention is to provide a kind of monoclonal antibodies of anti-H3N2 hypotype canine influenza virus nucleoprotein, hybridoma
Cell strain and application.
The technical solution used in the present invention is:
A kind of hybridoma cell strain for secreting anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody, is denoted as H3N2-
CIV-NP-2D10 was preserved in China typical culture collection center on August 12nd, 2016, and deposit number is CCTCC NO:
C2016155。
A kind of monoclonal antibody of anti-H3N2 hypotype canine influenza virus nucleoprotein is produced by hybridoma cell strain described above
It is raw.
Preferably, the monoclonal antibody is IgG2b subclass.
Preferably, the light chain of the monoclonal antibody is κ chain.
A kind of kit detecting H3N2 hypotype canine influenza virus, contains monoclonal antibody described above.
A kind of kit detecting H3N2 hypotype canine influenza virus antibody, contains monoclonal antibody described above.
The monoclonal antibody of anti-H3N2 hypotype canine influenza virus nucleoprotein described above is in identification A type canine influenza virus
Application in NP albumen.
The beneficial effects of the present invention are:
1, the hybridoma cell strain secretion yield that the present invention obtains is high, and it is mono- to secrete obtained anti-H3N2 hypotype dog influenza NP
Clonal antibody can identify H3N2 hypotype in conjunction with the recombinant proteins being coated on ELISA Plate, and in Western-blot detection
Dog influenza NP albumen.
2, the monoclonal antibody that the present invention obtains can identify natural NP albumen in Immunofluorescence test.
3, the present invention is for the monoclonal antibody specificity with higher of H3N2 hypotype canine influenza virus NP preparation and spirit
Sensitivity, and can ELISA and Western-blot detection in combine denaturation NP albumen, and in Immunofluorescence test combine have
The natural NP albumen of higher structure can mention for the research of structure function and the development of various hypotype external diagnosis reagents of NP albumen
For scientific basis, and experimental data is provided as the feasibility of disease marker for it and is supported.
Detailed description of the invention
The monoclonal antibody that Fig. 1 is hybridoma cell strain H3N2-CIV-NP-2D10 of the present invention is to H3N2 hypotype dog influenza disease
The result of malicious immunoblotting；
Fig. 2 is hybridoma cell strain H3N2-CIV-NP-2D10 monoclonal antibody of the present invention to H3N2 hypotype canine influenza virus
The result of immunofluorescence dyeing.
Specific embodiment
Below with reference to specific experiment, the present invention is further illustrated, and however, it is not limited to this.
The foundation of H3N2-CIV-NP-2D10 hybridoma cell strain and anti-H3N2 hypotype canine influenza virus NP monoclonal antibody
Preparation and identification
One, mouse immune
After being mixed after the inactivation of H3N2 hypotype canine influenza virus with isometric Freund's complete adjuvant, with every 50 μ g/500
The amount of μ l divide multi-point injection to BALB/C mice dorsal sc (Nanfang Medical Univ's Experimental Animal Center, 6-8 week old female, 3
Only) carry out first immunisation；Adjuvant is changed into incomplete Freund's adjuvant after 2 weeks carries out secondary routine immunization, immunization method and dosage
It is identical as first immunisation；Routine immunization acquires a small amount of tail vein afterwards three times, and indirect ELISA detects serum titer.It the results are shown in Table
1。
Titration after table 1:H3N2 mouse is immunized three times
As shown in Table 1: being detected using H3N2 hypotype canine influenza virus as envelope antigen, 3# Mouse titers obviously reach four
Ten thousand or more, it is detected by envelope antigen of recombinant proteins, 3# Mouse titers reach 20,000, reach fusion and require.Fusion preceding 3
It, is injected intraperitoneally booster immunization with same dose antigen.
Two, prepared by hybridoma
1. preparing feeder cells: selecting BALB/C mice peritoneal macrophage.First 1 day of fusion, by 2 BALB/C mices
It draws neck to put to death, is immersed in 75% alcohol 5 minutes；Under aseptic technique, mouse skin is cut off with scissors, exposure peritonaeum is used
Syringe injects the RPMI-1640 culture medium of 5ml pre-cooling, and flushing liquor is sucked out in repeated flushing, and 1000rpm is centrifuged 5 minutes；In abandoning
Clearly, it is resuspended, is added in 5 piece of 96 orifice plate, 100 holes μ l/, 37 DEG C with the HAT RPMI-1640 culture medium containing 20% fetal calf serum,
5%CO2Culture.
2. preparing SP2/0 myeloma cell: will be cultivated through the processed SP2/0 myeloma cell of 8-anaguanine (8-AG)
It to logarithmic growth phase, takes 8 wares that cell suspension is made, is centrifuged, sediment fraction is washed 3 times with RPMI-1640 culture medium, is counted.
3. preparing immune spleen cell: 3 days post-tensioning necks of booster immunization put to death mouse, sterile to take spleen, are cultivated with RPMI-1640
Base grinds spleen after washing once, crosses 200 mesh stainless steel mesh, cell suspension is made.Centrifugation, sediment fraction RPMI-1640
Culture medium is washed 3 times, is counted.
4. cell fusion: splenocyte and myeloma cell being mixed with 4:1 ratio, gently attack tube bottom, makes sedimentation cell
Loosely uniformly at paste after, be added 1ml polyethylene glycol (PEG, molecular weight 1500, Roche) merged, use RPMI-1640
Culture medium terminates reaction.Centrifuging and taking sediment fraction is added after being suspended with the HAT culture medium containing 20%FBS and has been covered with feeder layer
In 5 piece of 96 orifice plate of cell, 100 holes μ l/.Culture plate is placed in 37 DEG C, contains 5%CO2Culture in the incubator of saturated humidity.
5. the screening of hybridoma and the foundation of cell strain: screening cells and supernatant using indirect elisa method.With
0.05M pH9.6 carbonate coating buffer dilutes H3N2 hypotype canine influenza virus and recombination H3N2 hypotype dog influenza NP albumen, makes it
Final concentration of 2 μ g/ml and 4 μ g/ml, is added to 96 hole polystyrene plates, 100 holes μ l/, 37 DEG C 2 hours.With contain 5% degreasing
The 0.01M pH7.2PBS of milk powder is closed, 200 holes μ l/, 37 DEG C 2 hours.With washing lotion (Tween-20 of the PBS containing 0.05%v/v)
Board-washing three times, pats dry, for detecting.Fusion 10 days after, take 200 μ l of cell conditioned medium in above-mentioned ELISA Plate, 37 DEG C 1.5 hours,
Goat anti-mouse igg (the Wuhan doctor De Sheng of 1:10000 times of diluted horseradish peroxidase-labeled is added in washing lotion board-washing afterwards three times
Object Science and Technology Ltd.), 100 holes μ l/, 37 DEG C 1 hour, ibid after board-washing, be added TMB developing solution (the excellent enlightening biotechnology in Guangzhou
Co., Ltd's self-control) 100 holes μ l/, room temperature is protected from light 10 minutes, and 2MH is added2SO2Terminate liquid, 50 holes μ l/ terminate reaction, survey
450nm absorption value.Myeloma cell's culture supernatant must be positive next than >=2.1 with measured value and control value as negative control
Judge positive cell hole.The selection higher positive cell hole of potency is subcloned, and with sub- gram of limiting dilution assay serial dilution
It is grand at least twice more than, until monoclonal cell positive rate is 100%, obtaining one plant can the anti-H3N2 hypotype dog of stably excreting
The monoclonal cell strain of nucleoprotein influenza antibody is denoted as H3N2-CIV-NP-2D10, referred to as 2D10.Cell strain expands culture
Afterwards, conservation is frozen with frozen stock solution containing 10%DMSO.The hybridoma cell strain H3N2-CIV-NP- that the present embodiment is obtained
2D10 delivers China typical culture collection center and carries out preservation, and address is No. 299 forces of Wuhan City, Hubei Province Wuchang District Bayi Road
Chinese institution of higher education, deposit number are CCTCC NO:C2016155, and the deposit date is on August 12nd, 2016.
Three, the preparation and purification of monoclonal antibody
10-12 week old female BLAB/C mouse is selected, 500 μ l/ of intraperitoneal injection saxol is only.After 7-10 days, place is taken
Mouse peritoneal, every injection 5 × 10 are injected into the monoclonal hybridoma of logarithmic growth phase5-1×106A cell.Raising
After observation 7-10 days, ascites is collected with aseptic injection syringe needle, 5000rpm is centrifuged 20min, removal cell component and other precipitatings
Object collects supernatant.5ml ascites supernatant is taken, with 0.22 μm of filtering with microporous membrane after being diluted to 30ml with PBS.Extremely by filtrate loading
The Protein G affinity column (GE Healthcare) balanced with PBS, flow velocity 1ml/min are used after completion of the sample
PBS balance, then eluted with the glycine elution liquid of 0.1M pH3.0, flow velocity 4ml/min collects antibody, and with 1MpH9.0's
Tris-HCl neutralizer neutralizing antibody.With the purity of SDS-PAGE identification monoclonal antibody, purity reaches 90% or more.
Four, MAb concentration measures
Purified monoclonal antibody, concentration 1.5mg/ml are measured using BCA protein quantification kit (Thermo).
Five, monoclonal subtype identification
The 40 μ l of cell conditioned medium of hybridoma H3N2-CIV-NP-2D10 is taken to be added to mouse monoclonal hypotype fast typing glue
Identified on body gold test paper strip (self-control of Guangzhou You Di Biotechnology Co., Ltd), the antibody subtype of 2D10 secretion is IgG2b,
Light chain is κ chain.
Six, antibody titer detects
Indirect elisa method detection is carried out using H3N2 canine influenza virus and recombinant proteins as envelope antigen respectively, is as a result seen
Table 2.
Table 2: antibody purification bioactivity
As shown in Table 2: antibody titer prepared by the present invention can reach as 1:320000.
Seven, affinity detects
The affinity of antibody prepared by the present invention is detected with ELISA method, relative compatible constant is 1.58 × 108。
Eight, specific detection
Antibody prepared by the present invention is detected to the influenza viruses of tri- kinds of hypotypes of H5N1, H3N8, H7N9 using ELISA method
Reactivity.The result shows that cross reaction does not occur for antibody prepared by the present invention and H5N1, H3N8, H7N9 subtype influenza virus.
Nine, Western-blot is identified
The H3N2 canine influenza virus and recombinant proteins 20ul by denaturation treatment are taken, 12%SDS-PAGE point sample is added to
It, will be on protein delivery to pvdf membrane with BIO-RED transferring film instrument after conventional electrophoretic separation in hole；With diluted 5% defatted milk of TBST
Powder closes 1h, is washed film 3 times, every minor tick 10min with TBST, the antibody 1:1000 dilution prepared in the present invention is added, 37 DEG C incubate
1h is educated, the goat anti-mouse igg of 1:5000 times of diluted horseradish peroxidase-labeled is added after washing film 3 times, 1h is reacted, washes film 3
It is secondary, take ECL fluorogenic substrate (Pierce company, article No.: 37071) A liquid and each mL of B liquid 1, after mixing, by pvdf membrane impregnate wherein,
After incubation at room temperature 3-5 minutes, pvdf membrane surface liquid is dried with filter paper, and place it in darkroom, medical X exograph X is taken to cover
Lid after exposure 1 minute, successively develops, is fixed.The anti-H3N2 hypotype dog influenza of hybridoma H3N2-CIV-NP-2D10 preparation
There is single specific band in NP albumen, as a result as shown in Figure 1.Fig. 1 is hybridoma cell strain H3N2-CIV-NP- of the present invention
The monoclonal antibody of 2D10 is to H3N2 hypotype canine influenza virus immunoblotting as a result, it is about in conjunction with the molecular weight of band
60KDa.Wherein, swimming lane 1: recombinant proteins, swimming lane 2:H3N2 canine influenza virus, swimming lane M: albumen Marker.
Ten, Immunofluorescence test
Mdck cell in logarithmic growth phase is layered in the 6 orifice plates containing slide, every hole 2 × 105A cell, was cultivated
Night.After infecting 48h with 200 μ lH3N2 canine influenza virus, PBS is washed slide 3 times, each 5min.4% paraformaldehyde fixes cell
Afterwards, PBS is washed 3 times, and antibody prepared by the present invention is added in each 5min, 1:1000, and negative control adds antibody diluent, 4 DEG C of mistakes
Night.Goat anti-mouse igg secondary antibody (Zhong Shan Golden Bridge) the 1:100 dilution of FITC label, 37 DEG C of incubations are added in PBS after washing slide 3 times
1h, PBS are washed 3 times, and DAPI room temperature dyes 5min, and after PBS develops a film, inverted fluorescence microscope microscopic observation is simultaneously taken pictures.By this
Green fluorescence is observed in the mdck cell matter of the antibody dyeing of invention preparation, it was demonstrated that antibody prepared by the present invention can recognize day
Right NP albumen, as shown in Figure 2.Fig. 2 is the monoclonal antibody pair of hybridoma cell strain H3N2-CIV-NP-2D10 of the present invention
The result of H3N2 hypotype canine influenza virus immunofluorescence dyeing.H3N2 hypotype canine influenza virus infects mdck cell, and inoculation 48 is small
4% paraformaldehyde of Shi Houyong is fixed, and the goat-anti of H3N2-CIV-NP-2D10 monoclonal antibody and green fluorescence label is successively incubated for
Mouse secondary antibody, DAPI dyeing, fluorescence microscope microscopic observation result.Wherein Fig. 2A is the mdck cell core of DAPI dyeing, Fig. 2 B
It is the mdck cell matter dyed by H3N2-CIV-NP-2D10 antibody, Fig. 2 C is the coincidence pattern of Fig. 2A and Fig. 2 B.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (7)
1. a kind of hybridoma cell strain for secreting anti-H3N2 hypotype canine influenza virus nucleoprotein monoclonal antibody, is denoted as H3N2-
CIV-NP-2D10 was preserved in China typical culture collection center on August 12nd, 2016, and deposit number is CCTCC NO:
C2016155。
2. a kind of monoclonal antibody of anti-H3N2 hypotype canine influenza virus nucleoprotein, by hybridoma described in claim 1
Strain generates.
3. the monoclonal antibody of anti-H3N2 hypotype canine influenza virus nucleoprotein according to claim 2, which is characterized in that institute
Stating monoclonal antibody is IgG2b subclass.
4. the monoclonal antibody of anti-H3N2 hypotype canine influenza virus nucleoprotein according to claim 2, which is characterized in that institute
The light chain for stating monoclonal antibody is κ chain.
5. a kind of kit for detecting H3N2 hypotype canine influenza virus, anti-containing the described in any item monoclonals of claim 2-4
Body.
6. a kind of kit for detecting H3N2 hypotype canine influenza virus antibody, contains the described in any item Dan Ke of claim 2-4
Grand antibody.
7. the monoclonal antibody of the described in any item anti-H3N2 hypotype canine influenza virus nucleoprotein of claim 2-4 is identified in preparation
Application in the reagent of A type canine influenza virus NP albumen.
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