CN110240650A - The monoclonal antibody and its preparation method and application of anti-flounder rhabdovirus G-protein - Google Patents
The monoclonal antibody and its preparation method and application of anti-flounder rhabdovirus G-protein Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- G—PHYSICS
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
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Abstract
The invention discloses a kind of monoclonal antibodies of anti-flounder rhabdovirus G-protein, the monoclonal antibody is by title are as follows: hybridoma cell strain HIRRV-G-4D10, deposit number are as follows: CCTCC NO:C201869, depositary institution are as follows: China typical culture collection center, preservation date are as follows: the hybridoma secretion on March 29th, 2018.The monoclonal antibody proves to specifically bind with the virus of concentration through indirect enzyme-linked immunosorbent reaction experiment, micro virus neutralizing mensuration experimental result is shown, monoclonal antibody HIRRV-G-4D10 has neutralization activity to HIRRV, can significantly reduce the pathological effect of the EPC cell of infection flounder rhabdovirus.Immunoblotting combination mass spectral analysis is as the result is shown: the natural lefteye flounder Rhabdoviral G protein of 60 kDa of monoclonal antibody energy specific recognition of the invention.Indirect immunofluorescence shows that with the EPC cell of infection flounder rhabdovirus specific reaction can occur for monoclonal antibody HIRRV-G-4D10, and fluorescence signal is mainly distributed in cell membrane and cytoplasm.Monoclonal antibody of the invention can be used for preparing the immune formulation of anti-flounder rhabdovirus and the detection reagent of flounder rhabdovirus.
Description
Technical field
The present invention relates to a kind of monoclonal antibodies and preparation method thereof, and in particular to a kind of anti-lefteye flounder with neutralization activity
Monoclonal antibody of rhabdovirus and the preparation method and application thereof belongs to immunology and virology interleaving techniques field.
Background technique
Flounder rhabdovirus (HIRRV) is a kind of important viral cause of disease of aquaculture fish, has strong cause a disease
Property, which can infect a variety of sea water and fresh water economic fish such as lefteye flounder, sweetfish, stone flounder, perch, grayling.When water temperature is at 10 DEG C or so
The easy outbreak of epidemic of the sickly look can cause to infect the higher death rate of fish body, generate serious prestige to the healthy aquaculture of aquatic fish
The side of body.Therefore, the monoclonal antibody for developing specific recognition HIRRV can be applied to the quick diagnosis detection of disease, pathogenic mechanism is ground
Study carefully and the exploitation of disease-resistant biomolecule etc., there is important theory and practical application value.Flounder rhabdovirus gene
Group is made of a mononegavirale RNA, 5 kinds of structural proteins of codified, respectively nucleoprotein (N), phosphoprotein (P), stromatin
(M), glycoprotein (G) and RNA polymerase (L).Wherein, G-protein is the major structural protein for forming flounder rhabdovirus, in disease
Malicious surface forms tripolymer, it is in conjunction with cell-membrane receptor and mediate retroviral enters born of the same parents.The albumen contains multiple antigen sites, has
Very strong antigenicity can induce fish body and generate neutralizing antibody.
Thus, using flounder rhabdovirus G-protein as target antigen, its specific monoclonal antibody is developed, for screening viral neutrality
Monoclonal antibody and research HIRRV infection mechanism have even more important meaning.
Summary of the invention
The object of the present invention is to provide a kind of anti-flounder rhabdovirus monoclonal antibodies and its system for having viral neutralization activity
Preparation Method.
The purpose of the present invention is implemented by the following technical solutions: a kind of monoclonal of anti-flounder rhabdovirus G-protein is anti-
Body, the monoclonal antibody are by title are as follows: hybridoma cell strain HIRRV-G-4D10, deposit number are as follows: CCTCC NO:
C201869, depositary institution are as follows: China typical culture collection center, address: Wuhan City, Hubei Province Wuhan University, preservation date
Are as follows: the hybridoma secretion on March 29th, 2018.
It is located at flounder rhabdovirus G genetic fragment with the antigenic determinant that the monoclonal antibody is specifically bound
The polypeptide region of (61-1524 nt) gene coding.
A kind of preparation method of the anti-flounder rhabdovirus G-protein monoclonal antibody includes the following steps: firstly, benefit
CDNA is obtained after extracting the reversion of rhabdovirus total serum IgE with Trizol method, using cDNA product as template, PCR amplification G genetic fragment
(61-1524 nt) connects pET-28a vector construction recombinant expression plasmid pET-28a-G, is conducted into competent E.coli
Recombinant G protein is obtained by inducing expression, affinitive layer purification, Balb/ is immunized using the recombinant G protein of affinity purification as antigen
C small white mouse prepares hybridoma using cell fusion method, passes through microneutralization experiment and immunology detection screening technique, sieve
Choosing secretion has the hybridoma cell strain HIRRV-G- of anti-flounder rhabdovirus G-protein monoclonal antibody of viral neutralization activity
4D10 is expanded using conventional method and cultivates above-mentioned obtained hybridoma cell strain, cell culture supernatant is collected, after purified
Obtain the monoclonal antibody of anti-flounder rhabdovirus G-protein.
The immunology detection screening technique is: Dot-ELISA, transfers Western blot and is immunized indirectly glimmering
Light method.The wherein Dot-ELISA and the comprehensive determining monoclonal antibody of transfer Western blot and flounder rhabdovirus G egg
Necessary being is reacted in white specific binding；The transfer Western blot determines that the antigenic determinant of the monoclonal antibody is located at lefteye flounder G
The polypeptide region of genetic fragment (61-1524 nt) gene coding.
Monoclonal antibody can occur specifically monoclonal antibody of the invention with the virus of concentration as the result is shown through indirect enzyme-linked immunosorbent reaction experiment
Property combine, transfer immunoblotting and mass spectrometry results show: monoclonal antibody energy specific recognition lefteye flounder G gene of the invention
The natural lefteye flounder Rhabdoviral G protein that the recombinant protein and molecular weight of segment (61-1524 nt) gene coding are 60 kDa.
Indirect immunofluorescene assay shows that monoclonal antibody can be reacted with the EPC cell-specific after 24 h of infection, fluorescence signal
It is distributed in the cell membrane and cytoplasm that virus is felt.
Observed under inverted microscope, growth conditions good hybridoma division is vigorous, appearance is full, it is perfectly round,
Refractivity is strong, cell size is uniform, adherent good；The hybridoma has unlimited division and proliferation ability；This to grow fine is miscellaneous
It hands over oncocyte routine culture 2-3 days, culture medium is changed into yellow by pink, and the hybridoma point is contained in the culture medium
The a large amount of anti-flounder rhabdovirus G-protein monoclonal antibody secreted.
The monoclonal antibody of the anti-flounder rhabdovirus G-protein is preparing answering in anti-flounder rhabdovirus immune formulation
With.
The monoclonal antibody of the anti-flounder rhabdovirus G-protein is preparing answering in flounder rhabdovirus detection reagent
With.
Advantages of the present invention: anti-flounder rhabdovirus G-protein monoclonal antibody prepared by the present invention can be used for HIRRV sense
Dye blocks reagent exploitation and the conventional detection of HIRRV, has great application for the outburst of HIRRV in prevention aquatic fish
Value, and then ensured the safety of aquaculture.The preparation method is that obtaining recombination tooth by DNA recombinant expression
Flounder Rhabdoviral G protein prepares hybridoma using cell fusion, by micro- using recombinant protein as mice immunized with antigen
Amount neutralizes the measuring neutralization of flounder rhabdovirus G-protein monoclonal antibody, sieves using immunological detection method
Anti- flounder rhabdovirus G-protein monoclonal antibody is selected, such technology of preparing highway route design is novel, tight and reasonable.
Detailed description of the invention
Fig. 1 is the SDS-PAGE figure in the rhabdovirus recombinant G protein of expression in escherichia coli.Swimming lane M indicates standard scores
Son amount albumen；Swimming lane 1 indicates the negative control of no IPTG induction；Swimming lane 2 indicates the recombination pET-28a-G transfection of IPTG induction
Escherichia coli；Swimming lane 3 indicates the recombinant G protein of purifying.
Fig. 2 is the viral microneutralization measurement experiment figure of monoclonal antibody HIRRV-G-4D10.Rhabdovirus infection EPC is thin
After born of the same parents, HIRRV-G-4D10 and control group respectively indicate monoclonal antibody HIRRV-G-4D10 and myeloma cell's culture supernatant
With the cytopathy situation of different time points after virus incubation postoperative infection cell.
Fig. 3 is immunoblotting assay figure of the monoclonal antibody to recombinant G protein.Swimming lane M indicates standard molecular weight albumen；Swimming
Road 1 indicates the SDS-PAGE of the flounder rhabdovirus of purifying；Swimming lane 2: the lefteye flounder of monoclonal antibody HIRRV-G-4D10 and purifying
The immune response result of rhabdovirus；Swimming lane 3: negative control.
Fig. 4 is the 60kDa virus protein of the monoclonal antibody HIRRV-G-4D10 specific recognition of anti-flounder rhabdovirus
The mass spectrogram of matter.Sequence is and the consistent peptide fragment of rhabdovirus G sequence in box.
Fig. 5 is the EPC that infection HIRRV is detected using monoclonal antibody HIRRV-G-4D10 combination indirect immunofluorescence
Cellular immunofluorescence result.
Specific embodiment
Further illustrate the present invention with reference to the accompanying drawing and by specific embodiment.
Embodiment 1: building expression flounder rhabdovirus G-protein plasmid and protein expression
(1) with the total serum IgE of Trizol method extracting rhabdovirus.
(2) according to design of primers principle and the G gene of flounder rhabdovirus separation strains CNPo2015 of announcement a kind of
(GeneBank:KY363350), using primer-design software Primer5.0, in conjunction with the multiple cloning sites of pET28 (a) plasmid
Feature selects the insertion position of gene as a purpose at the restriction enzyme site of Bam H I and Xho I, designs flounder rhabdovirus G
Protein expression primer:
Upstream primer: 5 '-GAGAATTCCAAACCATCAA GCCTGGAG-3 '；
Downstream primer: 5 '-GAGTCGACTCGACTGGCGAGGTGGT-3 '.
(3) using the total serum IgE of extraction as template, first reverse transcription synthesizes the first chain of cDNA, then carries out standard PCR amplification, instead
Answer condition: 95 DEG C, 5 min；94 DEG C of denaturation 20s, 52 DEG C of annealing 30s, 72 DEG C of 1 min of extension, totally 30 recycle；Then 72 DEG C
Extend 10 min, PCR product carries out target gene after agarose gel electrophoresis detects, using DNA fragmentation QIAquick Gel Extraction Kit
Purification and recovery.
(4) by the pcr amplification product of plasmid pET28 (a) and purification and recovery use respectively restriction enzyme Bam H I and
Xho I carries out double digestion, is attached using T4 DNA ligase to digestion products, and plasmid pET28a-G is constructed after connection.It will
Recombinant plasmid transformed is to e. coli bl21 competent cell, bacterium colony to be grown, using T7 universal primer, using bacterium colony PCR
Method screens positive bacteria, and carries out sequence verification.
(5) bacterial strain of positive colony bacterium and the empty carrier containing pET28a is put into 37 in the LB liquid medium containing kanamycins
DEG C overnight shaking culture, next day by the bacterium solution of 1:100 dilution overnight incubation, continue culture to bacterium solution OD600When=0.4-0.6, add
Enter isopropylthiogalactoside (isopropyl β-D-thiogalactoside, IPTG) extremely final concentration of 1mmol/L, not
The bacterium solution for adding IPTG to induce continues after cultivating 6h as control, and thalline were collected by centrifugation carries out SDS-PAGE analysis.
The result shows that the bacterium solution albumen after induction is in relative molecular massMrThere is apparent band at=52 kDa, and does not lure
The bacterium solution albumen led is without respective strap (Fig. 1).And flounder rhabdovirus G-protein theoretical molecular weight and label protein molecular weight it
It is consistent with the destination protein molecular weight shown with PAGE gel.
(6) above method is used, is largely induced.Bacterium solution is collected, by somatic cells buffer solution B (50 mM Tris-
HCl, 1 mM EDTA, 100 mM NaCl, 1% NP-40, pH 8.0) be resuspended, be placed under condition of ice bath for multigelation 5 times into
8 min(Sonics & Materials of row ultrasonication；Amplitude 39%；Pulse on, 4 s；Pulse off, 1 s), and 1500
G is centrifuged 30 min and collects inclusion body precipitating；With the affinity column (HisTrap HP 1 ml, GE) of Ni ion chelating to recombination
Albumen is purified, and the destination protein for purifying acquisition detects purifying situation through SDS-PAGE, remaining to use PBS buffer solution (NaCl
137 mmol/L, KCl 2.7 mmol/L, Na2HPO410 mmol/L, KH2PO42 mmol/L, pH 7.2) it is resuspended, -80 DEG C
It saves backup.
The results show that it is the purpose band of 52 kDa, nothing that the recombinant protein after affinitive layer purification, which has a molecular weight,
Other obvious protein bands (Fig. 1).
Embodiment 2: the preparation of the anti-flounder rhabdovirus G-protein monoclonal antibody of mouse
One, it is immunized
Use the recombinant large-tooth flounder Rhabdoviral G protein of purifying as antigen.Immune to be divided into 4 progress, each immunization interval is 1 week,
Preceding is three times intraperitoneal injection, is for the last time tail vein injection:
(1) fundamental immunity: the recombinant protein and Freund's complete adjuvant equivalent (V/V) of 100 μ g purifying, which mix, is used as antigen；
(2) booster immunization: the recombinant protein and incomplete Freund's adjuvant equivalent (V/V) of 100 μ g purifying, which mix, is used as antigen；
(3) secondary booster immunization: the recombinant protein and incomplete Freund's adjuvant equivalent (V/V) of 100 μ g purifying, which mix, is used as antigen；
(4) amplification for merging first three days is immune: the recombinant large-tooth flounder Rhabdoviral G protein of 50 μ g purifying is as antigen.
Two, cell fusion
(1) preparation of immune spleen cell: cervical dislocation, which is put to death, is immunized mouse, after sterile taking-up spleen, crosses 200 mesh mesh screens, uses RPMI-
1640 solution are blown and beaten to form single cell suspension；1000rpm/ is centrifuged 5min, discards supernatant liquid, 10 mL RPMI-1640 of precipitating
Solution is resuspended, as far as possible suction tissue block, is placed on spare on one side；
(2) preparation of SP2/0 myeloma cell: taking -80 DEG C of SP2/0 cells frozen, and 37 DEG C of water-baths are instant；1000 r/min from
Supernatant is sucked out in 3 min of the heart, and 1mL GIT culture medium is added, is transferred in 24 orifice plates；After about cell covers with for 24 hours, divide hole；Wait divide
Supernatant is sucked out in hole to 16 holes, and every hole adds 1.5ml culture medium, and the cell in 4 holes is transferred to 25mm2In Tissue Culture Flask, CO at 37 DEG C2
Incubator culture.48 h or so enter logarithmic growth phase, and 12h replaces culture medium before merging, and select well-grown and form typical
It is in the SP2/0 cell of logarithm division stage；
(3) about 1.0 × 10 are taken8A splenocyte and 2.0 × 107A myeloma cell's suspension carries out cell fusion；
(4) myeloma cell of 4 bottles of logarithmic growth phases is taken out, waste liquid is sucked out in dropper, and 1640 culture medium of RPMI is added, blows repeatedly
Beating completely falls off cell；
(5) myeloma is mixed with immune spleen cell, and piping and druming mixes, 1200 rpm/min, 8 min；
(6) supernatant is sucked out in dropper as far as possible, flicks centrifuge tube bottom, and two kinds of cells is made to be sufficiently mixed into paste.It is placed in prior preparation
In 37 DEG C of good water-baths；
(7) 50% PEG1500 is placed in 37 DEG C of biochemical cultivation cases and is incubated, drawn 1 mL PEG1500, be slowly added into mixed
It closes on cell, side edged is gently mixed, and (is added in 90 s, slowly plus, side edged is slowly stirred, and bottom cell is allowed all to hang
Come, not blow and beat), water-bath stands 90 s, and timing is using 10 s as boundary；
(8) from CO2Incubator taking-up is incubated to the RPMI-1640 basic culture solution of 37 DEG C of 50Ml, is added dropwise 10 in 5 min
ML culture medium dilutes PEG.It is slowly added on fused cell with liquid-transfering gun along wall, side edged is gently mixed, and disperses cell mass；
(9) Adding Way: preceding 2min respectively adds 1mL, the 3rd, 4min add 1.5 min, 5min to add 5 mL.Timing is using 10s as boundary, and one
As will it is first slow after it is fast.40 mL basal mediums are added along tube wall, lid is tightened, blows afloat precipitating, mix cell.800rpm/ min
It is centrifuged 6 min；
(10) supernatant is abandoned, mixing is gently resuspended in the RPMI-1640 incomplete culture medium with 10 mL containing feeder cells.96 orifice plates are dripped,
2 drops/hole.37 DEG C of CO2Incubator culture；
(11) cell growth status is checked daily after merging and whether has pollution.After fused cell forms cell mass, culture is taken
Supernatant is screened.
Three, it screens and clones
(1) it screens: after fusion, starting to detect when hybridoma group grows to the hole floor space 1/3 of 96 well culture plates, use
Indirect enzyme-linked immunosorbent technology screening positive hybridoma cell.
It screens for the first time:
1. envelope antigen: the G-protein of purifying being diluted to 10 μ g/mL with carbonate coating buffer (pH 9.6) 1:10, is added 96
In hole elisa Plates (50 hole μ l/), 4 DEG C of coatings are overnight；
2. coating buffer is sucked out, washed with the phosphate buffer (PBST) containing 0.05% Tween-20, each 5min is washed three times；
3. the bovine serum albumin(BSA) (PBS matches) of 200 μ l 3%, 37 DEG C of closing 1h are added in every hole；
4. three times with the washing of 2. method；
5. ELISA Plate, 37 DEG C of incubation 1h are added by every 50 μ l of hole using Hybridoma Cell Culture supernatant as first antibody；
6. three times with the washing of 2. method；
7. the sheep anti-Mouse Ig(1:6000 of alkali phosphatase enzyme mark dilutes) enzyme mark is added by every 50 μ l of hole as secondary antibody
Plate, 37 DEG C of incubation 1h；
8. three times with the washing of 2. method；Then every hole is added 100 μ l 4- nitrophenols phosphate (pNPP) and applies liquid, and 5- is reacted in dark place
The NaOH of 50 μ l 2M is added in 20min, every hole, stablizes the i.e. available 405nm operation wavelength of 3-5min and measures OD value.
The label protein that bacterium colony to be coated with the empty carrier containing pET28a is expressed is as negative control, when survey wavelength is 405nm
Each hole absorbance value calculates the ratio between each experimental port and negative control absorbance value (P/N), and as P/N >=2.1, the hole is the positive.
Programmed screening:
1. envelope antigen: the virus that ultracentrifugation is concentrated and purified is added in 96 hole elisa Plates with the concentration in 1 hole μ g/
(50 hole μ l/), 4 DEG C of coatings are overnight.The EDTA solution processing of 1 mol/L is added, removes the influence of endogenous enzymes；
2. remaining step is the same as screening for the first time.
(2) it clones: the positive hybridoma cell detected being cloned using limiting dilution assay, steps are as follows:
1. cervical dislocation puts to death mouse, sterile taking-up thymus gland grinds on 200 mesh mesh screens, blows and beats to form list with RPMI-1640 solution
Cell suspension；
2. thymus cell suspension 1000rpm is centrifuged 3min, liquid, thymocyte precipitating 10ml RPMI-1640 are discarded supernatant
(containing 10% fetal calf serum), cell culture fluid was resuspended；
3. the cell in the positive cell hole to be cloned is counted with blood cell counting plate, it is then dilute with 10 times of gradients with culture solution
It releases, takes out 100 hybridomas, be put into thymus cell suspension；
4. cell suspension is blown and beaten uniformly with dropper, it is added drop-wise in 96 well culture plates, every 100 μ l of hole, average individual hole contains one
Hybridoma；
5. being put into CO2It is cultivated in incubator；
6. each hole Hybridoma Cell Culture supernatant is detected by indirect enzyme-linked immunosorbent technology after two weeks, gained positive colony hole
Hybridoma is cloned once again according to the above method, to guarantee to form monoclonal.
Four, it freezes
It takes and grows vigorous, the good hybridoma of form, cell suspension is made, 200g is centrifuged 5min, removes supernatant, add frozen stock solution
(+1 part of dimethyl sulfoxide of 9 parts of RPMI-1640 culture mediums), makes final cell density 5 × 106A/ml fills 1ml cell suspension
In 2ml cryopreservation tube, blind nut is tightened, then cryopreservation tube is packed into the capsule for filling cotton, is put in -80 DEG C of ultra low temperature freezers
Overnight after (8-12 hours), then immerse long-term preservation in liquid nitrogen.The growth that the present invention obtains is vigorous, the good 4 plants of hybridization of form
Oncocyte is respectively designated as HIRRV-G-3E5, HIRRV-G-1D2, HIRRV-G-6F3 and HIRRV-G-4D10.
Embodiment 3: has the screening of the anti-flounder rhabdovirus monoclonal antibody of viral neutralization activity
(1) EPC cell is cultivated in 96 orifice plates, grows to 90%；
(2) by Hybridoma culture supernatants (monoclonal antibody HIRRV-G-3E5, HIRRV-G-1D2, HIRRV-G-6F3,
HIRRV-G-4D10) contain 100 TCID with (50 μ L) in equal volume respectively50The flounder rhabdovirus diluent in/hole mixes, 20
Under the conditions of DEG C, it is incubated for 2 hours；
(3) virus after incubation is added on EPC cell monolayer with monoclonal antibody mixture, is incubated for 1 hour；
(4) it removes inoculum and the M199 culture medium containing 2%FBS is added, lefteye flounder bullet is incubated for myeloma cell's culture supernatant
Shape virus is set as control group, and experiment is in triplicate；
(5) after infection 1,2,3,4 and 5 day, the state of cell is observed daily by microscope.In shooting cytopathy situation
When, cell fixes 1 hour with the PBS containing 4% paraformaldehyde at room temperature, with 0.1% violet staining.
As a result, it has been found that monoclonal antibody HIRRV-G-4D10 and HIRRV is incubated compared with other three plants anti-flounder rhabdovirus G monoclonal antibodies
Infection EPC cell is educated, the lesion process of EPC cell can be obviously delayed, same time point cytopathy degree, which is substantially less than, to be compareed
Group shows that monoclonal antibody 4D10 has apparent HIRRV neutralization activity, but is unable to infection of the complete neutralization HIRRV to EPC, and other
Three plants of monoclonal antibodies do not show viral neutralization activity (Fig. 2) then.Secretion is had to the hybridoma of HIRRV neutralization activity monoclonal antibody
Cell strain send to China typical culture collection center and carries out preservation, title are as follows: hybridoma cell strain HIRRV-G-4D10 is protected
Hiding number are as follows: CCTCC-C201869, preservation date are as follows: on March 29th, 2018.
Embodiment 4: the transfer Western blot identification of monoclonal antibody of the present invention
(1) dodecyl sodium sulfate-polyacrylamide gel electrophoresis:
1. sample buffer of the equal proportion containing dodecyl sodium sulfate is added in the rhabdovirus of purifying, 3- is boiled in boiling water
5min；
2. will 1. processed sample be added in loading hole, 10 μ l of sample-adding product in every hole, under constant current conditions, with low electricity when starting
It flows (30-40mA), after sample after concentration glue partial concentration is into a line, high current (50-70mA), electrophoresis to bromophenol blue refers to
Electrophoresis can be stopped by showing when agent reaches bottom margin, take out gel；
3. one piece of clip (electric to turn with electrotransfer buffer with the nitrocellulose filter (0.22 μm of aperture) of running gel same size
Move buffer: 25mmol/L Tris-Base, 192mmol/L glycine, 20% methanol, pH 8.3) wetting, after being placed on electrophoresis
On gel.An angle is cut, on nitrocellulose filter to indicate the starting point of sampfe order.It is supported with the filter paper of wetting, it will
The filter paper of second piece of wetting is attached to the another side of gel film；By above-mentioned placement order, make blob of viscose and nitrocellulose filter and filter paper
Form a set of sandwich " sandwich "；
4. " gel sandwich " merging is filled in the electrophoresis tank of electrotransfer buffer, by nitrocellulose filter towards anode；Electricity
Swim constant current 200mA, is powered 5 hours；
5. transfer finishes, nitrocellulose filter is taken out.
(2) immunoblotting:
1. nitrocellulose filter is washed 10 minutes with PBS, then sets and closes 1h in 4% bovine serum albumin solution (PBS matches),
37 ℃；
2. being washed 3 times, every time 5 minutes with PBST；
3. nitrocellulose filter is placed in Hybridoma Cell Culture supernatant as first antibody, 37 DEG C are slowly shaken 1 hour；With
Myeloma cell's culture supernatant is incubated for nitrocellulose filter as negative control；
4. three times with the washing of 2. method；
5. nitrocellulose filter is added in the goat anti-mouse igg antibody (1:6000 dilution) of alkali phosphatase enzyme mark, 37 DEG C slow
It is slow to shake 1 hour；
6. three times with the washing of 2. method；
7. nitrocellulose filter is put into color development in alkaline phosphatase color development liquid (NBT-BCIP color development liquid), until color is clearly
Only；
8. being washed with deionized, to terminate reaction.It is dry between nitrocellulose filter is clipped in filter paper.Set dark place preservation.
As the result is shown: the flounder rhabdovirus that monoclonal antibody HIRRV-G-4D10 of the invention can be 60kDa with a molecular weight
Specific reaction occurs for specific protein, shows puce band, and negative control shows (Fig. 3) without band.
Embodiment 5: the mass spectral analysis of monoclonal antibody of the present invention
(1) specific with monoclonal antibody HIRRV-G-4D10 generation in transfer immunoblot experiment from being cut on SDS-PAGE glue
The protein band of the 60kDa flounder rhabdovirus of reaction；
(2) protein band cut being passed through into ABI5800(Applied Biosystems) mass spectrograph is analyzed by mass spectrometry.
Mass spectrometry results are shown: being lefteye flounder with the protein band that monoclonal antibody HIRRV-G-4D10 is immunoreacted
Rhabdoviral G protein, it was confirmed that monoclonal antibody HIRRV-G-4D10 of the invention can the natural lefteye flounder bullet shape disease of specific recognition
The G-protein (Fig. 4) of poison.
Embodiment 6: the indirect immunofluorescence method identification of monoclonal antibody of the present invention
(1) it is inoculated with flounder rhabdovirus with the ratio that the ratio of virus and cell quantity is 0.1, the cell after collecting infection for 24 hours,
PBS washed once；
(2) cell suspension drop is dripped 30 μ l, natural air drying on clean glass slide.It is put into acetone and fixes 15min, PBS is washed
It washs primary；
(3) 3% bovine serum albumin(BSA) (PBS matches) of glass slide is closed, 37 DEG C of incubation 1h；
(4) with hybridoma supematant (hybridoma culture fluids) for first antibody, 1h is incubated in 37 DEG C of wet box；
(5) glass slide is taken out, is washed three times with 0.01M PBS, each 5min；
(6) it using the goat anti-mouse antibody of marked by fluorescein isothiocyanate as secondary antibody, is added on cell sample, in 37 DEG C of wet box
45min is incubated for wash three times with (5) method；
(7) with 1% 4', 6- diamidino -2-phenylindone solution contaminates core, washes three times with (5) method；
(8) an anti-fluorescence decay quencher of drop is added dropwise on the cover slip, cell climbing sheet is tipped upside down on glass slide, it is aobvious to be inverted fluorescence
See whether fluorescence signal occur under micro mirror.
As the result is shown: monoclonal antibody HIRRV-G-4D10 of the present invention is specifically bound with flounder rhabdovirus, and fluorescence is presented
Positive signal, under 100 times of oil mirrors it can be seen that green florescent signal be distributed in virus infected cell cell membrane on and cell
In matter, and fluorescence signal (Fig. 5) is then not observed in negative control (non-infected cells).Illustrate monoclonal antibody HIRRV-G-
4D10 can the virion that is proliferated in EPC cell of specific recognition.
Those skilled in the art will appreciate that, within the scope of the present invention, above-described embodiment is carried out
Modification, it is all possible for adding and replacing, all without departing from protection scope of the present invention.
Claims (7)
1. a kind of monoclonal antibody of anti-flounder rhabdovirus G-protein, it is characterised in that: the monoclonal antibody is by title
Are as follows: hybridoma cell strain HIRRV-G-4D10, deposit number are as follows: CCTCC NO:C201869, depositary institution are as follows: Chinese Typical Representative culture
Object collection, address: Wuhan City, Hubei Province Wuhan University, preservation date are as follows: the hybridoma on March 29th, 2018 is secreted
's.
2. the monoclonal antibody of anti-flounder rhabdovirus G-protein as described in claim 1, it is characterised in that: the monoclonal
The antigenic determinant of antibody is located at the polypeptide region of lefteye flounder G genetic fragment (61-1524 nt) gene coding.
3. the monoclonal antibody of anti-flounder rhabdovirus G-protein as described in claim 1, it is characterised in that: the monoclonal
Resist the natural lefteye flounder Rhabdoviral G protein of 60 kDa of anti-energy specific recognition.
4. a kind of preparation method of the monoclonal antibody of anti-flounder rhabdovirus G-protein as described in claim 1, it is characterised in that
It includes the following steps: to obtain cDNA after extracting the reversion of rhabdovirus total serum IgE using Trizol method, using cDNA product as template,
PCR amplification G genetic fragment (61-1524 nt) connects pET-28a vector construction recombinant expression plasmid pET-28a-G, is led
Enter and obtain recombinant G protein through inducing expression, affinitive layer purification after competent E.coli, with the recombinant G protein of affinity purification
Balb/c small white mouse is immunized as antigen, hybridoma is prepared using cell fusion method, by viral microneutralization experiment and
Immunology detection screening technique, filtering out secretion has the monoclonal of the anti-flounder rhabdovirus G-protein of viral neutralization activity anti-
The hybridoma cell strain HIRRV-G-4D10 of body is expanded using conventional method and cultivates above-mentioned obtained hybridoma cell strain, is received
Collect cell culture supernatant, the monoclonal antibody of anti-flounder rhabdovirus G-protein is obtained after purified.
5. the preparation method of anti-flounder rhabdovirus G-protein monoclonal antibody as claimed in claim 4, it is characterised in that described
Immunology detection screening technique include Dot-ELISA, transfer Western blot and indirect immunofluorescence.
6. the monoclonal antibody of anti-flounder rhabdovirus G-protein as described in claim 1 is immunized prepare anti-flounder rhabdovirus
Application in preparation.
7. the monoclonal antibody of anti-flounder rhabdovirus G-protein as described in claim 1 is preparing flounder rhabdovirus detection examination
Application in agent.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608383A (en) * | 2021-01-08 | 2021-04-06 | 青岛农业大学 | Single-chain antibody for resisting paralichthys rhabdovirus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003155254A (en) * | 2001-09-06 | 2003-05-27 | Meiji Seika Kaisha Ltd | Dna vaccine for fish infected with hirame rhabdovirus |
-
2019
- 2019-06-04 CN CN201910481325.9A patent/CN110240650A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003155254A (en) * | 2001-09-06 | 2003-05-27 | Meiji Seika Kaisha Ltd | Dna vaccine for fish infected with hirame rhabdovirus |
Non-Patent Citations (4)
| Title |
|---|
| TANG,X ET AL.: "Hirame novirhabdovirus isolate CNPo2015 glycoprotein (G) mRNA, complete cds", 《GENBANK ACCESSION: KY363350.1》 * |
| 张家林等: "牙鲆原代肾细胞培养及其对牙鲆弹状病毒的易感性研究", 《中国海洋大学学报(自然科学版)》 * |
| 张小飞等: "牙鲆弹状病毒研究进展", 《江苏农业科学》 * |
| 阮红梅等: "鱼类弹状病毒分子生物学研究动态", 《中国水产科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608383A (en) * | 2021-01-08 | 2021-04-06 | 青岛农业大学 | Single-chain antibody for resisting paralichthys rhabdovirus |
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Application publication date: 20190917 |