CN110483638A - A kind of preparation method of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source - Google Patents
A kind of preparation method of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source Download PDFInfo
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- 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
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The present invention relates to a kind of preparation methods of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source, belong to antibody production techniques field.Preparation method of the present invention is by separating PBMCs out of immune A type FMDV inactivated vaccine ox body, use dyestuff mark A type FMDV 146S antigen, then PBMCs is dyed, it can be separated to the single B cell in conjunction with A type FMDV simultaneously by polychrome selected by flow cytometry apoptosis, and then obtain VH and VL gene, engineered antibody technology is expressed in conjunction with EXPI293, expression obtains the single-stranded genetic engineering antibody in A type FMDV specificity ox source.Preparation method of the present invention avoids the cell fusion screening process of traditional murine hybridoma technology complexity, has great importance to the identification of research A type FMDV host specificity antigen site, can lay the foundation for the development of aftosa molecular vaccine.
Description
Technical field
The present invention relates to antibody production techniques fields, and in particular to a kind of single-stranded gene work in anti-A type foot and mouth disease virus ox source
The preparation method of engineered antibody.
Background technique
Aftosa (foot-and-mouth disease, FMD) is the great animal epidemic for seriously endangering pig, Niu Yuyang.
Foot and mouth disease virus (FMDV) has seven serotypes, respectively A type, O-shaped, 1 type of Asia, c-type, the type of SAT1~3, wherein each blood
Clear type is again multiple topological types (VP1,85% amino acid identity) according to popular region division.China's Major Epidemic A type and
O-shaped FMDV, wherein A type FMDV is primarily present G1 the and G2 branch in the topological type of Asia in China.A type and O-shaped FMDV serum
It cannot provide in Cross immunogenicity or even same serotype between different topology type virus that there is also antigenic structures between type
Difference, good Cross immunogenicity cannot be provided to heterologous strain.Therefore, research A type FMDV antigenic structure is to vaccine virus
Strain great significance for design.Monoclonal antibody is undoubtedly the important tool for parsing antigenic structure.
Monoclonal antibody derives from B cell, and antibody germline gene VDJ segment is produced by gene rearrangement and somatic mutation
The raw single B cell clone of specificity, B cell are further differentiated into thick liquid cell, and final secretion generates monoclonal antibody specific.
It can be seen that antibody germline gene VDJ resets the diversity for determining antibody, however different plant species are in VDJ genetic fragment quantity
It is upper that there are great differences.It is reported that murine heavy chain IGH gene contains 101 V genetic fragments, 9 D genetic fragments, 4 J bases
Because of segment.There are about 20 functional V gene segments, 2 D genetic fragments and 1 J genetic fragments for pig heavy chain IGH gene.Mesh
Before, there are 12 V genetic fragments in Niu Chonglian IGH gene database, 23 D genetic fragments and 4J genetic fragment (http: //
www.imgt.org/IMGTveterinary/).Complementary determining region (CDRs) is the important of identification epitope in antibody molecule
Position, especially complementary determining region of heavy chain (HCDR3), influence affinity of antibody.About 10% sequence of heavy chain contains in ox antibody molecule
There is the HCDR3 of overlength, longest can be other species of about 60 amino acid, this characteristic, as not available for mouse and pig etc.
's.It is reported that epitope can be individually identified in Niu Chaochang HCDR3 structural domain.It in summary it can be seen, VDJ base between different plant species
Because of composition difference and CDR3 difference in length and K/L light chain composition ratio difference, caused antibody diversity, Shi Bike
It can lead to the generation of host specificity antigen site.Therefore, using natural reservoir (of bird flu viruses) source Identification of monoclonal, it identifies antigen site pair
Parsing A type FMDV antigenic structure and vaccine design has great importance.Currently, for studying A type FMDV antigenic structure
Monoclonal antibody be mainly mouse monoclonal antibody.However since ox is the natural infection host of FMDV, and mouse and ox
There are larger differences for B cell diversity, therefore generate A type FMDV specificity ox resource monoclonal antibody to parsing A type FMDV antigen
Structure, especially discovery FMDV host specificity antigen site are of great significance.Currently, there has been no A type FMDV specificity
The report of ox resource monoclonal antibody.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation sides of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source
Method.Preparation method of the present invention is simple, convenient, avoids the cell fusion screening of traditional murine hybridoma technology complexity
Process, can directly sort the single B cell of antigentic specificity by flow cytometer, and vivoexpression obtains single-stranded genetic engineering
Antibody has great importance to the identification of research A type FMDV host specificity antigen site, can be aftosa molecule epidemic disease
The development of seedling lays the foundation.
The present invention provides a kind of preparation methods of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source, including with
Lower step:
1) separating peripheral blood mononuclear cells from the bovine blood sample of immune A type FMDV inactivated vaccine；
2) ultrafiltration centrifugation is carried out to A type FMDV 146S antigen, the A type FMDV 146S antigen after being concentrated uses dye
Material dyes the A type FMDV 146S antigen after concentration, the antigen after dyeing is carried out ultrafiltration centrifugation, after being marked
A type FMDV 146S antigen；The dyestuff include FluoProbes 647H, Allophycocyanin, DyLight 633,
DyLight 650, Atto 633 or Cynaine Dye 5；
3) the A type FMDV 146S after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains is anti-
Former, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis A type FMDV specificity；
5) reverse transcription is carried out to the single B cell of A type FMDV specificity that step 4) obtains, is expanded after obtaining cDNA,
Obtain the heavy chain variable region gene and light-chain variable region gene of the single B cell IgG antibody of A type FMDV specificity；
6) heavy chain variable region gene and light-chain variable region gene obtained step 5) passes through the flexibility such as SEQ ID NO.1
Linker connection, the N-terminal of gene upon connection introduce the signal peptide sequence as described in SEQ ID NO.2, introduce in C-terminal
Flag label and His label carry out gene order optimization with reference to Homo sapiens (Human) codon, base after optimization
Because introducing KOZAK sequence before initiation codon, it is inserted into pcDNA3.4 expression vector, obtains ox source single-chain antibody expression vector；
7) step 6) is obtained into the expression that single-chain antibody expression vector transfection cell in ox source carries out antibody；
The step 1) and 2) the not restriction of chronological order.
Preferably, step 1) the bovine blood sample is the bovine blood sample of immune 14~30d of A type FMDV inactivated vaccine
This.
Preferably, step 2) the ultrafiltration centrifugation are as follows: be centrifuged using 4000 rpm of super filter tube of interception 100kDa
10min replaces the antigen after antigen or dyeing into PBS buffer solution.
Preferably, the step 4) sorting are as follows: the 96-PCR orifice plate that 10 μ L lysates are contained in every hole is put into sorting
Cabin is arranged every hole and sorts 1 cell pattern, loading；It draws door and irises out lymph and monocyte, be arranged according to FSC-A and FSA-H
Adhesion cells are excluded, diagonal line single cell is irised out；To CD21+IgM-A-FMDV+Cell is sorted.
Preferably, the step 5) amplification includes nested PCR amplification method.
Preferably, step 6) is inserted into pcDNA3.4 expression vector using Not1 and Nhel restriction enzyme site；
Preferably, the step 7) cell includes Expi293 suspension cell.
The present invention provides a kind of preparation methods of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source.The present invention
The preparation method uses dyestuff mark A type FMDV 146S by separating PBMCs out of immune A type FMDV inactivated vaccine ox body
Antigen, dye marker antigen of the present invention will not damage antigen particles, can guarantee 146S antigen particles integrality.
Then PBMCs is dyed, it is thin to be separated to the single B in conjunction with A type FMDV simultaneously by polychrome selected by flow cytometry apoptosis
Born of the same parents.In conjunction with monoclonal antibody gene sequencing, VH and VL gene is obtained respectively, passes through flexible linker
" GGGGSGGSGGGGSGGS " connection and EXPI293 express engineered antibody technology, and expression obtains A type FMDV specificity Niu Yuandan
Chain gene engineered antibody verifies preparation method of the present invention through ELISA, WB and immunofluorescence experiment and successfully obtains anti-A type FMDV spy
Anisotropic single-stranded genetic engineering antibody.The antibody gene diversity that preparation method of the present invention is prepared is good, high-efficient,
Full host (ox) source, needs cell concentration few；And the preparation method is simple, convenient, avoids traditional murine hybridoma
The cell fusion screening process of technology complexity can directly sort the single B cell of antigentic specificity, body by flow cytometer
Outer expression obtains single-stranded genetic engineering antibody.The screening technique has the identification of research A type FMDV host specificity antigen site
There is great importance, can lay the foundation for the development of aftosa molecular vaccine.
Detailed description of the invention
Fig. 1 prepares schematic diagram for the single-stranded genetic engineering antibody in ox source provided by the invention；
Fig. 2 is the A type FMDV antigen Electronic Speculum observation (30000 times of amplification) of fluorochrome label provided by the invention；
Fig. 3 is flow cytometer showed result provided by the invention；
Fig. 4 is IgG antibody variable region gene PCR amplification provided by the invention；
Fig. 5 is the single-stranded genetic engineering antibody in the type FMDV ox source A that purifying provided by the invention obtains；
Fig. 6 is IFA experimental result provided by the invention；
Fig. 7 is indirect ELISA experimental result provided by the invention；
Fig. 8 is WB experimental result provided by the invention.
Specific embodiment
The present invention provides a kind of preparation methods of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source, including with
Lower step:
1) separating peripheral blood mononuclear cells from the bovine blood sample of immune A type FMDV inactivated vaccine；
2) ultrafiltration centrifugation is carried out to A type FMDV 146S antigen, the A type FMDV 146S antigen after being concentrated uses dye
Material dyes the A type FMDV 146S antigen after concentration, the antigen after dyeing is carried out ultrafiltration centrifugation, after being marked
A type FMDV 146S antigen；The dyestuff include FluoProbes 647H, Allophycocyanin, DyLight 633,
DyLight 650, Atto 633 or Cynaine Dye 5；
3) the A type FMDV 146S after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains is anti-
Former, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis A type FMDV specificity；
5) reverse transcription is carried out to the single B cell of A type FMDV specificity that step 4) obtains, is expanded after obtaining cDNA,
Obtain the heavy chain variable region gene and light-chain variable region gene of the single B cell IgG antibody of A type FMDV specificity；
6) heavy chain variable region gene and light-chain variable region gene obtained step 5) passes through the flexibility such as SEQ ID NO.1
Linker connection, the N-terminal of gene upon connection introduce the signal peptide sequence as described in SEQ ID NO.2, introduce in C-terminal
Flag label and His label carry out gene order optimization with reference to Homo sapiens (Human) codon, base after optimization
Because introducing KOZAK sequence before initiation codon, it is inserted into pcDNA3.4 expression vector, obtains ox source single-chain antibody expression vector；
7) step 6) is obtained into the expression that single-chain antibody expression vector transfection cell in ox source carries out antibody；
The step 1) and 2) the not restriction of chronological order.
Present invention separating peripheral blood mononuclear cells from the bovine blood sample of immune A type FMDV inactivated vaccine.In this hair
In bright, the bovine blood sample is the bovine blood sample of immune 14~30d of A type FMDV inactivated vaccine, is more selected as immune 21d
Bovine blood sample.In the present invention, the blood sample is preferably ox peripheral blood.The present invention is to the single core of the peripheral blood
The separation method of cell is not particularly limited, using conventional separation methods well known to those skilled in the art.
The present invention carries out ultrafiltration centrifugation to A type FMDV 146S antigen, and the A type FMDV 146S antigen after being concentrated makes
The A type FMDV 146S antigen after concentration is dyed with dyestuff, the antigen after dyeing is subjected to ultrafiltration centrifugation, is marked
A type FMDV 146S antigen afterwards；The dyestuff includes FluoProbes 647H, Allophycocyanin, DyLight
633, DyLight 650, Atto 633 or Cynaine Dye 5.In the present invention, the ultrafiltration centrifugation is preferred are as follows: uses and cuts
The super filter tube 4000rpm of allowance 100kDa is centrifuged 10min, and the antigen after antigen or dyeing is replaced into PBS buffer.In
In the present invention, before dyeing, antigen is preferably concentrated into 1mg/ml, antigen concentration to be marked is in 1mg/ml, label
Efficiency highest, while not only can guarantee that fluorescein will not damage antigenic structure, but also can guarantee can on each antigen particles
Fluorescein molecule on label.The present invention is not particularly limited the method for the dyeing, is made using the corresponding routine of corresponding dyestuff
With method.The present invention is not particularly limited to the source of the dyestuff, it is preferable to use Lightning-LinkTM Rapid
FluoProbes 647H labelling kit (Innova Biosciences, San Diego, USA), Lightning-LinkTM
Allophycocyanin labelling kit (Innova Biosciences, San Diego, USA), Lightning-LinkTM
633 labelling kit of DyLight (Innova Biosciences, San Diego, USA), Lightning-LinkTM
650 labelling kit of DyLight (Innova Biosciences, SanDiego, USA), Lightning-LinkTMAtto
633 labelling kits (Innova Biosciences, San Diego, USA) or Lightning-LinkTM Cynaine Dye
5 labelling kits (Innova Biosciences, San Diego, USA).A type FMDV 146S antigen after being marked
Afterwards, the present invention is preferably added to isometric 100% glycerol and saves.
After A type FMDV 146S antigen after obtaining peripheral blood mononuclear cells and label, the present invention is single by peripheral blood
Nucleus is mixed with A type FMDV 146S antigen, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence antibody of mouse after label,
Cell after being dyed.In the present invention, by the way that the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence antibody of mouse is added,
CD21 is irised out for drawing door+IgM-B cell group, being then sorted in this group of cells can A type FMDV 146S after binding marker
The single B cell of the B cell of antigen, as antigentic specificity.
After cell after being dyed, the present invention is thin using the single B of polychrome selected by flow cytometry apoptosis A type FMDV specificity
Born of the same parents.The present invention is not particularly limited the model of the polychrome flow cytometer, and present invention preferably uses BD FACSAria II
U flow sorter, when using II u flow sorter of BD FACSAria, preferably instrument parameter is arranged by the present invention are as follows: spray
Mouth size: 100 μm；Sorting mode: single cell mode；Separation velocity: 10000 cells/second；Amplitude: 20psi；Oscillation frequency:
30kHz.The above parameter setting closes Sweat button after adjusting, and using Accudrop, (article No.: 642412) delay microballoon is executed
Calculate liquid delay time.In the present invention, the sorting is preferred are as follows: every hole is contained the 96-PCR orifice plate of 10 μ L lysates
It is put into sorting cabin, every hole is set and sorts 1 cell pattern, loading；Draw door iris out lymph and monocyte, according to FSC-A with
FSA-H setting excludes adhesion cells, irises out diagonal line single cell；To CD21+IgM-A-FMDV+Cell is sorted.At this
In invention, before sorting, the hole 96-PCR Board position in sorting cabin is preferably adjusted, until sorting cell accurately falls into plate hole centre
(by using 96 orifice plates for being stained with sealed membrane, 60 cell patterns of every hole sorting is set and are adjusted, it is ensured that cell can sort
To every hole center).
After sorting obtains the single B cell of A type FMDV specificity, the present invention carries out the single B cell of A type FMDV specificity
Reverse transcription is expanded after obtaining cDNA, obtains the heavy chain variable region gene of the single B cell IgG antibody of A type FMDV specificity
And light-chain variable region gene.The present invention is not particularly limited the reverse transcription method, using conventional reverse transcription method.
In the present invention, the amplification preferably includes nested PCR amplification method.Present invention preferably employs nucleotide sequence such as SEQ ID
Primer shown in NO.3~10 is expanded.It is specific as shown in table 1.
The primer of the amplification of table 1 ox IgG VH and VL gene
Degeneracy base annotation in sequence, S=C or G, Y=C or T, R=A or G.
After obtaining heavy chain variable region gene and the light-chain variable region gene of the single B cell IgG antibody of A type FMDV specificity,
The present invention connects heavy chain variable region gene with light-chain variable region gene by the flexible Linker of such as SEQ ID NO.1, even
The N-terminal of gene after connecing introduces the signal peptide sequence as described in SEQ ID NO.2, introduces Flag label and His label in C-terminal,
Gene order optimization is carried out with reference to Homo sapiens (Human) codon, is introduced before gene start codon after optimization
KOZAK sequence (GCCACC, SEQ ID NO.11), is inserted into pcDNA3.4 expression vector, obtains the expression of ox source single-chain antibody
Carrier.In the present invention, it is preferred to be inserted into pcDNA3.4 expression vector using Not1 and Nhel restriction enzyme site.
After obtaining ox source single-chain antibody expression vector, the present invention preferably transfects ox source single-chain antibody expression vector
The expression of cell progress antibody.In the present invention, the cell includes Expi293 suspension cell.Specifically, the present invention is preferred
Antibody is expressed by transiently transfecting EXPI293 suspension cell, by every 107Plasmid is added in the ratio of a 1 μ g plasmid of cell, is added
250μL OptiPROTMSFM culture medium is diluted, while taking equivalent OptiPROTMThe dilution of SFM culture medium
ExpiFectamineTM293 transfection reagents then mix the plasmid diluted and transfection reagent, and room temperature acts on 5min.
Combined with specific embodiments below to a kind of single-stranded genetic engineering in anti-A type foot and mouth disease virus ox source of the present invention
The preparation method of antibody is further described in detail, and technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
1.FMDV antigenic mark scheme
1.1A type FMDV 146S antigenic mark scheme
Use Lightning-LinkTMRapid FluoProbes 647H labelling kit (Innova
Biosciences, San Diego, USA) label A type FMDV 146S antigen.First with interception 100kDa super filter tube A type
FMDV 146S antigen is replaced into PBS buffer solution.4000rpm is centrifuged 10min, concentrated antigen to 1mg/ml (OD260=7.6 about
Equal to 1mg/ml).It takes 100 μ L146S antigens that 10 μ L LL-modifierreagent are added, mixes gently, then therefrom draw
100 μ L are added to a pipe Lightning-LinkTMMix reactive dye, gently aspirates and powder is resuspended, room temperature (20~25 DEG C)
Effect 3 hours.10 μ L LL-quencherreagent are added, act on 30 minutes, to terminate reaction.Then 100kDa ultrafiltration is used
Pipe centrifugation, 4000rpm are centrifuged 10min, the antigen after label are replaced into PBS buffer solution.It is sweet to be added isometric 100%
Oil, -70 DEG C of preservations.Antigen after label is named as A-FMDV 146S-FluoProbes 647H.(also can be used can quilt
Other fluorescent dyes of 633 lasers excitation, such as Lightning-LinkTM Allophycocyanin labelling kit
(Innova Biosciences, San Diego, USA), 633 labelling kit of Lightning-LinkTM DyLight
(Innova Biosciences, San Diego, USA), 650 labelling kit of Lightning-LinkTM DyLight
(Innova Biosciences, San Diego, USA), 633 labelling kit (Innova of Lightning-LinkTMAtto
Biosciences, San Diego, USA), 5 labelling kit (Innova of Lightning-LinkTM Cynaine Dye
Biosciences, San Diego, USA).)
The dyeing of 1.2 Electronic Speculum
To confirm that above-mentioned fluorochrome label A type FMDV antigen particles is selected not cause brokenly antigen particles integrality
It is bad, therefore negative staining and transmission electron microscope observing are carried out to the antigen after label.4 microlitres of antigen after taking label are gently added copper
On the net, room temperature acts on 2min, then laterally blots sample with filter paper.Then sample is clamped in 1% phosphoric acid tungsten dyeing liquor with tweezers
In carry out shuttle dyeing, using 3 drop methods, every drop dye liquor acts on 10 seconds, then draws dye liquor with filter paper, dry, and Electronic Speculum is observed.
2. polychrome streaming Staining Protocol
Immune A type FMDV vaccine 14 days ox peripheral bloods are taken, lymphocyte separation medium (density=1.083g/ is used
ML) therefrom Separation of Bovine PBMCs.
(1) 10 are taken7A above-mentioned ox PBMCs cell is resuspended in 200 μ L cell sorting liquid, and 0.5 μ g A-FMDV is added
146S-FluoProbes 647H antigen, the anti-ox CD21-RPE fluorescence antibody (Bio-Rad, USA) of 2 μ g mouse and the 1 anti-ox of μ g mouse
IgM-FITC fluorescence antibody (Bio-Rad, USA), acts on 25min on ice.
(2) it takes and A-FMDV 146S-FluoProbes 647H antigen Dyeing pipe is not added as the control that subtracts one.It is arranged simultaneously
Dan Yangguan, for adjusting compensation.Cell sorting liquid washes cell twice, 400 × g, 4 DEG C of centrifugation 5min.
(3) cell is resuspended in 500 μ L cell sorting liquid, room temperature acts on 5min, is protected from light on ice, and machine sorts in preparation
Cell.
3. sorting the single B cell of A type FMDV specificity
Use the single B cell of II u flow sorter of BD FACSAria sorting FMDV specificity.Parameter is arranged in instrument,
Jet size: 100 μm；Sorting mode: single cell mode；Separation velocity: 10000 cells/second；Amplitude: 20psi；Concussion frequency
Rate: 30kHz.The above parameter setting closes Sweat button after adjusting, and using Accudrop, (article No.: 642412) delay microballoon is held
Row calculates liquid delay time.Then the hole 96-PCR Board position in sorting cabin is adjusted, until sorting cell is accurately falling into plate hole just
Center (can be arranged 60 cell patterns of every hole sorting and be adjusted) by using 96 orifice plates for being stained with sealed membrane.It sets above
After the completion of setting, the 96-PCR plate containing 10 μ L lysates be put into sorting cabin, 1 cell sorting mode is set, start on
Sample.It draws door and irises out lymph and monocyte, adhesion cells are excluded according to FSC-A and FSA-H setting, it is single thin to iris out diagonal line
Born of the same parents.Sorting and CD21+IgM-A-FMDV+The single B cell of cell, as A type FMDV specificity.
4. N single B cell source IgG antibody variable region gene amplification
4.1 unicellular cDNA molecule preparations
After the completion of sorting, 1 μ L terminate liquid is added in every hole, and room temperature acts on 5min, terminates reaction.Then 4 μ L are added in every hole
SuperScriopt VILO mix solution and 6 μ L DNase/RNase-free water are mixed.1500rpm, 4 DEG C of centrifugations
5min.PCR instrument carries out post transcription cloning.
Reaction condition such as table 2 is set.
2 post transcription cloning condition of table
CDNA-20 DEG C of storage of acquisition, is used for subsequent PCR amplification.
The PCR amplification of 4.2 unicellular cDNA
Using primer shown in table 1, take nested PCR method to the heavy chain variable region of the single B cell IgG antibody of ox
(VH) gene and lambda light chain variable region (VL) gene.
First round PCR reaction system
The first round reaction system such as table 3 for expanding IgG VH and VL gene, in addition to primer is different with template, remaining ingredient
Is carried out according to the system
3 first round of table PCR reaction system
Because annealing temperature is different, response procedures involved in different primers are different, specific response procedures such as table 4
With table 5:
Table 4IgG VL gene first round amplification program
Table 5IgG VH gene first round amplification program
Second wheel PCR reaction system
The second wheel reaction system of IgG VH and VL gene is expanded, and except (template is first round PCR for primer and template
Amplified production) it is different, remaining ingredient is carried out according to system as shown in table 6:
Table 6 second takes turns PCR reaction system
The amplification program of second wheel PCR amplification IgG VH and VL gene is identical, and response procedures are as shown in table 7.
The amplification program of 7 IgG VH and VL gene of table
The sequencing of 4.3PCR product
The PCR product for taking 5 μ L second wheel amplification carries out electrophoresis with 1.5% Ago-Gel, observes amplification.Success
Remaining 45 μ L product is carried out DNA sequencing, uses DNASTAR and SnapGene software by the sample for amplifying VH and VL gene
Analysis compares sequencing result.
The preparation of 5 Ns of source single-chain antibody expression vectors
The building of 5.1 Ns of source single-chain antibody expression vectors
Ox IgG antibody variable region VH and the VL gene of amplification is passed through into flexibility Linker " GGGGSGGSGGGGSGGS "
(SEQ ID NO.1) connection, and ox source IgG heavy chain antibody secretion signal peptide sequence is introduced in fusion N-terminal
" MNPLWTLLFVLSAPRGVLS " (SEQ ID NO.2) introduces Flag label and 6 × His label in C-terminal, in favor of detection and
Purifying carries out gene order optimization then referring to Homo sapiens (Human) codon, and before gene start codon
It introduces KOZAK sequence " GCCACC " (SEQ ID NO.11), finally synthesis gene is inserted by Not1 and Nhel restriction enzyme site
To pcDNA3.4 expression vector (Thermo scientific, USA).Sequent synthesis and vector construction trust money only intelligence biology skill
Art company is synthesized.
5.2 endotoxin-free plasmids are extracted
Using a large amount of extraction agent boxes of endotoxin-free plasmid (TIANGEN, Beijing, China), illustrate in strict accordance with kit
Book carries out endotoxin-free plasmid preparation to the antibody expressing plasmid of building.
6. the expression and purification of the single-stranded genetic engineering antibody in N source
Antibody is expressed by transiently transfecting EXPI293 suspension cell, by every 107Matter is added in the ratio of a 1 μ g plasmid of cell
250 μ L OptiPRO are added in grainTMSFM culture medium is diluted, while taking equivalent OptiPROTMThe dilution of SFM culture medium
ExpiFectamineTM293 transfection reagents then mix the plasmid diluted and transfection reagent, and room temperature acts on 5min.It should
Mixture is slowly added to 1.5 × 108In a EXPI293 cell.EXPI293 cell is placed in 37 DEG C of constant temperature suspension cultures after transfection
Case suspends after culture 18h, adds 150 μ L Expi293TMEnhancer and 6mL EXPI293TMFeed.37 DEG C are continued to cultivate
After 9d, it is centrifuged 30min by 10000 × g, collects cells and supernatant, is filtered through 0.22 μm of filter, subsequent antibody purification is used for.
Antibody purification is carried out on AKAT protein purification instrument using HiTrap TALON pillar, is eluted using the PBS liquid of 250mM imidazoles
Antibody is then charged into bag filter and is placed in PBS liquid and dialyses 3 times, each 3h.Antibody after dialysis is carried out dense with PEG 8000
Contracting carries out SDS-PAGE electrophoretic analysis after concentration.
The activity verifying of the single-stranded genetic engineering antibody in the type FMDV ox source 7.A
7.1 indirect immunofluorescences (indirect immunofluorescence assay, IFA) test
BHK-21 cell is accessed in 24 orifice plates, when cell grows to 80%, in the ratio of MOI=1 by O-shaped FMDV
(O/HN/CHA93 strain) and A type FMDV (A/GDMM/CHA/2013 strain) is inoculated with 24 porocyte culture plates respectively, sets simultaneously
It sets and does not connect malicious normal cell controls, after being incubated for 4h in 37 DEG C of cell incubators, collect supernatant inactivation treatment.Then -20 DEG C are added
The fixed cell of Jia Chun ︰ acetone (1:1) fixer of pre-cooling, room temperature act on 20min.PBS is washed cell 3 times, by 5 μ g/mL concentration
The antibody of purifying, 37 DEG C of incubation 1h are added.PBS is washed 3 times, and the fluorescein label rabbit-anti ox IgG-FITC of working concentration is added
(Thermo Scientific, USA), 37 DEG C of incubation 30min.Then DAPI dyestuff is added, room temperature acts on 10min.PBS is washed carefully
Born of the same parents 5 times, fluorescence microscope fluorescence signal simultaneously photographs to record.
7.2 indirect ELISA
With PBS 1 μ g/mL will be diluted to by A type FMDV (A/GDMM/CHA/2013 strain) antigen after purification, by 100 μ L/
Hole, 4 DEG C of overnight coated elisa plates；After PBST washes 5 times, 1% gelatin closes 1h, after PBST washes 5 times, drying；Antibody to be checked is from 1 ︰
100, which start 2 times, is serially diluted, and is added in ELISA Plate by every 100 μ L of hole, 37 DEG C of incubation 1h；PBST is washed 5 times, and HRP is added in every hole
Anti- His tag antibody (1 ︰ 10000) 100 the μ l, 37 DEG C of incubation 1h of label；PBST is washed 5 times, and 100 μ L TMB developing solutions are added,
Color development at room temperature 10min；Terminate liquid color development stopping is added, with the light absorption value (D at microplate reader measurement 450nm450).S/C.O mode
Statistics, S are sample D450Value, C.O, that is, Cut Off value (positive decision content), (N is negative control D to C.O=2.1 × N450Value, when
When N is less than 0.05 based on 0.05), S/C.O >=1 is determined as the positive, and S/C.O < 1 is determined as feminine gender.
7.3 virus neutralization experiment
It is carried out using the single-stranded genetic engineering antibody in the ox source arrived of the A type FMDV (A/GDMM/CHA/2013 strain) to screening
Virus neutralization experiment.Steps are as follows for specific experiment:
A) 50 μ L domain antibodies to be checked are added in every hole, and doubling dilution is carried out in 96 orifice plates.Then, every hole is added 100 μ L and contains
100TCID50FMDV, 37 DEG C of effect 1h.Setting contains 10,100 and 1000 TCID simultaneously50Control wells (do not heal
Final proof sheet).
B) 100 μ L are added containing 5 × 10 in every hole4The complete medium of a BHK21 cell is put into 37 DEG C containing 5%CO2Culture
Case acts on 72h.
C) cell liquid is discarded supernatant, the fixer (methanol: acetone=1:1) of pre-cooling, -20 DEG C of fixed 20min are added.
D) fixer is discarded, every hole is added 100 μ L crystal violet solutions and is dyed.After 30min, 96 orifice plates, observation are rinsed
Ox source single-chain antibody minimum concentration (i.e. half effective inhibition concentration, the IC of the non-lesion of 50% cell50) the viral energy of instruction neutralization
Power, unit μ g/mL.Make IC50> 50 μ g/mL are determined as non-neutralization and lived by critical value when equal to 50 μ g/mL as neutralization
Property；50 μ g/mL of < is determined as with neutralization activity
7.4 western blots (WesternBlot, WB) test
It takes A type FMDV (A/GDMM/CHA/2013 strain) antigen containing about 1 μ g to carry out SDS-PAGE electrophoresis, then will divide
From protein band be transferred to cellulose nitrate (NC) film, with the TBST buffer blind 2h containing 5% skimmed milk power, after washing,
With the TBST buffer dilution antibody containing 5% skimmed milk power to working concentration (2 μ g/mL), it is incubated at room temperature 2h, after TBST washes film,
The anti-His tag antibody (1 ︰ 4000) that HRP label is added is incubated at room temperature 1h, and after TBST washes film, ECL chemiluminescence bottom is added
Object, pressure X-ray is exposed imaging in darkroom.
Experimental result:
1, the preparation flow of the single-stranded genetic engineering antibody in A type FMDV specificity ox source
The single-stranded genetic engineering antibody in ox source to prepare schematic diagram as shown in Figure 1.
2, the A type FMDV antigen particles integrality of fluorochrome label
After negative staining shown in Electronic Speculum observation result figure 2, A type FMDV antigen (A/GDMM/CHA/2013 strain) is in fluorescent dye
146S particle size is uniform after label, 20~30nm of diameter, and shape is intact.Morphologic observation is the result shows that the dye marker antigen
Antigen particles will not be damaged, can guarantee 146 antigen particles integralities, be consequently adapted to be sorted as bait antigen
Single B cell.
3, the single B cell of A type FMDV specificity is successfully obtained
Fig. 3 is the single B cell of airflow classification A type FMDV specificity, wherein A: machine on PBMCs after dyeing irises out state
Good P1 group；The shooting of B:P1 cell colony, irises out diagonal line single cell, excludes adhesion cells；C: CD21 is irised out+IgM-B
Cell；D: the FMO control of fluorescent antigen is not added；E: normal dyeing sample；Each cell colony ratio in F:FMO check sample
Cell (about 1,000,000 cells of record)；G: each cell colony ratio of normal dyeing sample (about 1,000,000 cells of record).
Flow cytometer showed result such as Fig. 3 shows that A type FMDV specific b cells group (Fig. 3 E) is high-visible, is present in CD21+
IgM-Cell colony, account for about total PBMCs ratio be 106/1000000.Compared to subtracting one, control (FMO) sample (is not added glimmering
The A type FMDV antigen of cursor), the mono- positive B-cells of A type FMDV (Fig. 3 D, P4 group) are about 4/1000000 (Fig. 3 G), are passed through
To positive sample Guan Zhong P4 group (Fig. 3 E, CD21+IgM-A-FMDV+B cell) circle door sorting, using unicellular sorting mode,
Success sorts the single B cell for obtaining A type FMDV specificity.
4, the acquisition of the single B cell ox IgG variable region gene of A type FMDV specificity
Fig. 4 is IgG antibody variable region gene PCR amplification, wherein Fig. 4-1:IgG heavy chain variable region PCR product electrophoresis；Figure
4-2:IgG light chain variable region PCR product electrophoresis.
Nested PCR product agarose gel electrophoresis analyze result as shown in figure 4, IgG VH segment 450 bp-500bp it
Between there is high-visible band (Fig. 4-1)；There is clear band (Fig. 4-2) at 450bp in IgG VL segment.Sequencing result is logical
Cross the comparison of Ig BLAST database, it was demonstrated that VH the and VL segment that nested PCR amplification obtains is the IgG antibody variable region gene of ox
Sequence.
5, the single-stranded genetic engineering antibody molecule in ox source that successful expression obtains
Fig. 5 is the single-stranded genetic engineering antibody in the type FMDV ox source A that purifying obtains, wherein M represents albumen marker, 1-5
Represent the single-stranded genetic engineering antibody 1,2,3,4,5 in ox source that purifying obtains.
SDS-PAGE the results show that the cell conditioned medium of expression antibody after affinitive layer purification, electrophoresis result and expected one
It causes, purpose band occurs at 30-35kDa in single-stranded genetic engineering antibody 1,2,3,5, is consistent with expection.Single-stranded genetic engineering is anti-
Body 4 contains overlength complementary determining region of heavy chain 3 (HCDR3), therefore occurs purpose band at 35-40kDa, is consistent with expection.This
A little results illustrate that this method successful expression and can be purified into the single-stranded genetic engineering antibody molecule in ox source.
6, the single-stranded genetic engineering antibody in anti-A type FMDV ox source is successfully obtained
6.1IFA result
Fig. 6 is IFA experimental result, wherein Fig. 6-1 is single-stranded genetic engineering antibody 1 and A/GDMM/CHA/2013 strain
Infect BHK21 cell experiment result；Fig. 6-2 is the single-stranded genetic engineering antibody 1 in ox source and normal BHK21 cell experiment result；Figure
6-3 is single-stranded genetic engineering antibody 2 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment as a result, Fig. 6-4 is Niu Yuan
Single-stranded genetic engineering antibody 2 and normal BHK21 cell experiment result；Fig. 6-5 is single-stranded genetic engineering antibody 3 and A/GDMM/
CHA/2013 virus strain infection BHK21 cell experiment result；Fig. 6-6 is that the single-stranded genetic engineering antibody 3 in ox source and normal BHK21 are thin
Born of the same parents' experimental result；Fig. 6-7 is single-stranded genetic engineering antibody 4 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment knot
Fruit, Fig. 6-8 are the single-stranded genetic engineering antibody 4 in ox source and normal BHK21 cell experiment as a result, Fig. 6-9 is that single-stranded genetic engineering is anti-
Body 5 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment are as a result, Fig. 6-10 is the single-stranded genetic engineering antibody 5 in ox source
With normal BHK21 cell experiment result.
IFA testing result shows that single-stranded genetic engineering antibody 1,2,3,4,5 can be with A/GDMM/CHA/2013 strain sense
The BHK-21 cell-specific of dye combines, and green fluorescence occurs around nucleus (blue), and do not connect malicious control cell without
Visible green fluorescence.Result confirmation has successfully been obtained can be anti-with the single-stranded genetic engineering in ox source of A type FMDV specific reaction
Body.
6.2 result of indirect ELISA
Indirect ELISA experimental result is as shown in fig. 7,5 plants of single-stranded genetic engineering antibodies that screening obtains can be with A type
FMDV antigen binding, these single-stranded genetic engineering antibodies are to A type FMDV antigen relative affinity in 0.01 μ g/mL-0.1 μ g/mL
Between, wherein single-stranded genetic engineering antibody 5 is to other a little higher than single-stranded genetic engineering antibodies of A type FMDV antigen relative affinity.
6.3 virus neutralization tests results
Micro virus neutralization tests is carried out on BHK21 cell whether to detect these single-stranded genetic engineering antibodies in ox source
Activity with A/GDMM/CHA/2013 strain.As a result IC is taken50To evaluate antibody neutralization, IC50Refer to be detected and resist
The 503nhibiting concentration of body, unit are μ g/mL, IC50Value it is lower, show that the neutralization ability of antibody is stronger.Use 50 μ g/
The IC of mL50Value is used as neutralization critical value, as test antibodies IC50>=50 μ g/mL, it is believed that the antibody, which does not have to neutralize, lives
Property.The viral neutralization titer of 5 plants of single-stranded genetic engineering antibodies in ox source is as shown in table 8, the single-stranded genetic engineering antibody in ox source 1,2,
3,5 have the ability for neutralizing A/GDMM/CHA/2013 strain, are A type FMDV neutralizing antibody, and the single-stranded genetic engineering in ox source is anti-
Body 4 is nonneutralizing antibody.
The neutralization titer of 8 Ns of single-stranded genetic engineering antibodies in source of table
| The single-stranded genetic engineering antibody strain in ox source | 1 | 2 | 3 | 4 | 5 |
| A/GDMM/CHA/2013 strain | 17.87μg/mL | 1.47μg/mL | 8.89μg/mL | ≥50μg/mL | 1.43μg/mL |
6.4WB result
Electrophoresis is carried out to A/GDMM/CHA/2013 strain antigen, WB result is as shown in Figure 8, wherein swimming lane 1-5 generation respectively
List chain gene engineered antibody 1,2,3,4,5.The results show that single-stranded genetic engineering antibody 1 is between 25kDa and 35kDa, about
Occurs specific bond band at 28kDa, corresponding FMDV Viral structural protein VP2, same single-stranded genetic engineering antibody 2 is also in this position
There is onesize band in place.And single-stranded 3,4,5 pairs of A type FMDV antigens of genetic engineering antibody are reactionless.Experimental result card
What real single-stranded genetic engineering antibody 1 and 2 identified is the linear epitope on A type FMDV Viral structural protein VP2.And single-stranded genetic engineering
What antibody 3,4,5 identified is the comformational epitope on A type FMDV antigen.
Brief summary:
IFA and ELISA experimental result confirms, screens 5 plants of single-stranded genetic engineering antibodies in ox source of acquisition to A type FMDV energy
It specifically binds.Further pass through virus neutralization experiment, it was demonstrated that during the single-stranded genetic engineering antibody 1,2,3,5 in ox source has
It is A type FMDV neutralizing antibody with the ability of A/GDMM/CHA/2013 strain.WB is it is experimentally confirmed that the single-stranded genetic engineering in ox source is anti-
What body 1 and 2 identified is the linear epitope on A type FMDV capsid protein VP2, and the single-stranded genetic engineering antibody 3,4,5 in ox source is known
The comformational epitope of other A type FMDV antigen.Based on the above results, this research provides a kind of single-stranded gene in anti-A type FMDV ox source
The method of engineered antibody, the screening technique provide important side to the identification of research A type FMDV host specificity antigen site
Method, and then the design for aftosa molecular vaccine provides guidance.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>preparation method of the single-stranded genetic engineering antibody in a kind of anti-A type foot and mouth disease virus ox source
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1 5 10 15
Val Leu Ser
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ccctcctctt tgtgctstca gccc 24
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Claims (7)
1. a kind of preparation method of the single-stranded genetic engineering antibody in anti-A type foot and mouth disease virus ox source, comprising the following steps:
1) separating peripheral blood mononuclear cells from the bovine blood sample of immune A type FMDV inactivated vaccine；
2) ultrafiltration centrifugation is carried out to A type FMDV 146S antigen, the A type FMDV 146S antigen after being concentrated uses dyestuff pair
A type FMDV 146S antigen after concentration is dyed, and the antigen after dyeing is carried out ultrafiltration centrifugation, the A type after being marked
FMDV 146S antigen；
The dyestuff includes FluoProbes 647H, Allophycocyanin, DyLight 633, DyLight 650, Atto
633 or Cynaine Dye 5；
3) by the A type FMDV 146S antigen after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains, mouse
Anti- ox CD21-RPE and the anti-ox IgM-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis A type FMDV specificity；
5) reverse transcription is carried out to the single B cell of A type FMDV specificity that step 4) obtains, is expanded after obtaining cDNA, obtains A
The heavy chain variable region gene and light-chain variable region gene of the single B cell IgG antibody of type FMDV specificity；
6) heavy chain variable region gene and light-chain variable region gene obtained step 5) passes through the flexibility such as SEQ ID NO.1
Linker connection, the N-terminal of gene upon connection introduce the signal peptide sequence as described in SEQ ID NO.2, introduce Flag in C-terminal
Label and His label carry out gene order optimization with reference to Homo sapiens (Human) codon, and gene after optimization rises
KOZAK sequence is introduced before beginning codon, is inserted into pcDNA3.4 expression vector, is obtained ox source single-chain antibody expression vector；
7) step 6) is obtained into the expression that single-chain antibody expression vector transfection cell in ox source carries out antibody；
The step 1) and 2) the not restriction of chronological order.
2. screening technique according to claim 1, which is characterized in that step 1) the bovine blood sample is immune A type
The bovine blood sample of 14~30d of FMDV inactivated vaccine.
3. screening technique according to claim 1, which is characterized in that step 2) the ultrafiltration centrifugation are as follows: use interception
The super filter tube 4000rpm of 100kDa is centrifuged 10min, and the antigen after antigen or dyeing is replaced into PBS buffer solution.
4. screening technique according to claim 1, which is characterized in that the step 4) sorting are as follows: 10 μ L are contained in every hole
The 96-PCR orifice plate of lysate is put into sorting cabin, and every hole is arranged and sorts 1 cell pattern, loading；It draws door and irises out lymph and monokaryon
Cell excludes adhesion cells according to FSC-A and FSA-H setting, irises out diagonal line single cell；To CD21+IgM-A-FMDV+Carefully
Born of the same parents sort.
5. screening technique according to claim 1, which is characterized in that the step 5) amplification includes nested PCR amplification side
Method.
6. screening technique according to claim 1, which is characterized in that step 6) is inserted into using Not1 and Nhel restriction enzyme site
To pcDNA3.4 expression vector.
7. screening technique according to claim 1, which is characterized in that the step 7) cell includes that Expi293 suspends carefully
Born of the same parents.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086652A (en) * | 2014-06-30 | 2014-10-08 | 中国农业科学院兰州兽医研究所 | Anti-O type foot and mouth disease virus specific single-domain antibody and recombinant expression vector thereof |
| CN107177556A (en) * | 2016-03-09 | 2017-09-19 | 中国农业科学院兰州兽医研究所 | Anti- sheep IgM μ chain monoclonal antibodies, the hybridoma cell strain for secreting the antibody and application thereof |
-
2019
- 2019-07-30 CN CN201910694887.1A patent/CN110483638A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086652A (en) * | 2014-06-30 | 2014-10-08 | 中国农业科学院兰州兽医研究所 | Anti-O type foot and mouth disease virus specific single-domain antibody and recombinant expression vector thereof |
| CN107177556A (en) * | 2016-03-09 | 2017-09-19 | 中国农业科学院兰州兽医研究所 | Anti- sheep IgM μ chain monoclonal antibodies, the hybridoma cell strain for secreting the antibody and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 代鹏等: "单链抗体的表达", 《生物技术通报》 * |
| 刘玉堂主编: "《动物细胞工程》", 31 December 2003, 东北林业大学出版社 * |
| 施红主编: "《生物化学》", 31 July 2015, 中国中医药出版社 * |
| 王省等: "B 细胞分离方法的研究进展", 《中国兽医科学》 * |
| 王省等: "口蹄疫病毒 VP2 蛋白特异性单克隆工程抗体的表达与活性鉴定", 《中国兽医科学》 * |
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