CN110372791A - A kind of preparation method of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source - Google Patents
A kind of preparation method of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source 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
- C07K16/1009—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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C07K2317/35—Valency
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C12N2800/00—Nucleic acids vectors
- 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 foot-and-mouth disease virus resistant pig source, belong to antibody production techniques field.PBMCs is separated out of foot-and-mouth disease virus antigen immune swine body, using dye marker FMDV 146S antigen, PBMCs is dyed, by the single B cell of polychrome selected by flow cytometry apoptosis combination FMDV；In conjunction with monoclonal antibody gene sequencing, VH and VL gene is obtained respectively, engineered antibody technology is expressed by flexible linker and CHO-S, expression obtains the single-stranded genetic engineering antibody in FMDV specificity pig source.The antibody that preparation method of the present invention obtains has gene diversity good, high-efficient, full host (pig) source, the advantages such as need cell concentration few.
Description
Technical field
The present invention relates to antibody production techniques fields, and in particular to a kind of single-stranded genetic engineering in foot-and-mouth disease virus resistant pig source
The preparation method of 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 O-shaped, A type, 1 type of Asia, c-type, the type of SAT1~3, each serotype
Between without cross-immune reaction.China Major Epidemic O type FMDV and A type FMDV.The each serotype of FMDV is again according to popular region
It is divided into multiple topological types (VP1,85% amino acid identity).Presently, there are the O-shaped FMDV of 3 pedigrees in China, respectively
O/Mya/98 (Southeast Asia topological type), O/PanAsia (middle Southeast Asia topological type) and O/Cathay (Chinese topological type), three
There are larger differences for the antigenic structure of pedigree virus, and Cross immunogenicity effect is weaker, cannot provide heterologous strain good
Cross immunogenicity.A type FMDV is primarily present SEA97 topological type in China, including G1 and G2 branch.It can be seen that
FMDV is considerably complicated in China's prevalence, and antigenic variation is larger.Therefore, research FMDV antigenic structure has vaccine strain design
Significance.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.Light chain
It is divided into K light chain and L light chain, the role with stabilization of antibodies molecular conformation, mouse antibodies K:L light chain ratio is 95%:5%, and
Pig antibody K:L light chain ratio is 50%:50%.It in summary it can be seen, VDJ gene composition difference and CDR3 between different plant species
It is anti-certainly will to may cause host specificity for difference in length and K/L light chain composition ratio difference, caused antibody diversity
The generation put in situ.To FMDV antigenic structure the study found that initially passing through section of synthesized peptide and immunization trial interpretation of result,
The antigen site 1 for thinking that G-H ring and C-terminal are formed on VP1 albumen is immunodominance antigen site.However, using antigen position
Complete immunity can not be provided after the synthetic peptide vaccine immune cattle of 1 preparation of point.On pig, comparative studies immune serum pair
The dilution factor difference that monoclonal antibody is escaped between strain and immune strain, it is believed that O-shaped FMDV antigen site 2 is immunodominance to pig
Property antigen site.However, using the O-shaped of the VP1 Protein G-H ring and C-terminal gene fusion expression for containing multiple pedigree FMDV
Or A type FDMV polyepitope vaccines, complete protection is each provided with after immune swine.These results of study explanation, there are places by FMDV
Main specific antigen site, using natural reservoir (of bird flu viruses) source Identification of monoclonal its identify antigen site to parsing FMDV antigenic structure and
Vaccine design has great importance.Currently, being mainly mouse list for studying the monoclonal antibody of FMDV antigenic structure
Clonal antibody.However since pig is the natural infection host of FMDV, and the B cell diversity of mouse and pig is there are larger difference,
Therefore FMDV specificity pig resource monoclonal antibody is generated to parsing FMDV antigenic structure, especially discovery FMDV host specificity
Antigen site is of great significance.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source.
The antibody that preparation method of the present invention obtains has gene diversity good, and high-efficient, full host (pig) source needs cell
Measure the advantages such as few.
The present invention provides a kind of preparations of the preparation method antibody of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source
Method, comprising the following steps:
1) separating peripheral blood mononuclear cells from the pig blood sample of immune foot-and-mouth disease virus antigen；
2) ultrafiltration centrifugation is carried out to FMDV 146S antigen, the FMDV 146S antigen after being concentrated, using dyestuff to dense
FMDV 146S antigen after contracting is dyed, and the antigen after dyeing is carried out ultrafiltration centrifugation, the FMDV 146S after being marked
Antigen；
3) the FMDV 146S antigen after the label for obtaining peripheral blood mononuclear cells that step 1) obtains and step 2),
The anti-pig IgM-PE of mouse and the anti-pig IgG-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis FMDV specificity；
5) reverse transcription is carried out to the single B cell of FMDV specificity that step 4) obtains, after obtaining cDNA, utilizes such as SEQ
Primer shown in NO.1~16 ID carries out nested PCR amplification, obtains pig IgG heavy chain variable region gene, Lambda light chain variable
Area's gene and Kappa light-chain variable region gene；
6) the pig IgG heavy chain variable region gene for obtaining step 5) respectively with Lambda light-chain variable region gene, Kappa
Light-chain variable region gene is distinguished by the flexibility Linker connection as shown in SEQ ID NO.17, the N-terminal of gene upon connection
The signal peptide sequence as described in SEQ ID NO.18 is introduced, introduces Myc label and His label respectively in C-terminal, is referred to
Cricetulus griseus codon carries out gene order optimization respectively, before gene start codon after optimization respectively
Introduce KOZAK sequence, be inserted respectively into pcDNA3.4 expression vector, obtain Lambda pig source single-chain antibody expression vector and
Kappa pig source single-chain antibody expression vector；
7) step 6) is obtained into Lambda pig source single-chain antibody expression vector and kappa pig source single-chain antibody expression vector
Transfection cell carries out the expression of antibody respectively；
The step 1) and 2) the not restriction of chronological order.
Preferably, the step 1) foot and mouth disease virus includes O-shaped FMDV, A type FMDV, C type FMDV, Asia1 type
FMDV, SAT1 type FMDV, SAT2 type FMDV and SAT3 type FMDV.
Preferably, step 1) the pig blood sample is the pig blood sample of immune 14~30d of foot-and-mouth disease virus antigen
This.
Preferably, the step 2) dyestuff includes FluoProbes 647H dyestuff or Pacific Blue dyestuff.
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, therefrom irises out IgG+IgM-Cell colony, circle door sort IgG+IgM-FMDV+
Cell.
Preferably, step 6) is inserted into pcDNA3.4 expression vector using Not1 and Nhel restriction enzyme site.
Preferably, the step 7) cell includes CHO-S suspension cell.
The present invention provides a kind of preparations of the preparation method antibody of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source
Method.PBMCs is separated out of foot-and-mouth disease virus antigen immune swine body, uses dye marker FMDV 146S antigen, the dyestuff mark
Note antigen will not damage antigen particles, can guarantee 146S antigen particles integrality.Then PBMCs is dyed, is borrowed
Help the single B cell of polychrome selected by flow cytometry apoptosis combination FMDV；In conjunction with monoclonal antibody gene sequencing, obtain respectively VH and
VL gene expresses engineered antibody technology by flexible linker " GGGGSGGSGGGGSGGS " and CHO-S, and expression obtains FMDV
The specific single-stranded genetic engineering antibody in pig source.It is verified through ELISA, WB and immunofluorescence experiment, screens FMDV specificity pig source
Single-stranded genetic engineering antibody.The antibody that preparation method of the present invention obtains has gene diversity good, high-efficient, full host
(pig) source, the advantages such as need cell concentration few, and the method for the present invention be simpler, conveniently, and it is multiple to avoid traditional murine hybridoma technology
Miscellaneous cell fusion screening process can directly sort the single B cell of antigentic specificity, vivoexpression by flow cytometer
Obtain single-stranded genetic engineering antibody；There is great importance to the identification of research FMDV host specificity antigen site, can be
The development of aftosa molecular vaccine lays the foundation.
Detailed description of the invention
Fig. 1 is the preparation flow of the single-stranded genetic engineering antibody in pig source；
Fig. 2 is the O-shaped FMDV antigen Electronic Speculum observation (30000 times of amplification) of fluorochrome label；
Fig. 3 is the single B cell of the O-shaped FMDV specificity of airflow classification；
Fig. 4 is resisting O-type FMDV pig IgG antibody variable gene PCR amplification result；
Fig. 5 is the single-stranded genetic engineering antibody in O-shaped FMDV pig source that purifying obtains；
Fig. 6 is the single-stranded genetic engineering antibody IFA testing result of resisting O-type FMDV；
Fig. 7 is the single-stranded genetic engineering antibody indirect ELISA experimental result of resisting O-type FMDV；
Fig. 8 is the single-stranded genetic engineering antibody WB result of resisting O-type FMDV；
Fig. 9 is the A type FMDV antigen Electronic Speculum observation (30000 times of amplification) of fluorochrome label；
Figure 10 is the single B cell of airflow classification A type FMDV specificity pig；
Figure 11 is anti-A type FMDV pig IgG antibody variable gene PCR amplification result；
Figure 12 is the single-stranded genetic engineering antibody in the type FMDV pig source A that purifying obtains；
Figure 13 is the single-stranded genetic engineering antibody IFA experimental result of anti-A type FMDV；
Figure 14 is the single-stranded genetic engineering antibody indirect ELISA experimental result of anti-A type FMDV；
Figure 15 is the single-stranded genetic engineering antibody WB experimental result of anti-A type FMDV.
Specific embodiment
The present invention provides a kind of preparations of the preparation method antibody of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source
Method, comprising the following steps:
1) separating peripheral blood mononuclear cells from the pig blood sample of immune foot-and-mouth disease virus antigen；
2) ultrafiltration centrifugation is carried out to FMDV 146S antigen, the FMDV 146S antigen after being concentrated, using dyestuff to dense
FMDV 146S antigen after contracting is dyed, and the antigen after dyeing is carried out ultrafiltration centrifugation, the FMDV 146S after being marked
Antigen；
3) the FMDV 146S antigen after the label for obtaining peripheral blood mononuclear cells that step 1) obtains and step 2),
The anti-pig IgM-PE of mouse and the anti-pig IgG-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis FMDV specificity；
5) reverse transcription is carried out to the single B cell of FMDV specificity that step 4) obtains, after obtaining cDNA, utilizes such as SEQ
Primer shown in NO.1~16 ID carries out nested PCR amplification, obtains pig IgG heavy chain variable region gene, Lambda light chain variable
Area's gene and Kappa light-chain variable region gene；
6) the pig IgG heavy chain variable region gene for obtaining step 5) respectively with Lambda light-chain variable region gene, Kappa
Light-chain variable region gene is distinguished by the flexibility Linker connection as shown in SEQ ID NO.17, the N-terminal of gene upon connection
The signal peptide sequence as described in SEQ ID NO.18 is introduced, introduces Myc label and His label respectively in C-terminal, is referred to
Cricetulus griseus codon carries out gene order optimization respectively, before gene start codon after optimization respectively
Introduce KOZAK sequence, be inserted respectively into pcDNA3.4 expression vector, obtain Lambda pig source single-chain antibody expression vector and
Kappa pig source single-chain antibody expression vector；
7) step 6) is obtained into Lambda pig source single-chain antibody expression vector and kappa pig source single-chain antibody expression vector
Transfection cell carries out the expression of antibody respectively；
The step 1) and 2) the not restriction of chronological order.
Present invention separating peripheral blood mononuclear cells from the pig blood sample of immune foot-and-mouth disease virus antigen.In this hair
In bright, the foot-and-mouth disease virus antigen include: O-shaped FMDV, A type FMDV, c-type FMDV, Asia1 type FMDV, SAT1 type FMDV,
SAT2 type FMDV and SAT3 type FMDV.In the present invention, the foot-and-mouth disease virus antigen is preferably foot and mouth disease virus inactivation epidemic disease
Seedling.In the present invention, the preferably immune 14~30d of foot-and-mouth disease virus antigen of the pig blood sample, the more preferably pig of 21d
Blood sample.In the present invention, the blood sample is preferably pig peripheral blood.The present invention is to the peripheral blood mononuclear cells
Separation method be not particularly limited, using conventional separation methods well known to those skilled in the art.
The present invention carries out ultrafiltration centrifugation to FMDV 146S antigen, and the FMDV 146S antigen after being concentrated uses dyestuff
FMDV 146S antigen after concentration is dyed, the antigen after dyeing is subjected to ultrafiltration centrifugation, the FMDV after being marked
146S antigen.Ultrafiltration of the present invention centrifugation can mild concentrated antigen lost simultaneously on antigen particles integrality without influence
It is small, the rate of recovery with higher.In the present invention, the small fluorescein molecule labelled antigen of selection molecular weight can avoid fluorescence dye
Material is to FMDV antigen site stopping effect.In the present invention, the dyestuff is preferably the institute removed except PE and FITC dyestuff
There are small molecule dyes, more preferably FluoProbes 647H dyestuff or Pacific Blue dyestuff, above two dyestuff hair
Ejected wave is long to be concentrated, and fluorescence signal is strong, and the single B cell group of antigentic specificity of both fluorescent dyes instruction is easily distinguishable, point
Select accuracy rate height.The FMDV antigen of FMDV antigen and dye marker that the present invention is immunized is the antigen of the same serotype, such as O
Type FMDV antigen is immune, that is, marks O-shaped FMDV 146S antigen；A type FMDV antigen is immune, i.e. label A type FMDV 146S is anti-
It is former；C-type FMDV antigen is immune, i.e. label c-type FMDV 146S antigen；Asia1 type FMDV antigen is immune, i.e. label Asia1 type
FMDV 146S antigen；SAT1 type FMDV antigen is immune, i.e. label SAT1 type FMDV 146S antigen；SAT2 type FMDV antigen is exempted from
Epidemic disease, i.e. label SAT2 type FMDV 146S antigen；SAT3 type FMDV antigen is immune, i.e. label SAT3 type FMDV 146S antigen.
In the present invention, ultrafiltration centrifugation are as follows: be centrifuged 10min using the super filter tube 4000rpm of interception 100kDa, by antigen or
Antigen after dyeing is replaced into PBS buffer solution.In the present invention, before using dyeing, antigen is preferably concentrated into 1mg/
Ml, antigen concentration to be marked is in 1mg/ml, labeling effciency highest, while both can guarantee that fluorescein will not make antigenic structure
At destruction, and it can guarantee and can mark fluorescein molecule on each antigen particles.In the present invention, the preferred packet of the dyeing
It includes following steps: taking 100 μ L 146S antigens that 10 μ L LL-modifier reagent are added, mix gently, then therefrom inhale
It takes 100 μ L to be added to a pipe Lightning-LinkTM mix reactive dye, gently aspirates and powder is resuspended, room temperature (20~25
DEG C) effect 3 hours.10 μ L LL-quencher reagent are added, act on 30 minutes, to terminate reaction.The present invention is to described
The source of dyestuff is not particularly limited, it is preferable to use conventional commercial dyestuff, such as Lightning-LinkTM Rapid
FluoProbes 647H labelling kit (Innova Biosciences, San Diego, USA) or Pacific BlueTMIt is anti-
Body labelling kit (Thermo Scientific, USA).
After FMDV 146S antigen after obtaining peripheral blood mononuclear cells and label, the present invention is thin by the single core of peripheral blood
Born of the same parents mix with FMDV 146S antigen, the anti-pig IgM-PE of mouse and the anti-pig IgG-FITC fluorescence antibody of mouse after label, are dyed
Cell afterwards.In the present invention, the anti-pig IgM-PE of mouse is added in the dyeing of polychrome fluidic cell, the anti-pig IgG-FITC fluorescence of mouse resists
The purpose of body is to iris out IgG first+IgM-B cell group, can be obtained with high-affinity, expression IgG positive B-cells, and
The positive weak affinity B cell of IgM can be removed simultaneously.The purpose of FMDV 146S antigen is added simultaneously is in IgG+IgM-B cell group
On the basis of body, sorting FMDV 146S positive B-cells are irised out, can be obtained the single B cell of high-affinity specificity.
After cell after being dyed, the present invention uses the single B cell of polychrome selected by flow cytometry apoptosis FMDV specificity.
The present invention is not particularly limited the model of the polychrome flow cytometer, and present invention preferably uses II u of BD FACSAria streams
Formula sorter, when using II u flow sorter of BD FACSAria, preferably instrument parameter is arranged by the present invention are as follows: nozzle is big
It is small: 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: first by using 96 orifice plates for being stained with sealed membrane, if
It sets 60 cell patterns of every hole sorting to be adjusted, adjusts the hole 96-PCR Board position in sorting cabin, until sorting cell is accurately fallen
Enter plate hole centre, records coordinate position.Then the 96-PCR orifice plate that 10 μ L lysates are contained in every hole is put into sorting cabin, if
It sets every hole and sorts 1 cell pattern, loading；It draws door and irises out lymph and monocyte, excluded according to FSC-A and FSA-H setting
Adhesion cells iris out diagonal line single cell, therefrom iris out IgG+IgM-Cell colony, circle door sort IgG+IgM-FMDV+Carefully
Born of the same parents.In the present invention before sorting is unicellular, is preconditioned, can visually be evaluated thin using 60 cell per wells sorting modes
Born of the same parents are sorted into plate hole locations locating for 96 orifice plates.To guarantee that subsequent unicellular sorting can accurately fall into 96 orifice plates center, protect
Demonstrate,prove the efficiency of separation.
After obtaining the single B cell of FMDV specificity, the present invention carries out reverse transcription to the single B cell of FMDV specificity, obtains
After cDNA, nested PCR amplification is carried out using the primer as shown in NO.1~16 SEQ ID, obtains pig IgG heavy chain variable region base
Cause, Lambda light-chain variable region gene and Kappa light-chain variable region gene.Primer sequence table is as shown in table 1:
Table 1 expands the primer of pig IgG heavy chain, Kappa light chain and Lambda light-chain variable region gene
aDegeneracy base annotation in sequence, S=C or G, Y=C or T, R=A or G, K=G or T.
In the present invention, pig IgG heavy chain first round amplification institute is using primer pairing scheme, outside the outer upstream IgG and IgG
Downstream pairing；Pig IgG heavy chain second takes turns amplification scheme, is expanded with downstream pairing primer first round PCR outside upstream outside IgG and IgG
Template is increased to, is expanded using downstream primer pairing in upstream in IgG and IgG.
In the present invention, lambda light chain first round amplification institute is the outer upstream Lambda V3 using primer pairing scheme
It is matched with downstream outside Lambda；The outer upstream Lambda V2-6 and the outer downstream Lambda are matched；The outer upstream Lambda V8 with
The outer downstream pairing of Lambda.Lambda light chain second takes turns amplification scheme, with downstream outside upstream outside Lambda V3 and Lambda
Pairing primer first round PCR amplification is template, is expanded using downstream primer pairing in upstream in Lambda V3 and Lambda；With
The outer upstream Lambda V2-6 and the outer downstream pairing primer first round PCR amplification of Lambda are template, using in Lambda V2-6
Upstream is expanded with downstream primer pairing in Lambda；The primer first round is matched with downstream outside upstream outside Lambda V8 and Lambda
PCR amplification is template, is expanded using downstream primer pairing in upstream in Lambda V8 and Lambda.
In the present invention, Kappa light chain first round amplification institute is using primer pairing scheme, the outer upstream Kappa with
The outer downstream pairing of Kappa；Kappa light chain second takes turns amplification scheme, matches primer with downstream outside upstream outside Kappa and Kappa
First round PCR amplification is template, is expanded using downstream primer pairing in upstream in Kappa and Kappa.
After obtaining pig IgG heavy chain variable region gene, Lambda light-chain variable region gene and Kappa light-chain variable region gene,
The present invention leads to pig IgG heavy chain variable region gene with Lambda light-chain variable region gene, Kappa light-chain variable region gene respectively
The flexibility Linker connection as shown in SEQ ID NO.17 is crossed, the N-terminal of gene upon connection introduces such as SEQ ID respectively
Signal peptide sequence described in NO.18 introduces Myc label and His label at the end C respectively, close with reference to Cricetulus griseus
Numeral carries out gene order optimization respectively, introduces KOZAK sequence before gene start codon after optimization respectively, inserts respectively
Enter to pcDNA3.4 expression vector, obtains Lambda pig source single-chain antibody expression vector and the expression of kappa pig source single-chain antibody carries
Body.In the present invention, pcDNA3.4 expression vector is inserted into using Not1 and Nhel restriction enzyme site.
After obtaining Lambda pig source single-chain antibody expression vector and kappa pig source single-chain antibody expression vector, the present invention will
Lambda pig source single-chain antibody expression vector and kappa pig source single-chain antibody expression vector transfect cell respectively and are resisted
The expression of body.In the present invention, the cell includes CHO-S suspension cell.Specifically, the present invention preferably passes through transient transfection
CHO-S suspension cell expresses antibody, takes 15~30 μ g, preferably 20 μ g plasmids (Lambda pig source single-chain antibody expression vector or
Kappa pig source single-chain antibody expression vector) 250 μ L OptiPRO are addedTMSFM culture medium is diluted, while taking equivalent
OptiPROTMSFM culture medium dilutes ExpiFectamineTMCHO-S transfection reagent then tries the plasmid diluted and transfection
Agent mixing, room temperature act on 5min；The mixture is slowly added to 1.5 × 108In a CHO-S cell.CHO-S cell after transfection
37 DEG C of constant temperature suspension incubators are placed in, add 150 μ L CHO-S after the culture 18h that suspendsTMEnhancer and 6mL CHO-
STMFeed.Purifying, dialysis obtain pig source single-chain antibody.
Combined with specific embodiments below to a kind of single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source of the present invention
The preparation method of preparation method antibody be further described in detail, technical solution of the present invention includes but is not limited to following
Embodiment.
Embodiment 1
1.FMDV antigenic mark scheme
1.1 O-shaped FMDV 146S antigenic mark schemes
Selection uses Lightning-LinkTM Rapid FluoProbes 647H labelling kit (Innova
Biosciences, San Diego, USA) the O-shaped FMDV 146S antigen of label.First with interception 100kDa super filter tube O-shaped
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-modifier reagent are added, mixes gently, then therefrom inhale
It takes 100 μ L to be added to a pipe Lightning-LinkTM mix reactive dye, gently aspirates and powder is resuspended, room temperature (20-25
DEG C) effect 3 hours.10 μ L LL-quencher reagent are added, act on 30 minutes, to terminate reaction.Then 100kDa is used
Super filter tube centrifugation, 4000rpm are centrifuged 10min, the antigen after label are replaced into PBS buffer solution.It is added isometric 100%
Glycerol, -70 DEG C of preservations.Antigen after label is named as O-FMDV 146S-FluoProbes 647H.
The dyeing of 1.2 Electronic Speculum
To confirm that the above-mentioned O-shaped FMDV antigen particles of fluorochrome label 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
2.1 PBMCs separation
Take O-shaped foot-and-mouth disease virus antigen is immunized 21 days (- 30 days 14 days sections) pig peripheral blood, use lymphocyte
Separating liquid (density=1.083g/mL) therefrom separates PBMCs, and concrete operations are as follows:
(1) 6mL lymphocyte separation medium is added into 15mL centrifuge tube in lymphocyte separation medium and PBS equilibrium at room temperature.
(2) 1:1 dilution proportion ox EDTA anticoagulation is pressed with PBS, whole blood is added to lymphocyte point after drawing 8mL dilution
Chaotropic upper layer, 1200 × g are centrifuged 30min.
(3) it draws milky-white layer PBMCs cell and is added and (contain 1% BSA, 2mM containing 1/2 volume cells sorting liquid
EDTANa2PBS) 15mL centrifuge tube in, 600 × g be centrifuged 5min.
(4) supernatant liquid is discarded, 1~2mL erythrocyte cracked liquid is added, cell sorting is added after 1~2min of lysis at room temperature
Liquid, 250 × g are centrifuged 10min.
(5) supernatant liquid is discarded, is washed twice of cell with cell sorting liquid, 400 × g is centrifuged 5min.Supernatant liquid is discarded,
Cell sorting liquid is added to dispel for individual cells, the cell finally obtained is PBMCs, is counted.
The dyeing of 2.2 fluidic cells
Take 107A B cell is resuspended in 200 μ L cell sorting liquid, and 0.5 μ g O-FMDV 146S-FluoProbes is added
647H antigen, the anti-pig IgG-FITC fluorescence antibody (MyBioSource, USA) of 2 μ g mouse and the 1 anti-pig IgM-PE fluorescence antibody of μ g mouse
(Bio-Rad, USA), acts on 25min on ice.Take same cell setting Isotype control and the control that subtracts one.Dan Yangguan is set simultaneously,
It is compensated for adjusting.
(2) 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, is protected from light on ice, machine sorts cell in preparation.
3. sorting the single B cell of O-shaped 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 is put into sorting cabin, starts 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.Then it further therefrom irises out
IgG+IgM-Cell colony, gating sort IgG+IgM-O-FMDV+Cell, the as single B cell of O-shaped FMDV specificity.
4. 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 is set are as follows: 25 DEG C, 5min；42 DEG C, 120min；85 DEG C, 5min.It obtains
CDNA-20 DEG C storage, be used for subsequent nested PCR amplification.
The nested PCR amplification of 4.2 unicellular cDNA
The primer of amplification pig IgG heavy chain, Kappa light chain and Lambda light-chain variable region gene is as used shown in table 1, benefit
With the primer, nested PCR method, i.e. two-wheeled PCR amplification are taken, to pig single B cell IgG heavy chain variable region (VH) gene
It is expanded with Lambda light chain variable region (VL) and Kappa light chain variable region (VL) gene.
(1) first round PCR amplification scheme
First round PCR reaction system:
Expand pig IgG heavy chain variable region (VH) gene and Lambda light chain variable region (VL) and Kappa light chain variable region
(VL) first round reaction system of gene, in addition to primer is different with template, the addition of remaining component is carried out according to table 2:
2 first round of table PCR reaction system
First round PCR amplification program are as follows: 95 DEG C of initial denaturation 15min first；Then 94 DEG C of 0.5 min of denaturation, annealing (annealing
Temperature is shown in Table 1) 0.5min, 72 DEG C of extension 1min, totally 32 circulations；Last 72 DEG C re-extend 10min.Wherein annealing temperature is pressed
It is chosen shown in table 1.Second wheel PCR reaction system
(2) second wheel PCR amplification schemes
Second wheel reaction system:
Using first round amplified production as template, add in corresponding trip primer amplification pig IgG heavy chain variable region (VH) gene and
Lambda light chain variable region (VL) and Kappa light chain variable region (VL) gene, the second wheel PCR reaction system component is according to table 3
It carries out:
Table 3 second takes turns PCR reaction system component
Second wheel PCR amplification program are as follows: 95 DEG C of initial denaturation 15min first；Then 94 DEG C of 0.5 min of denaturation, annealing (annealing
Temperature is shown in Table 1) 0.5min, 72 DEG C of extension 1min, totally 36 circulations；Last 72 DEG C re-extend 10min.
The sequencing of 4.3 PCR products
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.
5. expression vector establishment and plasmid extraction
The building of 5.1 pig source single-chain antibody expression vectors
The pig IgG antibody variable region VH of amplification and VL gene are passed through into flexibility Linker " GGGGSGGSGGGGSGGS "
(SEQ ID NO.17) connection, and pig source IgG heavy chain secretion signal peptide sequence is introduced in fusion N-terminal
" MEFRLNWVVLFALLQGVQG " (SEQ ID NO.18) introduces Myc label and 6 × His label in fusion C-terminal, with benefit
In detection and purifying, gene order optimization is carried out then referring to Cricetulus griseus (CHO) codon, and in gene
Introduce KOZAK sequence " GCCACC " (SEQ ID NO.19) before initiation codon, finally synthesis gene by Not1 and
Nhel restriction enzyme site is inserted into pcDNA3.4 expression vector (Thermo scientific, USA).Sequent synthesis and vector construction
Trust money Wei Zhi biotech 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 antibody
Antibody is expressed by transiently transfecting CHO-S suspension cell, takes 15 μ g-30 μ g plasmids that 250 μ L are added
OptiPROTMSFM culture medium is diluted, while taking equivalent OptiPROTMSFM culture medium dilutes ExpiFectamineTMCHO-
S transfection reagent then mixes the plasmid diluted and transfection reagent, and room temperature acts on 5min.By the mixture be slowly added to
1.5×108In a CHO-S cell.CHO-S cell is placed in 37 DEG C of constant temperature suspension incubators after transfection, suspends after culture 18h, mends
Add 150 μ L CHO-STMEnhancer and 6mL CHO-STMFeed.37 DEG C are continued after cultivating 9d, are centrifuged by 10000 × g
30min collects cells and supernatant, filters through 0.22 μm of filter, is used for subsequent antibody purification.Use HiTrap TALON column
Son carries out antibody purification on AKAT protein purification instrument, using the PBS liquid antibody elution of 250mM imidazoles, is then charged into bag filter
It is placed in PBS liquid and dialyses 3 times, each 3h.Antibody after dialysis is concentrated with PEG 8000, and SDS-PAGE is carried out after concentration
Electrophoretic analysis.
7. the activity verifying of the single-stranded genetic engineering antibody in O-shaped FMDV pig source
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) is inoculated with 24 porocyte culture plates, while setting does not connect malicious normal cell controls, 37 DEG C of cell incubators
After middle incubation 4h, supernatant inactivation treatment is collected.Then the fixed cell of Jia Chun ︰ acetone (1:1) fixer of -20 DEG C of pre-coolings is added,
Room temperature acts on 20min.PBS is washed cell 3 times, and the antibody of purifying, 37 DEG C of incubation 1h are added by 5 μ g/mL concentration.PBS is washed 3 times,
The anti-Myc-FITC of fluorescence secondary antibody mouse (Thermofisher, USA), 37 DEG C of incubation 30min of working concentration is added.Then PBS is used
It washes cell 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 O-shaped FMDV (O/HN/CHA93 strain) antigen after purification, by 100 holes μ L/, 4 DEG C
Coated elisa plate overnight；After PBST washes 5 times, 1% gelatin closes 1h, after PBST washes 5 times, drying；Antibody to be checked is opened from 1 ︰ 100
Begin 2 times to be serially diluted, be added in ELISA Plate by every 100 μ L of hole, 37 DEG C of incubation 1h；PBST is washed 5 times, and HRP label is added in every hole
Anti- His tag antibody (1 ︰ 10000) 100 μ l, 37 DEG C of incubation 1h；PBST is washed 5 times, and 100 μ L TMB developing solutions are added, and room temperature is aobvious
Color 10min；Terminate liquid color development stopping is added, with the light absorption value (D450) at microplate reader measurement 450nm.S/C.O mode counts, S
For sample D450 value, C.O, that is, Cut Off value (positive decision content), C.O=2.1 × N (N is negative control D450 value, when N not
When foot 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
Virus is carried out using the single-stranded genetic engineering antibody in the pig source arrived of the O-shaped FMDV (O/HN/CHA93 strain) to screening
Neutralize 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
Pig 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 (Western Blot, WB) test
O-shaped FMDV (O/HN/CHA93 strain) antigen containing about 1 μ g is taken to carry out SDS-PAGE electrophoresis, then by separation
Protein band is transferred to cellulose nitrate (NC) film, with the TBST buffer blind 2h containing 5% skimmed milk power, after washing, with containing
The TBST buffer of 5% skimmed milk power dilutes antibody to working concentration (2 μ g/mL), is incubated at room temperature 2h, after TBST washes film, is added
The anti-His tag antibody (1 ︰ 4000) of HRP label is incubated at room temperature 1h, after TBST washes film, ECL chemiluminescent substrate is added, secretly
X-ray is pressed to be exposed imaging in room.
Experimental result:
1, the preparation flow of the single-stranded genetic engineering antibody in O-shaped FMDV specificity pig source
The preparation flow of the single-stranded genetic engineering antibody in pig source is as shown in Figure 1.
2, the O-shaped FMDV antigen particles integrality of fluorochrome label
After negative staining shown in Electronic Speculum observation result figure 2, O-shaped FMDV antigen (O/HN/CHA93 strain) is in fluorochrome label
146S particle size is uniform afterwards, diameter 20-30nm, and shape is intact.Morphologic observation is the result shows that the dye marker antigen will not be right
Antigen particles damage, and can guarantee 146S antigen particles integrality, are consequently adapted to carry out sorting single B as bait antigen
Cell.
3, the O-shaped single B cell of FMDV specificity pig is successfully obtained
Fig. 3 is the O-shaped single B cell of FMDV specificity pig of airflow classification, wherein A: from PBMCs after the dyeing of normal sample pipe
Centre circle does well good P1 group；B: P1 cell colony is shot in sample tube, irises out diagonal line single cell, and it is thin to exclude adhesion
Born of the same parents；C: IgG+IgM-B cell is irised out in sample tube；D: normal dyeing sample；E: each cell colony ratio of normal dyeing sample
(about 1,000,000 cells of record)；It is irised out from PBMCs after F:FMO control (the O-shaped FMDV antigen of fluorescence target is not added) dyeing
P1 group in good condition；The shooting of P1 cell colony, irises out diagonal line single cell in G:FMO control, excludes adhesion cells；H:
IgG is irised out in FMO control+IgM-B cell；Each cell colony ratio cell (about 1,000,000 cells of record) in I:FMO control.
Flow cytometer showed result is present in IgG as shown in figure 3, O-shaped FMDV specific b cells group (Fig. 3 D) is high-visible
The cell colony of+IgM-, the ratio for accounting for about total PBMCs is 140/1000000.Compared to subtracting one, control (FMO) sample (is not added
The O-shaped FMDV antigen of fluorescence target), the O-shaped mono- positive B-cells of FMDV (Fig. 3 I, P4 group) are about 0/1000000 (Fig. 3 G), are passed through
To normal sample Guan Zhong P4 group (Fig. 3 D, IgG+IgM-O-FMDV+B cell) circle door sorting, using unicellular sorting mode,
Success sorts the single B cell of pig for obtaining O-shaped FMDV specificity.
The acquisition of the single B cell IgG variable region gene of 4.O type FMDV specificity pig
Fig. 4 is pig IgG antibody variable gene PCR amplification result, wherein Fig. 4-1: pig IgG heavy chain variable region PCR produces
Object electrophoresis result；Fig. 4-2: pig kappa light chain variable region PCR product electrophoresis result；Fig. 4-3: pig Lambda light chain variable region
PCR product electrophoresis result.
Nested PCR product agarose gel electrophoresis analyze result as shown in figure 4, pig IgG heavy chain 450 bp-500bp it
Between there is high-visible band (Fig. 4-1)；There is clear band (Fig. 4-2) at about 475bp in pig kappa light chain variable region.Pig
There is clear band (Fig. 4-3) at about 475bp in Lambda light chain variable region.Sequencing result passes through Ig BLAST database ratio
It is right, it was demonstrated that the above variable region sequences that nested PCR amplification obtains are the IgG antibody variable region gene sequence of pig.
5, the single-stranded genetic engineering antibody molecule in pig source that successful expression obtains
Fig. 5 is the single-stranded genetic engineering antibody in O-shaped FMDV pig source that purifying obtains, wherein M represents albumen marker, 1-5
Represent the single-stranded genetic engineering antibody 1,2,3,4,5,6 in pig 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 35kDa in single-stranded genetic engineering antibody 1,2,3,5,6, is consistent with expection.These results explanation should
Method successful expression and can be purified into the single-stranded genetic engineering antibody molecule in pig source.
6. successfully obtaining the single-stranded genetic engineering antibody in resisting O-type FMDV pig source
6.1 IFA results
IFA testing result is as shown in Figure 6, wherein Fig. 6-1 is single-stranded genetic engineering antibody 1 and the sense of O/HN/CHA93 strain
Contaminate BHK21 cell experiment result；Fig. 6-2 is that single-stranded genetic engineering antibody 2 and O/HN/CHA93 virus strain infection BHK21 cell are real
Test result；Fig. 6-3 be the single-stranded genetic engineering antibody in pig source 3 with O/HN/CHA93 virus strain infection BHK21 cell experiment result；
Fig. 6-4 is the single-stranded genetic engineering antibody 4 in pig source and normal BHK21 cell experiment result；Fig. 6-5 is single-stranded genetic engineering antibody
5 with O/HN/CHA93 virus strain infection BHK21 cell experiment result；Fig. 6-6 is the single-stranded genetic engineering antibody 6 in pig source and O/HN/
CHA93 virus strain infection BHK21 cell experiment result；Fig. 6-7 is not connect malicious normal BHK21 cell controls.
Single-stranded genetic engineering antibody 1,2,3,4,5,6 can be with the BHK-21 cell-specific of O/HN/CHA93 virus strain infection
Property combine, there is obvious green fluorescence, and do not connect poison control cell without visible green fluorescence.Result confirmation successfully obtains
It can with the single-stranded genetic engineering antibody in pig source of O-shaped FMDV specific reaction.
6.2 result of indirect ELISA
Indirect ELISA experimental result as shown in fig. 7,6 plants of single-stranded genetic engineering antibodies can and O-shaped FMDV antigen binding,
Wherein single-stranded genetic engineering antibody 2 other single-stranded genetic engineering antibodies a little higher than to O-shaped FMDV antigen relative affinity.It is single-stranded
1,2,3,4,5 pair of O-shaped FMDV antigen relative affinity of genetic engineering antibody is between 0.01 μ g/mL-0.1 μ g/mL, single-stranded base
Because engineered antibody 6 to O-shaped FMDV antigen relative affinity between 0.1 μ g/mL-0.5 μ g/mL.
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 pig source
Activity with O/HN/CHA93 strain.As a result IC is taken50To evaluate antibody neutralization, IC50Refer to detected antibody
503nhibiting concentration, unit are μ g/mL, IC50Value it is lower, show that the neutralization ability of antibody is stronger.Use 50 μ g/mL's
IC50Value is used as neutralization critical value, as test antibodies IC50>=50 μ g/mL, it is believed that the antibody does not have neutralization activity.6 plants
The viral neutralization titer of the single-stranded genetic engineering antibody in pig source is as shown in table 4, and the single-stranded genetic engineering antibody 1,3,4,5,6 in pig source has
There is the ability for neutralizing O/HN/CHA93 strain, is O-shaped FMDV neutralizing antibody, and the single-stranded genetic engineering antibody 2 in pig source is non-neutralization
Antibody.
The neutralization titer of the 4 single-stranded genetic engineering antibody in pig source of table
6.4WB result
Electrophoresis is carried out to O/HN/CHA93 strain antigen, WB result is as shown in Figure 8, wherein swimming lane 1-6 respectively represents list
Chain gene engineered antibody 1,2,3,4,5,6.The results show that single-stranded genetic engineering antibody 1 is between 25kDa and 35kDa, about
Occur specific bond band, corresponding FMDV Viral structural protein VP2 at 28kDa.Single-stranded genetic engineering antibody 3 and 4 25kDa with
Between 35kDa, occur specific bond band, corresponding FMDV Structural protein VP1 at about 32kDa.The experimental result confirms single-stranded
What genetic engineering antibody 1 identified is the linear epitope on O-shaped FMDV Viral structural protein VP2, and single-stranded genetic engineering antibody 3 and 4 identifies
Be linear epitope on O-shaped FMDV Structural protein VP1.And that single-stranded genetic engineering antibody 2,5,6 identifies is O-shaped FMDV anti-
Comformational epitope in original.
Brief summary:
IFA and ELISA experimental result confirms, screens 6 plants of single-stranded genetic engineering antibodies in pig source of acquisition to O-shaped FMDV energy
It specifically binds.Further pass through virus neutralization experiment, it was demonstrated that the single-stranded genetic engineering antibody 1,3,4,5,6 in pig source has
The ability of O/HN/CHA93 strain is neutralized, is O-shaped FMDV neutralizing antibody.WB is it is experimentally confirmed that the single-stranded genetic engineering antibody 1 in pig source
What is identified is the linear epitope on O-shaped FMDV capsid protein VP2, and what antibody 3 and 4 identified is on O-shaped FMDV capsid protein VP1
Linear epitope, and the comformational epitope for the O-shaped FMDV antigen that the single-stranded genetic engineering antibody 2,5,6 in pig source identifies.In summary it ties
Fruit, this research provide a kind of method of the single-stranded genetic engineering antibody in resisting O-type FMDV pig source, and the screening technique is O-shaped to studying
The identification of FMDV host specificity antigen site provides important method, and then the design for aftosa molecular vaccine provides finger
It leads.
Embodiment 2
1. FMDV antigenic mark scheme
1.1 A type FMDV 146S antigenic mark schemes
Choose Pacific BlueTMAntibody labeling kit (Thermo Scientific, USA) marks A type FMDV
146S antigen.A type FMDV 146S antigen is replaced into PBS buffer solution with interception 100kDa super filter tube first, 4000rpm
It is centrifuged 10min, concentrated antigen concentration to 1mg/ml (OD260=7.6 are approximately equal to 1mg/ml).Take 10 μ L sodium bicarbonate buffer liquid
100 μ L146S antigens are added in (1M, pH=8.3), mix gently, then therefrom draw 100 μ L and be added to a tube reaction dyestuff,
It turns upside down 10 times, with abundant dissolving dye.(20~25 DEG C) of room temperature are incubated for 1 hour, during which run every about 10~15 minutes
3 times.Then it is centrifuged with 100kDa super filter tube, 4000rpm is centrifuged 10min, and the antigen after label is replaced to PBS buffer solution
In.Isometric 100% glycerol, -70 DEG C of preservations are added.Antigen after label is named as A-FMDV 146S-Pacific Blue.
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
2.1 PBMCs separation
Take 10 days pig peripheral bloods of immune A type FMDV inactivated vaccine, using lymphocyte separation medium (density=
PBMCs 1.083g/mL) is therefrom separated, concrete operations are as follows:
(1) 6mL lymphocyte separation medium is added into 15mL centrifuge tube in lymphocyte separation medium and PBS equilibrium at room temperature.
(2) 1:1 dilution proportion pig EDTA anticoagulation is pressed with PBS, whole blood is added to lymphocyte point after drawing 8mL dilution
Chaotropic upper layer, 1200 × g are centrifuged 30min.
(3) it draws milky-white layer PBMCs cell and is added and (contain 1% BSA, 2mM containing 1/2 volume cells sorting liquid
EDTANa2PBS) 15mL centrifuge tube in, 600 × g be centrifuged 5min.
(4) supernatant liquid is discarded, 1~2mL erythrocyte cracked liquid is added, cell sorting is added after 1~2min of lysis at room temperature
Liquid, 250 × g are centrifuged 10min.
(5) supernatant liquid is discarded, is washed twice of cell with cell sorting liquid, 400 × g is centrifuged 5min.Supernatant liquid is discarded,
Cell sorting liquid is added to dispel for individual cells, the cell finally obtained is PBMCs, is counted.
The dyeing of 2.2 fluidic cells
(1) 10 are taken7A B cell is resuspended in 200 μ L cell sorting liquid, and 0.5 μ g A-FMDV 146S-Pacific is added
Blue antigen, the anti-pig IgG-FITC fluorescence antibody (MyBioSource, USA) of 2 μ g mouse and the 1 anti-pig IgM-PE fluorescence antibody of μ g mouse
(Bio-Rad, USA), acts on 25min on ice.Take same cell setting Isotype control and the control that subtracts one.Dan Yangguan is set simultaneously,
It is compensated for adjusting.
(2) 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, is protected from light on ice, machine sorts cell in preparation.
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 is put into sorting cabin, starts 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.Then it further therefrom irises out
IgG+IgM-Cell colony, gating sort IgG+IgM-A-FMDV+The single B cell of cell, as A type FMDV specificity.
4. 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 is set are as follows: 25 DEG C, 5min；42 DEG C, 120min；85 DEG C, 5min.It obtains
CDNA-20 DEG C storage, be used for subsequent nested PCR amplification.
The nested PCR amplification of 4.2 unicellular cDNA
Table 1 is the primer for expanding pig IgG heavy chain, Kappa light chain and Lambda light-chain variable region gene, using in table 1
Shown primer takes nested PCR method, i.e. two-wheeled PCR amplification, to single B cell IgG heavy chain variable region (VH) gene of pig and
Lambda light chain variable region (VL) and Kappa light chain variable region (VL) gene are expanded.
(1) first round PCR amplification scheme
First round PCR reaction system:
Expand pig IgG heavy chain variable region (VH) gene and Lambda light chain variable region (VL) and Kappa light chain variable region
(VL) first round reaction system of gene, in addition to primer is different with template, the addition of remaining component is carried out according to table 2.
First round PCR amplification program are as follows: 95 DEG C of initial denaturation 15min first；Then 94 DEG C of 0.5 min of denaturation, annealing (annealing
Temperature is shown in Table 1) 0.5min, 72 DEG C of extension 1min, totally 32 circulations；Last 72 DEG C re-extend 10min.Wherein annealing temperature is pressed
It is chosen shown in table 1.Second wheel PCR reaction system
(2) second wheel PCR amplification schemes
Second wheel reaction system:
Using first round amplified production as template, add in corresponding trip primer amplification pig IgG heavy chain variable region (VH) gene and
Lambda light chain variable region (VL) and Kappa light chain variable region (VL) gene, the second wheel PCR reaction system component is according to table 3
It carries out.
Second wheel PCR amplification program are as follows: 95 DEG C of initial denaturation 15min first；Then 94 DEG C of 0.5 min of denaturation, annealing (annealing
Temperature is shown in Table 1) 0.5min, 72 DEG C of extension 1min, totally 36 circulations；Last 72 DEG C re-extend 10min.
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.
5. expression vector establishment and plasmid extraction
The building of 5.1 pig source single-chain antibody expression vectors
The pig IgG antibody variable region VH of amplification and VL gene are passed through into flexibility Linker " GGGGSGGSGGGGSGGS "
(SEQ ID NO.17) connection, and pig source IgG heavy chain secretion signal peptide sequence is introduced in fusion N-terminal
" MEFRLNWVVLFALLQGVQG " (SEQ ID NO.18) introduces Flag label and 6 × His label in fusion C-terminal, with
Conducive to detecting and purifying, gene order optimization is carried out then referring to Cricetulus griseus (CHO) codon, and in base
Because introducing KOZAK sequence " GCCACC " (SEQ ID NO.19) before initiation codon, finally synthesis gene by Not1 and
Nhel restriction enzyme site is inserted into pcDNA3.4 expression vector (Thermo scientific, USA).Sequent synthesis and vector construction
Trust money Wei Zhi biotech 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 antibody
Antibody is expressed by transiently transfecting CHO-S suspension cell, takes 20 μ g plasmids that 250 μ L OptiPRO are addedTMSFM culture
Base is diluted, while taking equivalent OptiPROTMSFM culture medium dilutes ExpiFectamineTMCHO-S transfection reagent, then
The plasmid diluted and transfection reagent are mixed, room temperature acts on 5min.The mixture is slowly added to 1.5 × 108A CHO-
In S cell.CHO-S cell is placed in 37 DEG C of constant temperature suspension incubators after transfection, suspends after culture 18h, adds 150 μ L CHO-
STMEnhancer and 6mL CHO-STMFeed.37 DEG C are continued after cultivating 9d, are centrifuged 30min by 10000 × g, collect cell training
Supernatant is supported, is filtered through 0.22 μm of filter, subsequent antibody purification is used for.Using HiTrap TALON pillar in AKAT protein purification
Antibody purification is carried out on instrument, using the PBS liquid antibody elution of 250mM imidazoles, is then charged into bag filter and is placed in PBS liquid and dialyse 3
It is secondary, each 3h.Antibody after dialysis is concentrated with PEG 8000, and SDS-PAGE electrophoretic analysis is carried out after concentration.
7. the activity verifying of the single-stranded genetic engineering antibody in the type FMDV pig source 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 A type FMDV
(A/GDMM/CHA/2013 strain) is inoculated with 24 porocyte culture plates, while setting does not connect malicious normal cell controls, 37 DEG C of cells
After being incubated for 4h in incubator, supernatant inactivation treatment is collected.Then Jia Chun ︰ acetone (1:1) fixer that -20 DEG C of pre-coolings are added is solid
Determine cell, room temperature acts on 20 min.PBS is washed cell 3 times, and the antibody of purifying, 37 DEG C of incubation 1h are added by 5 μ g/mL concentration.PBS
It washes 3 times, the anti-FLAG monoclonal antibody (Sigma, USA) of mouse, 37 DEG C of incubation 30min is added.PBS is washed 3 times, and the rabbit-anti of working concentration is added
Mouse IgG-Cy3 (health is reagent, Beijing) fluorescence secondary antibody, 37 DEG C of incubation 1h.Then it is washed cell 5 times with PBS, fluorescence microscope is seen
It examines fluorescence signal and 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 pig 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
Pig 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 pig source
The preparation flow of the single-stranded genetic engineering antibody in pig source is 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 9, 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 146S antigen particles integrality, be consequently adapted to be sorted as bait antigen
Single B cell.
3, the single B cell of A type FMDV specificity pig is successfully obtained
The single B cell of airflow classification A type FMDV specificity pig is as shown in Figure 10, wherein A: machine on PBMCs after dyeing, circle
Do well good P1 group；The shooting of B:P1 cell colony, irises out diagonal line single cell, excludes adhesion cells；C: IgG is irised out+
IgM-B cell；D: the FMO control of fluorescent antigen is not added；E: normal dyeing sample；Each cell colony in F:FMO check sample
Ratio cell (about 1,000,000 cells of record)；G: each cell colony ratio of normal dyeing sample is (record about 1,000,000 thin
Born of the same parents).
Flow cytometer showed result such as Figure 10 shows that A type FMDV specific b cells group (Figure 10 D) is high-visible, is present in IgG+IgM-Cell colony, account for about total PBMCs ratio be 56/1000000.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 (Figure 10 E, P4 group) are about 0/1000000 (Figure 10 G), are passed through
To P4 group in normal dyeing sample tube (Figure 10 D, IgG+IgM-A-FMDV+B cell) circle door sorting, use unicellular sorting
Mode, successfully sorting obtains the single B cell of pig of A type FMDV specificity.
The acquisition of the single B cell IgG variable region gene of 4.A type FMDV specificity pig
Pig IgG antibody variable gene PCR amplification result is as shown in figure 11, wherein Figure 11-1: pig IgG heavy chain variable region
PCR product electrophoresis result；Figure 11-2: pig kappa light chain variable region PCR product electrophoresis result；Figure 11-3: pig Lambda light chain
Variable region PCR product electrophoresis result.
Nested PCR product agarose gel electrophoresis analyze result it is as shown in figure 11, pig IgG heavy chain 450 bp-500bp it
Between there is high-visible band (Figure 11-1)；There is clear band (Figure 11-2) at about 475bp in pig kappa light chain variable region.
There is clear band (Figure 11-3) at about 475bp in pig Lambda light chain variable region.Sequencing result passes through Ig BLAST database
It compares, it was demonstrated that the above variable region sequences that nested PCR amplification obtains are the IgG antibody variable region gene sequence of pig.
5, the single-stranded genetic engineering antibody molecule in pig source that successful expression obtains
It is as shown in figure 12 to purify the single-stranded genetic engineering antibody in the type FMDV pig source A obtained, wherein M represents albumen
Marker, 1-5 represent the single-stranded genetic engineering antibody 1,2,3,4,5,6 in pig 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 35kDa in single-stranded genetic engineering antibody 1,2,3,5,6, is consistent with expection.These results explanation should
Method successful expression and can be purified into the single-stranded genetic engineering antibody molecule in pig source.
6. successfully obtaining the single-stranded genetic engineering antibody in anti-A type FMDV pig source
6.1IFA result
IFA testing result shows that single-stranded genetic engineering antibody 1,2,3,4,5,6 can be with A/GDMM/CHA/2013 strain
The BHK-21 cell-specific of infection combines, and obvious red fluorescence occurs, and the control cell for not connecing poison is glimmering without red color visible
Light.Result confirmation has successfully been obtained can be with the single-stranded genetic engineering antibody in pig source of A type FMDV specific reaction.
IFA experimental result is as shown in figure 13, wherein Figure 13-1 is single-stranded genetic engineering antibody 1 and A/GDMM/CHA/
2013 virus strain infection BHK21 cell experiment results；Figure 13-2 is single-stranded genetic engineering antibody 2 and A/GDMM/CHA/2013 strain
Infect BHK21 cell experiment result；Figure 13-3 be the single-stranded genetic engineering antibody in pig source 3 with A/GDMM/CHA/2013 strain sense
Contaminate BHK21 cell experiment result；Figure 13-4 is the single-stranded genetic engineering antibody 4 in pig source and normal BHK21 cell experiment result；Figure
13-5 is single-stranded genetic engineering antibody 5 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment result；Figure 13-6 is pig
The single-stranded genetic engineering antibody 6 in source and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment result；Figure 13-7 is not connect poison
Normal BHK21 cell controls.
6.2 result of indirect ELISA
Indirect ELISA experimental result is as shown in figure 14, and 6 plants of single-stranded genetic engineering antibodies can be with A type FMDV antigen knot
It closes, wherein single-stranded genetic engineering antibody 6 is to other a little higher than single-stranded genetic engineering antibodies of A type FMDV antigen relative affinity.It is single
For chain gene engineered antibody 5 and 6 pair A type FMDV antigen relative affinity between 0.01~0.1 μ g/mL, single-stranded genetic engineering is anti-
1,2,3,4 pair of A type FMDV antigen relative affinity of body is between 0.1~0.5 μ g/mL.
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 pig 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 6 plants of single-stranded genetic engineering antibodies in pig source is as shown in table 4, the single-stranded genetic engineering antibody in pig source 2,3,
4,6 have the ability for neutralizing A/GDMM/CHA/2013 strain, are A type FMDV neutralizing antibody, and the single-stranded genetic engineering in pig source is anti-
Body 1 and 5 is nonneutralizing antibody.
The neutralization titer of the 4 single-stranded genetic engineering antibody in pig source of table
6.4WB result
Electrophoresis is carried out to A/GDMM/CHA/2013 strain antigen, WB result is as shown in figure 15, wherein swimming lane 1-5 difference
Represent single-stranded genetic engineering antibody 1,2,3,4,5,6.The results show that single-stranded genetic engineering antibody 1 25kDa and 35kDa it
Between, occur specific bond band, corresponding FMDV Viral structural protein VP2 at about 28kDa.Single-stranded genetic engineering antibody 2,3 is in 25kDa
Between 35kDa, occur specific bond band, corresponding FMDV Structural protein VP1 at about 32kDa.The experimental result confirms single
What chain gene engineered antibody 1 identified is the linear epitope on A type FMDV Viral structural protein VP2, and single-stranded genetic engineering antibody 2,3 is known
Other is the linear epitope on A type FMDV Structural protein VP1.And that single-stranded genetic engineering antibody 4,5,6 identifies is A type FMDV
Comformational epitope on antigen.
Brief summary:
IFA and ELISA experimental result confirms, screens 6 plants of single-stranded genetic engineering antibodies in pig 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 2,3,4,6 in pig 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 pig source is anti-
What body 1 identified is the linear epitope on A type FMDV capsid protein VP2, and what antibody 2 and 3 identified is A type FMDV capsid protein
Linear epitope on VP1, and the comformational epitope for the A type FMDV antigen that the single-stranded genetic engineering antibody 4,5,6 in pig source identifies.It is comprehensive
Result above, this research provide a kind of method of the single-stranded genetic engineering antibody in anti-A type FMDV pig source, and the screening technique is to grinding
The identification for studying carefully A type FMDV host specificity antigen site provides important method, and then mentions for the design of aftosa molecular vaccine
For 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 foot-and-mouth disease virus resistant pig source
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<170> SIPOSequenceListing 1.0
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gtttcggctg aactgggtgg tc 22
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<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtcactgrc tcggggaagt agc 23
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggtggagtst ggrggaggcc tg 22
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cagggggcca gagggtagac c 21
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atggcctgga yccctctcct gctc 24
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<211> 25
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atggcccggg cttggctcct tgtca 25
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<400> 7
atggcctgga cggtgcttct gatc 24
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<211> 19
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<213>artificial sequence (Artificial Sequence)
<400> 8
cctccaggtc acsgtcacg 19
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<211> 20
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<213>artificial sequence (Artificial Sequence)
<400> 9
tcctgtgagc tgactcagcc 20
<210> 10
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<400> 10
cagkctgysc tgactcagc 19
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<213>artificial sequence (Artificial Sequence)
<400> 11
tctcagactg tgatccagga g 21
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<213>artificial sequence (Artificial Sequence)
<400> 12
gtcacttatt agacacacca gggtg 25
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<213>artificial sequence (Artificial Sequence)
<400> 13
atgagggccc ccrtgcagct cct 23
<210> 14
<211> 21
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<213>artificial sequence (Artificial Sequence)
<400> 14
tgtccttgct gtcctgctct g 21
<210> 15
<211> 21
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<213>artificial sequence (Artificial Sequence)
<400> 15
tcctcctgct ctgggtccca g 21
<210> 16
<211> 21
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<213>artificial sequence (Artificial Sequence)
<400> 16
gatgaagacg gatggcttgg c 21
<210> 17
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ggggsggsgg ggsggs 16
<210> 18
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
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Met Glu Phe Arg Leu Asn Trp Val Val Leu Phe Ala Leu Leu Gln Gly
1 5 10 15
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<210> 19
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gccacc 6
Claims (8)
1. a kind of preparation method of the single-stranded genetic engineering antibody in foot-and-mouth disease virus resistant pig source, comprising the following steps:
1) separating peripheral blood mononuclear cells from the pig blood sample of immune foot-and-mouth disease virus antigen；
2) ultrafiltration centrifugation carried out to FMDV 146S antigen, FMDV 146S antigen after being concentrated, using dyestuff to concentration after
FMDV 146S antigen dyed, the antigen after dyeing is subjected to ultrafiltration centrifugation, FMDV 146S antigen after being marked；
3) the FMDV 146S antigen after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains, mouse is anti-
Pig IgM-PE and the anti-pig IgG-FITC fluorescence antibody mixing of mouse, the cell after being dyed；
4) using the single B cell of polychrome selected by flow cytometry apoptosis FMDV specificity；
5) reverse transcription is carried out to the single B cell of FMDV specificity that step 4) obtains, after obtaining cDNA, utilizes such as SEQ ID
Primer shown in NO.1~16 carries out nested PCR amplification, obtains pig IgG heavy chain variable region gene, Lambda light chain variable region base
Cause and Kappa light-chain variable region gene；
6) the pig IgG heavy chain variable region gene for obtaining step 5) respectively can with Lambda light-chain variable region gene, Kappa light chain
Become area's gene to introduce respectively such as by flexibility Linker connection, the N-terminal of gene upon connection as shown in SEQ ID NO.17
Signal peptide sequence described in SEQ ID NO.18 introduces Myc label and His label in C-terminal, with reference to Cricetulus respectively
Griseus codon carries out gene order optimization respectively, introduces KOZAK sequence before gene start codon after optimization respectively
Column, are inserted respectively into pcDNA3.4 expression vector, obtain Lambda pig source single-chain antibody expression vector and kappa pig source is single-stranded anti-
Body expression vector；
7) step 6) is obtained into Lambda pig source single-chain antibody expression vector and kappa pig source single-chain antibody expression vector turns respectively
Contaminate the expression that cell carries out antibody；
The step 1) and 2) the not restriction of chronological order.
2. preparation method according to claim 1, which is characterized in that the step 1) foot and mouth disease virus include O-shaped FMDV,
A type FMDV, c-type FMDV, Asial type FMDV, SAT1 type FMDV, SAT2 type FMDV and SAT3 type FMDV.
3. preparation method according to claim 1, which is characterized in that step 1) the pig blood sample is immune aftosa
The pig blood sample of 14~30d of viral antigen.
4. preparation method according to claim 1, which is characterized in that the step 2) dyestuff includes FluoProbes
647H dyestuff or Pacific Blue dyestuff.
5. preparation method 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.
6. preparation method 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, therefrom iris out IgG+IgM-Cell
Group, circle door sort IgG+IgM-FMDV+Cell.
7. preparation method according to claim 1, which is characterized in that step 6) is inserted into using Notl and Nhel restriction enzyme site
To pcDNA3.4 expression vector.
8. preparation method according to claim 1, which is characterized in that the step 7) cell includes CHO-S suspension cell.
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