CN108250295A - A kind of method using phage antibody library screening estrogen receptor alpha single chain antibody - Google Patents
A kind of method using phage antibody library screening estrogen receptor alpha single chain antibody Download PDFInfo
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- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
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Abstract
The present invention discloses a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody, includes the following steps：ER α albumen is as immunogen immune new zealand white rabbit and spleen Total RNAs extraction；RT PCR amplification VH and VL segments and scFv gene splicings；Build PCANTAB5E ScFv recombinant plasmids, recombinant plasmid electricity Transformed E .coli TG1 competent cells structure phage antibody library；The breast tumor tissue sections and ER α albumen of the ER α positives carry out affine enrichment isolation as immobilization antigen to antibody library；Positive clone molecule is identified using the method that Phage ElLSA methods and breast cancer cell smear immunochemistry are combined.The present invention establishes a kind of method for carrying out estrogen receptor specificity screening to phage antibody library using ER α positive breast cancers histotomies, and identify positive clone molecule using the method that phage ElLSA methods and breast cancer cell smear immunochemistry are combined, the screening technique for phage antibody library provides new thinking.
Description
Technical field
The invention belongs to phage antibody library screening technique technical fields, and in particular to a kind of to be sieved using phage antibody library
The method for selecting estrogen receptor alpha single chain antibody.
Background technology
Estrogen receptor (estrogen receptor, ER) is used as a kind of biomarker, it is both category nuclear hormone receptor
One kind of (nuclear receptor, NR) albumen, and be to belong to the transcription factor with activation, presently found ER tools
There are tri- kinds of ER α, ER β, ER γ hypotypes.In recent years, researcher has found breast cancer disease of the estrogen receptor alpha (ER α) about 70%
High expression is all shown in example and high activity is horizontal.The expression of ERa and breast cancer are closely related, particularly the development of tumour.Example
Such as, ER alpha expressions are reduced in breast cancer treatment and often has better therapeutic effect.The effect of ER expressions and endocrine therapy
Closely related, ER detections help to predict reaction of the patient to endocrine therapy, and testing result will directly determine therapeutic scheme
Selection.Accurate detection and report ER states are extremely important to the clinical treatment and Index for diagnosis of breast cancer, it has also become breast cancer
Essential content in pathological diagnosis report.Immunohistochemistry is the best approach of current ER detections.
The antigen gene sequences of foreign protein were passed through technique for gene engineering by U.S. doctor Smithm first time in 1985
It is assembled in inside bacteriophage, as Phage Infection host strain completes self assembly, the case surface of bacteriophage illustrates egg
White antigenic determinant (with the secondary coat protein PIII amalgamation and expressions of bacteriophage), while propose the general of display technique of bacteriophage
It reads.Nowadays display technique of bacteriophage has become the screening destination protein and strong tool of target gene, be it is a kind of after
Polyclonal technology, even more important technique for gene engineering after monoclonal technigue.The technology is now widely used for screening polypeptide
With the substances such as protein, antibody, have many advantages, such as it is easy, efficiently, low cost, which can be from extensive and random library
Filter out the antibody with target proteins combination high-affinity, high specific.
Phage antibody library washes in a pan the committed step that sieved journey is library, classical antigen selection method, that is, solid-phase screening,
Purifying antigen is coated on one plant of specific bacteriophage of screening, this method on such as ELISA Plate, affinity column solid-phase media
It is to elute nonspecific bacteriophage, the process that specific recombinant phage is enriched with.But sometimes due to position
Point closing is incomplete, it may appear that situation that Non-specific phage the combines and positive colony filtered out fails in immunohistochemistry knot
Preferable binding ability is shown on fruit.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of single using phage antibody library screening estrogen receptor alpha (ER α)
The method of chain antibody
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of method using phage antibody library screening estrogen receptor alpha single chain antibody includes the following steps：
(1) exempted from three times as immunogen immune experimental animal new zealand white rabbit using estrogen receptor alpha (ER α) albumen
Auricular vein takes blood after epidemic disease, and using ELISA method detection serum titer, booster immunization is after three days after potency reaches requirement, place
Dead experimental animal extracts spleen total serum IgE, reverse transcription synthesis cDNA under aseptic condition；
(2) design degenerate primer amplification heavy chain variable region gene (VH) and chain variable region gene (VL), using SOE-PCR
Splicing VH and VL is scFv genes；
(3) double digestion processing is carried out respectively to PCANTAB5E phagemids and scFv genes, enzyme even builds PCANTAB-
ScFv recombinant plasmids, the primary phage antibody library of recombinant plasmid electricity Transformed E .coli TG1 competent cells structure, conversion
Liquid gradient dilution is simultaneously coated with 2 × YT-A tablets calculating storage capacity, and picking monoclonal bacterium solution PCR calculates recombination fraction；
(4) primary phage antibody library is incubated together with the breast tumor tissue sections of the ER α positives, carries out three-wheel enrichment
Screening, the three-level phage antibody library being enriched with and the ER α albumen being incorporated on solid phase carrier carry out two-wheeled enrichment again
Screening obtains the specific bacteriophage single-chain antibody library of affinity height, high specificity；
(5) specific bacteriophage single-chain antibody library infects E.coli TG1, gradient dilution coating 2 × YT-A, picking Dan Ke
Grand preparation bacteriophage supernatant carries out Preliminary Identification using phage-ElLSA methods, and the monoclonal for choosing OD450 higher is prepared again
Bacteriophage supernatant is incubated together with the breast cancer cell (T47D) being fixed on glass slide, positive gram of immunocytochemistry identification
Longzi.
In step (1), the ER α albumen is re-used as immunogene after the hot repair process of EDTA antigens in advance, which makes
The antibody that must be prepared is more targeted to immunohistochemistry detection, and antibody nuclear location is apparent, and background is cleaner, without non-specific
Property coloring.
In step (2), 9 pairs of primer amplification VL genes, 4 pairs of primer amplification VH genes, in VL genes and VH genes are designed altogether
Between addition one section of flexible polypeptide chain Linker be connected, the polypeptide chain Linker amino acid sequences be GGGGSGGGGSGGGGS.
In step (3), the PCANTAB5E phagemids and scFv genes are carried out at sfiI and NotI double digestions respectively
Reason turns special ligase connection structure PCANTAB-ScFv recombinant plasmids using T4DNA electricity, and electricity conversion Escherichia coli electricity turns special
Competent cell TG1, according to MOi=100:1, which adds in titre, is more than 1012The helper phage M13KO7 of pfu/ml, structure are primary
Phage antibody library, primary phage antibody library calculate phage titre, random picking list using double layer agar method
It clones bacterium solution PCR and calculates recombination fraction.
In step (4), primary phage antibody library is incubated together with the breast tumor tissue sections of the ER α positives, is discarded
The bacteriophage not combined with tissue, combining bacteriophage is eluted with Gly-HCL to be expanded, and carries out three-wheel enrichment sieve
Choosing obtains three-level phage antibody library；Three-level phage antibody library is together with the ER α albumen being incorporated on ELISA Plate
It is incubated, increases washing steps and wash away unbonded bacteriophage, enrichment and the protein bound bacteriophages of ER α, carry out to greatest extent
Two-wheeled enrichment isolation obtains the specific bacteriophage single-chain antibody library of affinity height, high specificity.
In step (5), specific bacteriophage single-chain antibody library infects E.coli TG1, and gradient dilution is coated with 2 × YT-A, chooses
30 monoclonals is taken to prepare bacteriophage supernatant as primary antibody, the HRP anti-M13KO7 of label mouse are secondary antibody, using phage-ElLSA methods
Preliminary Identification is carried out, 7 plants of monoclonals for choosing OD450 higher prepare bacteriophage supernatant again, with being fixed on mammary gland on glass slide
Cancer cell (T47D) is incubated together, and the anti-M13KO7 of HRP label mouse is secondary antibody, is dyed through DAB, haematoxylin is redyed, and PBS returns blue step
Afterwards, observation cell smear identification positive clone molecule.
Method of the present invention using phage antibody library screening estrogen receptor alpha single chain antibody, it is specific as follows：
1) the immune and spleen Total RNAs extraction of ER α Protein Assavs rabbit：ER α recombinant proteins are prepared by laboratory and purified, and are used
The ER α albumen that the hot repair process of EDTA antigen retrieval buffers has diluted, labeled as ER α-AR.The ER α albumen repaired is as immune
The immune new zealand white rabbit of original, it is negative control that vestibule edge venous blood sampling, which is immunized,.It is carried out every two weeks after first immunisation primary
Immune, auricular vein takes blood, ELISA method detection antibody titer after third time is immune, and potency reaches 105After carry out last
Secondary booster immunization, puts to death animal after three days, extract spleen cell, obtains total serum IgE, is template with Oligo (dT) using the RNA of purifying
The first chain cDNA is synthesized for primer reverse transcription.
2) RT-PCR expands VH and VL segments and scFv gene splicings：Rabbit source heavy chain of antibody and light chain according to having delivered can
Become area's gene order, design 9 pairs of primer amplification VL genes, 4 pairs of primer amplification VH genes altogether.PCR response procedures are：95 DEG C of pre- changes
Property 5min, 95 DEG C denaturation 30s, 58 DEG C annealing 45s, 72 DEG C extension 1min, 35 cycle after 72 DEG C of 5min, 4 DEG C preservation.PCR is produced
Object becomes scFv after the recycling of 1% Ago-Gel using the splicing of over-lap PCR method.PCR product is put through the recycling of 1% Ago-Gel
In -20 DEG C of preservations.
3) structure of primary phage antibody library and identification:It is right according to NEB companies of Britain double digestion system
PCABTAB5E phagemids and scFv segments carry out sfiI and NotI digestions processing respectively, make after the recycling of digestion products electrophoresis
Turn special ligase with T4DNA electricity to connect.Connection product electricity conversion Escherichia coli electricity turns special competent cell TG1, conversion fluid
2 × YT culture mediums are added in, 37 DEG C of shaking table recovery concussion 1h take out 100ul bacterium solutions, 10 gradient doubling dilutions are coated with 2 × YT-A and put down
Plate, remaining conversion fluid is according to MOi=100:1 adds in helper phage M13KO7,37 DEG C of water-bath 30min, 37 DEG C of shaking table cultures
30min.Supernatant is abandoned in centrifugation, is resuspended and precipitated with 100ml2 × YT-AK culture mediums, and 37 DEG C of shaking table shake cultures are stayed overnight.It is collected by centrifugation
Supernatant obtains primary phage antibody library through PEG/Nacl ice bath precipitating 1h.Recombinant phage titre is measured, and at the beginning of picking
Grade 20 monoclonals of phage antibody library, bacterium solution PCR calculate recombination fraction.
4) enrichment isolation of phage antibody library：The breast tumor tissue sections of the ER α positives are placed in 65 DEG C of roasting piece machines
Roasting piece processing 20min is carried out, the histotomy being baked is immersed in 10min in dimethylbenzene I, dimethylbenzene II, completes dewaxing successively
Process.After dewaxing treatment, histotomy is immersed in absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethyl alcohol I, 95% second successively
Slice is immersed in tap water by each 5min in alcohol II, 85% ethyl alcohol after completing hydration process；20ul EDTA are drawn to be added to
It in 1L ultra-pure waters, is poured into pot after mixing, histotomy is put into and is submerged, 1000W power boils 20min processing, boils
After, room temperature is naturally cooled to, 3min is impregnated in ultra-pure water, gets rid of excessive moisture, with PAP oil pens along histotomy
Profile is drawn a circle, and being sure not dry plate causes tissue to fall, and completes antigen retrieval process.In aseptic operating platform, even 2~3 drop endogenous of drop
Peroxidase blocking reagent is put into 37 DEG C of incubators and is incubated 10min.PBS is washed away, and animal non-immune serum is added dropwise, is put into 37
20min is incubated in DEG C incubator, PBS is washed away.Primary phage antibody library is added dropwise in histotomy, 37 DEG C of sterile incubations
The PBST of 1.5h, 0.05%Tween-20 develop a film 5 times, and rear two-wheeled is respectively 10 times, and 15 times, PH2.2,0.2M Gly-HCl are washed
It is de-, after pH7.4,2.0M Tris-HCl are neutralized, add in host strain TG1,37 DEG C of water-baths 30min, 37 DEG C of shaking table culture 30min.This
Operation is repeated 3 times, and obtains three-level phage antibody library.According to the principle of solid-phase screening, using ELISA Plate to three-level phagocytosis
Body single-chain antibody library carries out two-wheeled enrichment isolation again.ER α antigen coat concentration is respectively 15ug/ml and 10ug/ml, package amount
50ul, board-washing number are respectively 15 times, 20 times.Confining liquid be the PBS buffer solution containing 3% defatted milk, cleaning solution 0.05%
The PBST of Tween-20.Bacteriophage uses pH2.2, and 0.2M Gly-HCl elutions are neutralized using pH7.4,2.0M Tris-HCl
Afterwards, host strain TG1,37 DEG C of water-baths 30min, 37 DEG C of shaking table culture 30min are added in.Each round screening will calculate input bacteriophage
With the output phagocytosis scale of construction.
5) phage-ELISA methods Preliminary Identification positive clone molecule：30 lists are selected on the tablet terminated from the 5th wheel screening
Clone expands culture, and Prepare restructuring phagocytosis body fluid, using ER α as antigen, using the principle of ELISA, will be coated with overnight enzyme respectively
After target is using the PBS Seal treatments of 3%BSA, the bacteriophage supernatant prepared is added in, 37 DEG C combine 1.5h, PBST washings 3
It is secondary, the HRP label anti-M13 monoclonal antibodies of mouse diluted, 37 DEG C of incubation 45min are added in, TMB developing solutions are protected from light colour developing 20min,
Microplate reader reads OD450.It chooses the higher monoclonal of reading and carries out cellular immunity group.
6) T47D cellular immunities group specificity identification positive clone molecule：With the adherent T47D cells of trypsin digestion, training
Base pressure-vaccum is supported, obtains single cell suspension, and be collected in 1.5ml centrifuge tubes；1000g, 4 DEG C of centrifugation 5min, discards culture medium；Add
Enter 1ml PBS and cell precipitation, 1000g is resuspended, 4 DEG C of centrifugation 5min discard PBS, are repeated twice, clean the culture of cell surface
Base；The paraformaldehyde of addition 4% gives fixed 20min, and suspension is coated on glass slide, is placed in 60 DEG C of freeze-day with constant temperature incubators,
Dry piece 5-10min；It draws 20ul EDTA to be added in 1L ultra-pure waters, is poured into pot after mixing, cell smear is put into and is immersed
Not yet, 1000W power boils 20min processing, after boiling, naturally cools to room temperature, impregnates 3min in ultra-pure water, get rid of more
Remaining moisture content is drawn a circle with PAP oil pens along the profile of histotomy, and being sure not dry plate causes cell to fall, and completes antigen retrieval mistake
Journey；Even 2~3 drop endogenous peroxydase blocking agent is dripped, is put into 37 DEG C of incubators and is incubated 10min, PBS is washed away；It is added dropwise dynamic
Object non-immune serum is put into 37 DEG C of incubators and is incubated 20min, and PBS is washed away；Bacteriophage supernatant is added dropwise, 37 DEG C of incubators are incubated
1.5h；PBS washes away bacteriophage, and the HRP label mouse antiphagin secondary antibody 100ul diluted are added dropwise, is put into 37 DEG C of incubators and incubates
Educate 30min；PBS washes away secondary antibody, adds in prepared DAB100ul, is stored at room temperature 5min, and PBS is washed away；Haematoxylin dyeing 20s,
Tap water rinses, and PBS returns blue 20s, and tap water rinses, and is placed in 60 DEG C of freeze-day with constant temperature incubators, dries moisture；It is neutral that 2 drops are added dropwise
Resin, the resinene for gently being contacted and being spread apart using coverslip is slowly close, until whole combinations, dries tissue, makes
It is observed and made film with inverted microscope, find positive stronger clone.
The present invention is established a kind of utilization ER α positive breast cancer histotomies and bacteriophage is resisted using above technical scheme
The method that body library carries out estrogen receptor specificity screening, and utilize phage-ElLSA methods and breast cancer cell (T47D) smear
The method that immunochemistry is combined identifies positive clone molecule, and the screening technique for phage antibody library provides new thinking.
The advantage of the invention is that：This method makes phage library be combined enrichment, enriched antibody library with the breast tumor tissue sections of the ER α positives
Monoclonal bacterial strain is screened using breast cancer cell (T47D), and specific single-chain antibody is incorporated in T47D cell surfaces, without with its
His cell combination enhances the specificity of screening.
Description of the drawings
Fig. 1 is extracts spleen rna agarose gel electrophoresis figure, M：Marker DL 2000；1：Extract RNA.
Fig. 2 be VL gene PCR product agarose gel electrophoresis figures, M：Marker DL 2000；1-2：VL gene PCRs expand
Product.
Fig. 3 be VH gene PCR product agarose gel electrophoresis figures, M：Marker DL 2000；1-2：VH gene PCRs expand
Product.
Fig. 4 be scFv gene PCR product agarose gel electrophoresis figures, M：Marker DL 2000；1-2：ScFv gene PCRs
Amplified production.
Fig. 5 identifies agarose gel electrophoresis figure, M for antibody library recombination fraction bacterium solution PCR：Marker DL 2000；1-18：18
Monoclonal bacterium solution PCR results.
Fig. 6 detects block diagram for anti-ER alpha single chain antibodies (ScFv) ELISA.
Fig. 7 screens positive monoclonal immunohistochemistry figure for cell smear, and a, b figures are No. 19 monoclonals；C, d figure are No. 13 lists
Clone；E, f figure are No. 9 monoclonals；G, h figure are No. 22 monoclonals；I, j figure are negative control；K, l figure are positive control.
Specific embodiment
The specific embodiment of the present invention is described in detail with reference to embodiment, it is to be understood that the present invention
Protection domain is not restricted by specific implementation.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc. unless otherwise specified, commercially obtain.
Material：Escherichia coli Ecoli.TG1, phage vector pCANTAB5E, helper phage M13KO7, HRP label mouse
Anti- M13 antibody is purchased from Beijing Bao Kewei food peaces.New zealand white rabbit is purchased from Foochow Wu Shi animals.The primer is given birth to by Shanghai
Work biotech firm synthesizes.The breast tumor tissue sections of the ER α positives, T47D breast carcinoma cell strains, TransZol Up RNA Kit,
CDNA synthetic agent box, RT-PCR kit, purchased from the full formula gold in Beijing.Restriction enzyme sfiI, NotI-HF, T4DNA are connected
Enzyme, purchased from NEB companies.SanPrep pillar DNA plastic recovery kits, SanPrep pillar plasmids, DNA Mini Kits,
Purchased from OMEGA companies.Other reagents are that domestic analysis is pure.
Embodiment 1
A kind of method using phage antibody library screening estrogen receptor alpha single chain antibody：
1) the immune and spleen Total RNAs extraction of ER α Protein Assavs rabbit：ER α recombinant proteins are prepared by laboratory and purified, and are used
The ER α albumen that the hot repair process of EDTA antigen retrieval buffers has diluted, labeled as ER α-AR.The albumen repaired is exempted from as immunogene
Epidemic disease new zealand white rabbit, it is negative control that vestibule edge venous blood sampling, which is immunized,.Primary immunization is carried out after first immunisation every two weeks,
Auricular vein takes blood, ELISA method detection antibody titer after third time is immune, and potency reaches 105Last time is carried out afterwards to add
It is strong immune, animal is put to death after three days, extracts spleen cell, obtains total serum IgE, is to draw with Oligo (dT) using the RNA of purifying as template
The synthesis of object reverse transcription the first chain cDNA, RNA are template reverse transcription reaction system, add in RNA templates, RNase-free Water,
Anchored Oligo(dT)18Primer, 65 DEG C of incubation 5min, places two minutes, adds 2 × TS Reaction on ice
Mix (R-Mix), TransScript RT/RI Enzyme Mix (E-Mix), gDNA Remover, 42 DEG C of incubation reverse transcriptions
30min.85 DEG C of heating, 5 seconds inactivation E-Mix and gDNA Remover.
2) RT-PCR expands VH and VL segments and scFv gene splicings：Rabbit source heavy chain of antibody and light chain according to having delivered can
Become area's gene order, design 9 pairs of primer amplification VL genes, 4 pairs of primer amplification VH genes altogether.PCR response procedures are：95 DEG C of pre- changes
Property 5min, 95 DEG C denaturation 30s, 58 DEG C annealing 45s, 72 DEG C extension 1min, 35 cycle after 72 DEG C of 5min, 4 DEG C preservation.PCR is produced
Object becomes scFv after the recycling of 1% Ago-Gel using the splicing of over-lap PCR method.PCR product is put through the recycling of 1% Ago-Gel
In -20 DEG C of preservations.
3) primary phage antibody library：According to NEB companies of Britain double digestion system, to pCABTAB5E plasmids and
ScFv segments carry out sfiI and NotI digestions processing respectively, turn special ligase using T4DNA electricity after the recycling of digestion products electrophoresis
Connection.Connection product electricity conversion Escherichia coli electricity turns special competent cell TG1, and conversion fluid adds in 37 DEG C of 2 × YT culture mediums and shakes
Bed recovery concussion 1h takes out 100ul bacterium solutions, and 10 gradient doubling dilutions coating 2 × YT-A tablets, remaining conversion fluid is according to MOi=
100:1, which adds in titre, is more than 1012The helper phage M13KO7,37 DEG C of water-baths 30min, 37 DEG C of shaking table culture 30min of pfu/ml.
Centrifugation abandons supernatant 100ml2 × YT-AK culture mediums and precipitation is resuspended, and 37 DEG C of shaking table shake cultures are stayed overnight.Supernatant warp is collected by centrifugation
PEG/Nacl ice bath precipitating 1h obtain primary phage antibody library.Measure recombinant phage titre, and picking primary phagocytosis
20 monoclonals of body single-chain antibody library, bacterium solution PCR calculate recombination fraction.
4) enrichment isolation of phage antibody library：The breast tumor tissue sections of the ER α positives are placed in 65 DEG C of roasting piece machines
Roasting piece processing 20min is carried out, the histotomy being baked is immersed in 10min in dimethylbenzene I, dimethylbenzene II, completes dewaxing successively
Process.After dewaxing treatment, histotomy is immersed in absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethyl alcohol I, 95% second successively
Slice is immersed in tap water by each 5min in alcohol II, 85% ethyl alcohol after completing hydration process；20ul EDTA are drawn to be added to
It in 1L ultra-pure waters, is poured into pot after mixing, histotomy is put into and is submerged, 1000W power boils 20min processing, boils
After, room temperature is naturally cooled to, 3min is impregnated in ultra-pure water, gets rid of excessive moisture, with PAP oil pens along histotomy
Profile is drawn a circle, and being sure not dry plate causes tissue to fall, and completes antigen retrieval process.In aseptic operating platform, even 2~3 drop endogenous of drop
Peroxidase blocking reagent is put into 37 DEG C of incubators and is incubated 10min.3 × 3min of PBS are washed away, and the nonimmune blood of animal is added dropwise
Clearly, it is put into 37 DEG C of incubators and is incubated 20min, PBS3 × 3min is washed away.The dropwise addition of primary phage antibody library is being organized
Slice, the PBST of 37 DEG C of sterile incubation 1.5h, 0.05%Tween-20 develops a film 5 times, and rear two-wheeled is respectively 10 times, 15 times,
PH2.2,0.2M Gly-HCl are eluted, and after pH7.4,2.0M Tris-HCl are neutralized, add in host strain TG1,37 DEG C of water-bath 30min,
37 DEG C of shaking table culture 30min.This operation is repeated 3 times, and obtains three-level phage antibody library.According to the principle of solid-phase screening,
Two-wheeled enrichment isolation is carried out using ELISA Plate again to three-level phage antibody library.ER α antigen coat concentration is respectively 15ug/
Ml and 10ug/ml, package amount 50ul, board-washing number are respectively 15 times, 20 times.Confining liquid is the PBS bufferings containing 3% defatted milk
Liquid, cleaning solution are the PBST of 0.05%Tween-20.Bacteriophage uses pH2.2,0.2M Gly-HCl elutions, using pH7.4,
After 2.0M Tris-HCl are neutralized, host strain TG1,37 DEG C of water-baths 30min, 37 DEG C of shaking table culture 30min are added in.Each round is screened
Input bacteriophage and the output phagocytosis scale of construction will be calculated.
5) phage-ELISA methods Preliminary Identification positive clone molecule：30 lists are selected on the tablet terminated from the 5th wheel screening
Clone expands culture, and Prepare restructuring phagocytosis body fluid, using ER α as antigen, using the principle of ELISA, will be coated with overnight enzyme respectively
After target is using the PBS Seal treatments of 3%BSA, the bacteriophage supernatant prepared is added in, 37 DEG C combine 1.5h, PBST washings 3
It is secondary, the HRP label anti-M13 monoclonal antibodies of mouse diluted, 37 DEG C of incubation 45min are added in, TMB developing solutions are protected from light colour developing 20min,
Microplate reader reads OD450.It chooses the higher monoclonal of reading and carries out milling immunohistochemistry screening.
6) T47D cellular immunities group specificity identification positive clone molecule：With the adherent T47D cells of trypsin digestion, training
Base pressure-vaccum is supported, obtains single cell suspension, and be collected in 1.5ml centrifuge tubes；1000g, 4 DEG C of centrifugation 5min, discards culture medium；Add
Enter 1ml PBS and cell precipitation, 1000g is resuspended, 4 DEG C of centrifugation 5min discard PBS, are repeated twice, clean the culture of cell surface
Base；The paraformaldehyde of addition 4% gives fixed 20min, and suspension is coated on glass slide, is placed in 60 DEG C of freeze-day with constant temperature incubators,
Dry piece 5-10min；It draws 20ul EDTA to be added in 1L ultra-pure waters, is poured into pot after mixing, cell smear is put into and is immersed
Not yet, 1000W power boils 20min processing, after boiling, naturally cools to room temperature, impregnates 3min in ultra-pure water, get rid of more
Remaining moisture content is drawn a circle with PAP oil pens along the profile of histotomy, and being sure not dry plate causes cell to fall, and completes antigen retrieval mistake
Journey；Even 2~3 drop endogenous peroxydase blocking agent is dripped, incubation 10minPBS in 37 DEG C of incubators is put into and washes away；Animal is added dropwise
Non-immune serum is put into 37 DEG C of incubators and is incubated 20min, and PBS is washed away；Bacteriophage supernatant is added dropwise, 37 DEG C of incubators are incubated
1.5h；PBS washes away bacteriophage, and the HRP label mouse antiphagin secondary antibody 100ul diluted are added dropwise, is put into 37 DEG C of incubators and incubates
Educate 30min；PBS washes away secondary antibody, adds in prepared DAB100ul, is stored at room temperature 5min, and PBS is washed away；Haematoxylin dyeing 20s,
Tap water rinses, and PBS returns blue 20s, and tap water rinses, and is placed in 60 DEG C of freeze-day with constant temperature incubators, dries moisture；It is neutral that 2 drops are added dropwise
Resin, the resinene for gently being contacted and being spread apart using coverslip is slowly close, until whole combinations, dries tissue, makes
It is observed and made film with inverted microscope, find positive stronger clone.
1 rabbit immunologic process of table
2 ER alpha immunization rabbit anteserum titres of table
Table 3 tests the primer sequence
Note：Light chain primer is according to VL-FP1 and VL-RP1, VL-FP1 and VL-RP2, VL-FP1 and VL-RP3, VL-FP2
With VL-RP1, VL-FP2 and VL-RP2, VL-FP2 and VL-RP3, VL-FP3 and VL-RP1, VL-FP3 and VL-RP2, VL-FP3 with
VL-RP3 is combined, totally 9 pairs；
Heavy chain primer is according to VH-RP and VH-FP1, VH-RP and VH-FP2, VH-RP and VH-FP3, VH-RP and VH-FP4
Combination, totally 4 pairs.
The titre of bacteriophage and output bacteriophage is put into 4 phage selection of table
Analysis of experimental results：Rabbit potency is immunized as seen from Table 2 and reaches 105, reach and RNA taken to require.3 primer of experimental applications table
Successful amplification goes out VH and VL sequences, and size meets expected size and successful stitch scFv, size is on a 750bp left sides in 350bp or so
It is right.Structure recombinant phage titer determination is reaches 1012Pfu/ml, storage capacity are calculated as 1.9 × 108.18 single bacteriums of random picking
It falls, is detected using scFv splicing primers, wherein having the specific band that size 750bp is amplified in 17 bacterium solutions, explanation
There is gene insertion.Gene insertion rate is about 94%.Five wheel enrichment isolation result such as tables 4 find out that the output phagocytosis scale of construction is by the first round
10-7It is increased to the 10 of the 5th wheel-5, 100 times are improved, phage antibody library obtains specific enrichment.Utilize phage-
ELISA takes the preferable 7 plants of monoclonals of qualification result to prepare bacteriophage supernatant, with T47D cells 30 plants of monoclonal Preliminary Identifications
It is incubated together, cellular immunity groupization detection binding ability.Compared with negative control and positive control, one plant of binding ability is filtered out
It is good, the high monoclonal of specificity.
The above content is combine specific preferred embodiment to the further description of the invention made, it is impossible to assert this
The specific implementation of invention is confined to these explanations, for those of ordinary skill in the art to which the present invention belongs, not
Be detached from present inventive concept under the premise of, several simple deduction or replace can also be made, should all be considered as belonging to the present invention carried
The protection domain that claims of friendship determine.
Claims (7)
- A kind of 1. method using phage antibody library screening estrogen receptor alpha single chain antibody, it is characterised in that：It includes following Step：（1）Using ER α albumen as immunogen immune experimental animal, auricular vein takes blood after being immunized three times, using ELISA Method detects serum titer, and booster immunization puts to death experimental animal after three days after potency reaches requirement, and extraction spleen is total under aseptic condition RNA, reverse transcription synthesis cDNA；（2）Design degenerate primer amplification heavy chain variable region gene VH and chain variable region gene VL, using SOE-PCR splicing VH and VL is scFv genes；（3）Carry out double digestion processing respectively to PCANTAB5E phagemids and scFv genes, enzyme even builds PCANTAB-ScFv Recombinant plasmid, the primary phage antibody library of recombinant plasmid electricity Transformed E .coli TG1 competent cells structure, conversion fluid ladder Degree, which dilutes and is coated with 2 × YT-A tablets, calculates storage capacity, and picking monoclonal bacterium solution PCR calculates recombination fraction；（4）Primary phage antibody library is incubated together with the breast tumor tissue sections of the ER α positives, carries out three-wheel enrichment sieve Choosing, the three-level phage antibody library being enriched with and the ER α albumen being incorporated on solid phase carrier carry out two-wheeled enrichment and sieve again Choosing obtains the specific bacteriophage single-chain antibody library of affinity height, high specificity；（5）Specific bacteriophage single-chain antibody library infects E.coli TG1, and gradient dilution is coated with 2 × YT-A, picking monoclonal system Standby bacteriophage supernatant carries out Preliminary Identification using phage-ElLSA methods, and the monoclonal for choosing OD450 higher prepares phagocytosis again Body supernatant is incubated together with the breast cancer cell T47D being fixed on glass slide, immunocytochemistry identification positive clone molecule.
- 2. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（1）In, the experimental animal is new zealand white rabbit.
- 3. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（1）In, the ER α albumen is re-used as immunogene after the hot repair process of EDTA antigens in advance.
- 4. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（2）In, 9 pairs of primer amplification VL genes, 4 pairs of primer amplification VH genes, in VL genes and VH genes are designed altogether Between add a segment polypeptide chain Linker and be connected, the polypeptide chain Linker amino acid sequences are GGGGSGGGGSGGGGS.
- 5. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（3）In, the PCANTAB5E phagemids and scFv genes carry out sfiI and NotI double digestions respectively Processing turns special ligase connection structure PCANTAB-ScFv recombinant plasmids using T4DNA electricity, and electricity conversion Escherichia coli electricity turns special With competent cell TG1, according to MOi=100:1, which adds in titre, is more than 1012The helper phage M13KO7 of pfu/ml, structure are primary Phage antibody library, primary phage antibody library calculate phage titre, random picking list using double layer agar method It clones bacterium solution PCR and calculates recombination fraction.
- 6. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（4）In, primary phage antibody library is incubated together with the breast tumor tissue sections of the ER α positives, is discarded The bacteriophage not combined with tissue, combining bacteriophage is eluted with Gly-HCL to be expanded, and carries out three-wheel enrichment sieve Choosing obtains three-level phage antibody library；Three-level phage antibody library is together with the ER α albumen being incorporated on ELISA Plate It is incubated, increases washing steps and wash away unbonded bacteriophage, enrichment and the protein bound bacteriophages of ER α, carry out to greatest extent Two-wheeled enrichment isolation obtains specific bacteriophage single-chain antibody library.
- 7. a kind of method using phage antibody library screening estrogen receptor alpha single chain antibody according to claim 1, It is characterized in that：Step（5）In, specific bacteriophage single-chain antibody library infects E.coli TG1, and gradient dilution is coated with 2 × YT-A, 30 monoclonals of picking prepare bacteriophage supernatant as primary antibody, and the HRP anti-M13KO7 of label mouse are secondary antibody, using phage-ElLSA Method carries out Preliminary Identification, and 7 plants of monoclonals for choosing OD450 higher prepare bacteriophage supernatant again, newborn with being fixed on glass slide Adenocarcinoma cell T47D is incubated together, and the anti-M13KO7 of HRP label mouse is secondary antibody, is dyed through DAB, haematoxylin is redyed, and PBS returns blue step Afterwards, observation cell smear identification positive clone molecule.
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