CN103130893A - Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody - Google Patents
Diethylstilbestrol single-chain antibody screening method and purpose of diethylstilbestrol single-chain antibody Download PDFInfo
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Abstract
The invention relates to a diethylstilbestrol single-chain antibody screening method and a purpose of a diethylstilbestrol single-chain antibody, and belongs to the technical field of biology. The single-chain antibody is screened from a ribosome showing mouse source natural antibody base and is subjected to soluble expression. The invention further relates to a diethylstilbestrol antibody screening method with a ribosome showing technology. The diethylstilbestrol single-chain antibody screened through the method can be used for pollution detection, development of immune quick test kits used for quick test of diethylstilbestrol residual and treatment of relevant diseases, and a foundation is laid for future detection and research on the diethylstilbestrol single-chain antibody.
Description
Technical field
The present invention relates to a kind of screening method of stilboestrol single-chain antibody, belong to biological technical field, in the purposes for the preparation of can the rapid detection stilboestrol residual immune quick testing reagent box of the efficient antibody of specific recognition stilboestrol and development and treatment relative disease.
Background technology
Stilboestrol (Diethylstilbestrol, DES) is first oral activated synthetic non steroidal estrogen material.Be mainly used in clinically at present treating the various diseases that the low disease of oestrogenic hormon and hormonal equilibrium imbalance cause and be used for the treatment of hypoovarianism or various diseases that pituitary function causes extremely.On veterinary clinic, DES be mainly used in treating the weary feelings of sow infertile, induce female rabbit to oestrus, treat laying hen stopping production, fattening of goats etc.Stilboestrol is at aspect successfuls such as the anabolism that promotes animal protein, raising animal day weight gain and minimizing fat in addition.But, DES can be residual in animal derived food such as liver, muscle, egg, milk, can be rich long-pending in water source and soil, cause the Environmental Hormone Pollution vicious cycle, and cause animal and human's canceration by food chain, cause the harm such as juvenile's growth precocity and feminization.Now international cancer research institution has confirmed that DES is transplacental carcinogenesis, and it is classified as human carcinogen.For above-mentioned situation, it is the important technical of effectively containment abuse stilboestrol and the necessary guarantee of safeguarding rules and regulations that development detects residual easy, quick, the sensitive method of stilboestrol.Immunological analysis method detects DES and has fast and convenient characteristics, and immune analysis method detects the specific antibody that DES need to be arranged.But DES is the small molecules haptens, and immunogenicity is not strong, and more difficult by the method Dispersal risk of traditional immune animal, the cycle of preparation is also longer.
Genetic engineering antibody single-chain antibody (Singe chain variable fragment, ScFv) has low or non-immunogenicity, the characteristics such as molecular weight is little, tissue penetration is strong, cost is low, can be mass-produced.At present, mainly using ribosomal display technology and display technique of bacteriophage screens and prepares single-chain antibody.The ribosomal display technology is a kind of external display technique that does not rely on cell fully, the library storage capacity is far longer than the phage single-chain antibody library, the ribosomal display technology also has the storehouse of building and screening method is easy in addition, is convenient to introduce the advantages such as avidity that sudden change and recombinant technology improve target proteins.The antibody gene in natural antibody storehouse derives from the animal or human's body B cell without immunity.Theoretically, can represent the diversity of the antibody gene that all elementary B cells contain, use any antigen all may therefrom screen corresponding antibody, have stronger versatility.Utilize the ribosomal display technology screening less for the research of small molecules hapten antibody, especially screening is more rare from the natural antibody library.
Summary of the invention
Main purpose of the present invention is to provide a kind of potential detection and the anti-stilboestrol single-chain antibody of medical use.
Second purpose of the present invention provides a kind of preparation method of anti-stilboestrol single-chain antibody.
The 3rd purpose of the present invention provides the purposes of anti-stilboestrol single-chain antibody in the preparation detection kit.
Technical scheme of the present invention is summarized as follows:
That anti-stilboestrol single-chain antibody provided by the invention is that screening obtains from the natural single-chain antibody library in ribosomal display mouse source and carried out the soluble functional expression.
The preparation method of anti-stilboestrol single-chain antibody provided by the invention is:
1. the structure of the natural single-chain antibody library in ribosomal display mouse source
Extract total RNA from the splenocyte without the 6-8 of immunity Balb/c in age in week, C57/BL6 mouse, after synthetic cDNA the first chain of reverse transcription, amplification VH, VL gene fragment, size is respectively 380bp, the 350bp left and right; Synthetic (G4S) 3linker, through sequencing, in full accord with theoretical sequence.Utilize SOE-PCR to build the single-chain antibody library, size be 750bp, and through sequence alignment, 80% the single-chain antibody of having an appointment in the library of single-chain antibody can correctly be translated, without phase shift mutation and lethal mutation, and nothing termination codon; The complementary determining region of correct single-chain antibody of checking order is all different CDR, and wherein aminoacid sequence changes variously, illustrate that the single-chain antibody library diversity of structure is good, is suitable for further carrying out the screening of single-chain antibody.
Amplify size and (comprise the T7 promotor for 102bp ribosomal display elements T, 5 ' stem ring, ribosome bind site) and size be 298bp intervening sequence P (phage P protein part sequence), contain 3 ' stem ring, as intervening sequence, through sequencing, in full accord with theoretical sequence respectively.With overlapping primer extension (splicing by overlap extension, SOE), the T fragment is connected with the scfv library, then with P fragment and its splicing, is built into ribosomal display single-chain antibody library, size is the 1200bp left and right.
Its small mouse can be other Strains of Mouses, the combination of preferred several Strains of Mouses
The link linker that wherein connects VH, VL is the combination of several G4S, preferred (G4S)
3
Wherein the promotor in the ribosomal display element can be prokaryotic promoter (T7 startup, tac promotor etc.) according to the difference of external translating system, and the preferred prokaryotic promoter of the present invention is the T7 promotor particularly
2. the screening of anti-stilboestrol single-chain antibody
After the in-vitro transcription translation is carried out in the ribosomal display single-chain antibody library that builds, with stilboestrol-BSA and stilboestrol-magnetic bead as antigen solid phase and liquid phase alternately screening be specific to the single-chain antibody of stilboestrol, by change Mg
2+Single-chain antibody-rrna that concentration obtains screening-mRNA triplet dissociates, and obtains corresponding mRNA, the DNA library after being screened through RT-PCR.Then repeat in-vitro transcription-In Vitro Translation-affine screening-RT-PCR amplification procedure, obtain the single-chain antibody DNA library of repeated screening youngster wheel.After taking turns the ribosomal display screening through seven, the DNA band brightness that the RT-PCR amplification obtains obviously increases, and illustrates that the antibody of antigen positive in the antibody library after screening has obtained enrichment.
Can adopt stilboestrol-BSA to carry out solid-phase screening, can also adopt stilboestrol-magnetic bead to carry out solid-phase screening, the preferred intersection screens.
Mg
2+Concentration is adjusted according to the preciseness difference of screening conditions.
3. the soluble functional of anti-stilboestrol single-chain antibody is expressed
Take turns screening with seven and carry out Sequence Identification in the single-chain antibody library afterwards, 43 strains are wherein arranged without lethal mutation, 26 clone's of random choose, single-chain antibody gene and pTIG-TRX are connected into expression vector, express in e. coli bl21 (DE3), identify through SDS-PAGE and Western Blot, at the 30KDa place, the target protein band is arranged, with theoretical albumen in the same size, determine in the supernatant liquor after expression product is present in broken thalline.
The expression of antibody can be adopted prokaryotic expression carrier such as pBV220, pET serial carrier, preferred pTIG-TRX.
Label protein can be maltose binding protein, 6 * histidine-tagged etc., and is preferred 6 * histidine-tagged.
4. the purifying of anti-stilboestrol single-chain antibody and evaluation
The expression supernatant liquor of 26 strain clone is identified respectively with indirect ELISA and indirect competitive ELISA, and result filters out 5 strain clone has specific combination to DES; 5 strain clone are carried out sequence alignment, and result shows that there is similarity in the CDR district, infers that the antibody recognition site of anti-DES single-chain antibody is similar.The avidity of analyzing the single-chain antibody of a strain 30-3 who sifts out with SPR is 3.79 * 10
-6M。Utilize Anti-His tag affinity column to carry out purifying to the single-chain antibody of expressing, the purpose band has been obtained significantly concentrated.
The purifying of single-chain antibody can adopt ion exchange chromatography, gel chromatography, metal chelate chromatography etc., preferable alloy chelating chromatography.
The metal that metal chelate chromatography is sent out can be selected Ni
2+, Cu
2+, Zn
2+, Ca
2+Deng, preferred Ni
2+Ion.
The present invention can also provide a kind of most critical detection reagent---antibody of measuring in the residual detection kit of stilboestrol in environment and food, although HUMAN HEALTH in the residual serious threat of DES, but its application in livestock industry, can bring certain economic worth, this makes and still has socially illegal use phenomenon.Particularly, the illegal phenomenon of using stilboestrol ubiquity still in aquaculture.For above-mentioned situation, development detects residual easy, quick, the sensitive method of stilboestrol and is very important, and the stilboestrol single-chain antibody of the present invention's preparation can partly replace measures the required reagent of the residual mensuration of stilboestrol in Food and environment sample.
The advantage of the anti-stilboestrol single-chain antibody that the present invention is prepared is: (1) utilizes stilboestrol-BSA and stilboestrol-magnetic bead as antigen solid phase and alternately screening of liquid phase, can overcome the interference that BSA causes, and can obtain the antibody of DES high specific; (2) the single-chain antibody sequence that obtains is clear, is convenient to genetically engineered operation, is easy in intestinal bacteria functional expression in a large number; (3) molecular weight is little, a little less than immunogenicity, for clinical application is in the future laid a good foundation.
Description of drawings
Fig. 1 ribosomal display antibody library full length DNA electrophorogram.
M: molecular weight standard Marker 1-2: ribosomal display antibody Kuku DNA
Fig. 2 PCR identifies that fourth, fifth takes turns positive colony that (A), seven takes turns the rear RT-PCR product of (B) screening
A:1-10: the scfv DNA that after the fourth round screening, pcr amplification obtains; 13-22: the 5th takes turns the scfv DNA that the rear pcr amplification of screening obtains, 11 positive controls, and 12 negative contrasts, M is molecular weight standard
B. bacterium colony PCR identifies that the 7th takes turns positive colony of the rear RT-PCR product of screening, 1 positive contrast, and 2 negative contrasts, M is molecular weight standard, 3-24 is: the scfv DNA that after the fourth round screening, pcr amplification obtains
Fig. 3 Western-blot analyzes the single-chain antibody of expressing
1,2:SDS-PAGE induces rear full cell lysate; 3,4:Western blotting analyzes the full cell lysate 5 after inducing, and 6:Western blotting analyzes the full cell lysate supernatant after inducing
Fig. 4 seven takes turns in the 43 correct sequences of strain that screening obtains by order-checking random choose 26 strain clone sublists and reaches rear ELISA to measure single-chain antibody active
The competition of Fig. 5 single-chain antibody is in conjunction with the competitive ELISA analysis of DES.
Fig. 6 SPR is combined with antigen after analyzing anti-DES single-chain antibody 30-1 gradient dilution
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Reagent and material used in following embodiment if no special instructions, all can be bought from conventional reagent company and obtain.
The structure of embodiment 1 ribosomal display mouse source single-chain antibody
One .RNA extraction is synthetic with cDNA's
Get 6 age in week healthy BALB/c mouse, each 3 of C57/BL6 mouse, male and female half and half.The TRIZOL method is extracted the total RNA of splenocyte, adopts the synthetic cDNA of reverse transcription test kit.
The amplification of two .VH, VL gene
1. design of primers:
VH/for amplification heavy chain VH variable region upstream primer:
VH/for?5’TATATCC
ATGGCCCAGGTSMARCTGCAG?3’
VH/back amplification heavy chain VH variable region downstream primer:
VH/back?5’TGA?GGA?GAC?GGT?GAC?CGT?GGT?GCC?TTG?GCC?CC?3’
VL/for amplification heavy chain VH variable region upstream primer:
VL/for?5’GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCA?3’
VL/back amplification heavy chain VH variable region downstream primer:
VL/back?5’CGCG?GTTGCGGTCCG?TTT?BAK?YTC?CAR?CTT?KGT?SCC?3’
The single-letter representation of various nucleosides in degenerated primer: M=A or C R=A or G; S=G or C; W=A or T; B=T or C, G; K=T or G; Y=C or T.
Liner：
5‘ACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGTGGAGGTGGCTCTGGCGGTGGCGGATCTGACA?TCGAGCTCACTCAG-3’
The upstream and downstream primer of amplification connection peptides:
GS/for?5’ACGGTCACCGTCTCCTCA?3’
GS/back?5’CTGAGTGAGCTCGATGTC?3’
2.VH gene fragment pcr amplification system:
Reaction conditions: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 53 ℃ of annealing 30s; 72 ℃ are extended 1min; 30cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, and glue reclaims the purpose fragment.
3.VL gene fragment pcr amplification system and amplification condition
VL gene fragment pcr amplification system
Reaction conditions: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 63 ℃ of annealing 30s; 72 ℃ are extended 1min; 30cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, and glue reclaims the purpose fragment
4.linker junction fragment pcr amplification system
Reaction conditions: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s; 50 ℃ of annealing 20s; 72 ℃ are extended 30s; 30cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, and glue reclaims the purpose fragment.
The structure in three .scFv libraries
Utilize the method for SOE-PCR to be built into the scfv library VH, the VL gene fragment that reclaims and the correct linker fragment that checks order, obtain the fragment of 750bp size.Reaction system and the reaction conditions of SOE-PCR are as follows:
The reaction conditions of above-mentioned system is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 1min40s is extended in 72 ℃ of annealing; 15cycles; 72 ℃ are extended 7min; Add wherein each 1 μ L of primer VH/back, VL/for, 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 55 ℃ of annealing 30s; 72 ℃ are extended 1min20s; 20cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, can see the approximately fragment of 1200bp (Fig. 1), and glue reclaims the purpose fragment.
The screening of embodiment 2 stilboestrol single-chain antibodies
One. the affine screening of solid phase
Reagent bottle, reagent and enzyme plates all in this step all will be used the DEPC water treatment, to guarantee not having RNase to pollute.
(1) coated screen holes: get the DES-BSA coating antigen and be diluted to 20 μ g/mL with the carbonate buffer solution of sterilizing, every hole adds 100 μ L on enzyme plate, in 4 ℃ of coated spending the night;
(2) coating buffer that inclines, with sterilization PBS washing closed pores 3 times, each 3min.Then use cold WBT washed twice, each 3min.At last, fill with screen holes with cold WBT, place 20min at least on ice;
(3) get on ice the translation product (adopting promega Escherichia coli (E.coli) S30 Extract System In Vitro Translation) of placing and transfer in ready screen holes, jolting 1 hour gently in icehouse.Make In Vitro Translation product and the abundant combination of solid phase DES-BSA, catch the scfv-ribosome mRNA complex.
(4) raffinate in the hole that inclines is washed 3 times each 1min with the WBT of ice precooling.
(5) Xiang Kongzhong adds the elution buffer damping fluid EB (the Rnasin inhibitor that contains 5U) of 100 μ l ice precoolings, continues on ice jolting 10min gently, and the large small subunit of rrna is separated, and discharges mRNA.
(6) repeat 5 steps.
(7) twice elutriant all added Dnase I, hatch 15min for 37 ℃, to remove DNA profiling.The mRNA liquid nitrogen that obtains is preserved or purifying immediately.
Two. the affine screening of liquid phase
(1) get the good magnetic bead of coupling of 100 μ L, PBS washs closed pores 3 times with sterilization, each 3min.Then use cold WBT washed twice, each 3min put into icehouse some minutes with magnetic bead, made magnetic bead ice-cold.
(2) In Vitro Translation product solution is added in ice-cold magnetic bead jolting 1 hour gently in icehouse.
(3) use magnetic frame that liquid is removed, all the other steps are same. (4) of the affine screening of solid phase-(7).
Three. rebuilding with next round of template screened
(1) mRNA that adopts the RNeasy clean up kit purifying screening of Qiagen company to obtain, with M-MLV First Strand Synthesis System for RT-PCR (Invitrogen, USA), carry out reverse transcription, and further pcr amplification obtains the scFv library, and and T, the coupling successively of P fragment builds next round complete ribosomal display library.
With four-wheel, five take turns with seven and take turns the RT-PCR product that obtains after screening and be connected respectively on cloning vector pMD18-T, are transformed in intestinal bacteria the evaluation of checking order of random choose positive colony.Along with the increase of screening wheel number, mutation rate also increases sequencing result as can be known.The 7th take turns screening after, sequencing result shows 60% library sequence sudden change has occured, but CDR district diversity diminishes, and higher similarity occurred.Prove specific sequence highly enriched (Fig. 2).
Functional expression, purifying and the evaluation of the anti-stilboestrol single-chain antibody of embodiment 3
One. the functional expression of anti-stilboestrol single-chain antibody
1. the amplification of single-chain antibody gene after the screening
Will be through the primer amplification that contain restriction enzyme site of the right-on 26 strain single-chain antibody genes of order-checking translation with redesign.Obtain scfv gene with restriction enzyme site from pMD18-T-scfv cloning vector amplification.Primer SF-Trx-EcoRI and SR-Xhol-1 contain respectively EcoRI, XhoI restriction enzyme site.
SF-Trx-EcoRI (introducing the EcoI restriction enzyme site): CGCGAATTCTAAATGGCCCAGGT
SR-XhoI-1 (introducing the XhoI restriction enzyme site): AATCTACTCGAGCGCGGTTGCGGTCCGTTT
Reaction conditions: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 56 ℃ of annealing 30s; 72 ℃ are extended 1min20s; 10cycles; 94 ℃ of sex change 30s; 50 ℃ of annealing 30s; 72 ℃ are extended 1min20s; 20cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, and glue reclaims the purpose fragment.
2. restructuring scfv prokaryotic expression plasmid builds:
With EcoRI, XhoI double digestion plasmid pTIG-TRX.Enzyme is cut system 50 μ L:EcoRI 2.5 μ L, XhoI 2.5 μ L, 10 * buffer, 5 μ L, ddH
2O 10 μ L; Plasmid pTIG-TRX 30 μ L.37 ℃ of enzymes that spend the night are cut.1.5% agarose gel electrophoresis, the plasmid pTIG-TRX that scfv fragment and the enzyme of being connected of EcoRI, XhoI double digestion purifying are cut connects with the T4DNA ligase enzyme, linked system: pTIG-TRX 1 μ L, scfv 3 μ L, dd H
2O 4 μ L, after mixing, 45 ℃ of temperature are bathed 5min, put into immediately 0 ℃.Add again T4 ligase enzyme (Takara) 1 μ L, 10 * buffer, 1 μ L, 16 ℃ connect 3h.Transform Host Strains BL21 (DE3).
3. the abduction delivering of single chain antibody protein
The positive bacterium colony that step 2 is identified is inoculated in the LB liquid nutrient medium that 5ml contains Amp, 37 ℃, the 200r/min shaking culture is spent the night, and next day the bacterium liquid of incubated overnight is transferred again in the ratio of 1: 100 to contain the LB liquid nutrient medium of Amp in 5mL, and establish negative control, 30 ℃ of shaking culture are to A
600Be about 0.6-0.8, add IPTG to final concentration be 1mmol/L, then continue shaking culture, induce 4h.Centrifugal collection thalline, the centrifugal rear thalline of binding buffer liquid 50mL piping and druming with dilution, be crushed to bacterium liquid with the ultrasonic cell disintegration machine limpid, after refrigerated centrifuge was centrifugal, the centrifugal 20min of 10000r/min got respectively cleer and peaceful precipitation and carries out SDS-PAGE, and carry out the detection of Western Blot, as shown in Figure 3, the anti-DES single-chain antibody of purpose expression product mainly exists in supernatant, can be combined with mouse-anti 6 * His tag monoclonal antibody.
Two. the evaluation of anti-stilboestrol single-chain antibody
1. single-chain antibody is in conjunction with the detection of activity
Random choose 26 strains are used for colibacillary expression from the right-on 43 strain single-chain antibodies that check order, and all carry out the mensuration of indirect ELISA with expressing supernatant, filter out the supernatant liquor that the active expression strain of combination is arranged with DES.Use the coated enzyme of DES-OVA to join the hole, and the OVA of coated same concentrations simultaneously, supernatant liquor is diluted four gradients as primary antibodie solution, the monoclonal antibody of anti-his is anti-as two, sheep anti mouse-HRP is as ELIAS secondary antibody, after TMB colour developing, microplate reader is measured 450nm, and the value of DES-OVA is deducted the light absorption value that the value of OVA is combined with DES as single-chain antibody.As shown in Figure 4, be the column diagram that the light absorption value that supernatant liquors are combined with DES is expressed in 26 strains, take the expression supernatant of empty plasmid as contrast.
2. the detection of the competition activity of single-chain antibody after the screening
The elisa assay that will be at war with through the expression supernatant liquor of 26 strain bacterium of indirect ELISA analysis, take DES-OVA as envelope antigen, the methanol solution dilution DES solubility haptens with 10% is as the competition small molecules, and concentration is 0.5-500000ng/ml.Through the competitive ELISA analysis, wherein there are as can be known five strains that the variation of obvious OD value is arranged.
3. the purifying of single-chain antibody
Through the competitive ELISA analysis, will have and can carry out affinity chromatography with the single-chain antibody 30-3 of DES specific combination, the 30-3 supernatant liquor is carried out separation and purification of protein through the Ni post, albumen by its with 6x Histag and Ni
2+As seen sequestering action between ion, specific adsorption go out to have bright band in the target protein size after wash-out on chromatography column.
4.SPR the avidity of analysis list chain antibody
The supernatant liquor of 30-3 is cooked the dilution of 5 gradients and the fixing DES-OVA reaction 30min of chip surface, and SPR analyzes affinity of antibody.Fig. 6 be after SPR analysis list chain antibody gradient dilution with the antigenic action situation.Through after wash-out, the variation by angle single-chain antibody and envelope antigen as can be known has combination.With data acquisition software Autolab ESPRIT Data Acquisition 4.3, data analysis software Kinetic Evaluation5.0 measures its kinetic constant.K
D3.79 * 10 through calculating
-6M。
Claims (8)
1. anti-stilboestrol single-chain antibody, it is characterized in that can specific recognition small-molecule substance stilboestrol
2. the anti-stilboestrol single-chain antibody of claim 1 preparation method is comprised of following steps:
(1) structure of the natural single-chain antibody library in ribosomal display mouse source
Extract total RNA from the splenocyte without the 6-8 of immunity Balb/c in age in week, C57/BL6 mouse, after synthetic cDNA the first chain of reverse transcription, amplification VH, VL gene fragment, size is respectively 380bp, the 350bp left and right; Synthetic (G4S) 3linker, through sequencing, in full accord with theoretical sequence.Utilize SOE-PCR to build the single-chain antibody library, size be 750bp, and through sequence alignment, 80% the single-chain antibody of having an appointment in the library of single-chain antibody can correctly be translated, without phase shift mutation and lethal mutation, and nothing termination codon; The complementary determining region of correct single-chain antibody of checking order is all different CDR, and wherein aminoacid sequence changes variously, illustrate that the single-chain antibody library diversity of structure is good, is suitable for further carrying out the screening of single-chain antibody.
Amplify size and (comprise the T7 promotor for 102bp ribosomal display elements T, 5 ' stem ring, ribosome bind site) and size be 298bp intervening sequence P (phage P protein part sequence), contain 3 ' stem ring, as intervening sequence, through sequencing, in full accord with theoretical sequence respectively.With overlapping primer extension (splicing by overlap extension, SOE), the T fragment is connected with the scfv library, then with P fragment and its splicing, is built into ribosomal display single-chain antibody library, size is the 1200bp left and right.
(2) screening of anti-stilboestrol single-chain antibody
After the in-vitro transcription translation is carried out in the ribosomal display single-chain antibody library that builds, with stilboestrol-BSA and stilboestrol-magnetic bead as antigen solid phase and liquid phase alternately screening be specific to the single-chain antibody of stilboestrol, by change Mg
2+Single-chain antibody-rrna that concentration obtains screening-mRNA triplet dissociates, and obtains corresponding mRNA, the DNA library after being screened through RT-PCR.Then repeat in-vitro transcription-In Vitro Translation-affine screening-RT-PCR amplification procedure, obtain the single-chain antibody DNA library of repeated screening youngster wheel.After taking turns the ribosomal display screening through seven, the DNA band brightness that the RT-PCR amplification obtains obviously increases, and illustrates that the antibody of antigen positive in the antibody library after screening has obtained enrichment.
Can adopt stilboestrol-BSA to carry out solid-phase screening, can also adopt stilboestrol-magnetic bead to carry out solid-phase screening, the preferred intersection screens.
Mg
2+Concentration is adjusted according to the preciseness difference of screening conditions.
(3) soluble functional of anti-stilboestrol single-chain antibody is expressed
Single-chain antibody gene and pTIG-TRX are connected into expression vector, express in e. coli bl21 (DE3), identify through SDS-PAGE and Western Blot, at the 30KDa place, the target protein band is arranged, with theoretical albumen in the same size, determine in the supernatant liquor after expression product is present in broken thalline.
The expression of antibody can be adopted prokaryotic expression carrier such as pBV220, pET serial carrier, preferred pTIG-TRX.
Label protein can be maltose binding protein, 6 * histidine-tagged etc., and is preferred 6 * histidine-tagged.
(4) purifying of anti-stilboestrol single-chain antibody and evaluation
The expression supernatant liquor of 26 strain clone is identified respectively with indirect ELISA and indirect competitive ELISA, and result filters out 5 strain clone has specific combination to DES; The avidity of analyzing the single-chain antibody of a strain 30-3 who sifts out with SPR is 3.79 * 10
-6M。Utilize Anti-His tag affinity column to carry out purifying to the single-chain antibody of expressing.
3. mouse described in claim 2 can be other Strains of Mouses, the combination of preferred several Strains of Mouses.
4. the link linker that connects VH, VL described in claim 2 is the combination of several G4S, preferred (G4S)
3
5. the promotor in the element of ribosomal display described in claim 2 can be prokaryotic promoter (T7 startup, tac promotor etc.) according to the difference of external translating system, and preferred prokaryotic promoter is the T7 promotor particularly.
6. screening method described in claim 2 can adopt stilboestrol-BSA to carry out solid-phase screening, can also adopt stilboestrol-magnetic bead to carry out solid-phase screening, preferably adopts different methods to intersect in difference screening round and screens.
7. the expression of antibody described in claim 2 can be adopted prokaryotic expression carrier such as pBV220, pET serial carrier, and expressive host different from the carrier different choice, preferred pTIG-TRX carrier.
8. label protein described in claim 2 can be maltose binding protein, 6 * histidine-tagged etc., and is preferred 6 * histidine-tagged.
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