CN107057683B - A kind of preparation method of fibroin fluorescence probe - Google Patents
A kind of preparation method of fibroin fluorescence probe Download PDFInfo
- Publication number
- CN107057683B CN107057683B CN201611234585.9A CN201611234585A CN107057683B CN 107057683 B CN107057683 B CN 107057683B CN 201611234585 A CN201611234585 A CN 201611234585A CN 107057683 B CN107057683 B CN 107057683B
- Authority
- CN
- China
- Prior art keywords
- fibroin
- bacteriophage
- silk fibroin
- silk
- obtains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Optics & Photonics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种丝素荧光探针的制备方法。用丝素材料对噬菌体库进行筛选，得到与丝素蛋白特异性结合的噬菌体，进行基因测序，将测序获得的基因对应的氨基酸序列作为丝素亲和肽一级序列，通过多肽合成技术对丝素亲和肽一级序列进行多肽合成，得到丝素亲和肽粉，将丝素亲和肽粉和荧光素结合，得到丝素荧光探针。本发明所筛选的噬菌体对丝素材料具有很高的亲和性，探针灵敏度高，检测速度快，应用前景广泛，为丝素蛋白及丝素材料的高效靶向、鉴定提供更多的选择。The invention discloses a preparation method of a silk fibroin fluorescent probe. The phage library was screened with silk fibroin materials to obtain phages that specifically bind to silk fibroin proteins, and gene sequencing was performed. The amino acid sequence corresponding to the gene obtained by sequencing was used as the primary sequence of silk fibroin affinity peptide. The primary sequence of the fibroin affinity peptide is used for polypeptide synthesis to obtain the silk fibroin affinity peptide powder, and the silk fibroin affinity peptide powder is combined with fluorescein to obtain the silk fibroin fluorescent probe. The phage screened by the invention has high affinity for silk fibroin materials, high probe sensitivity, fast detection speed, wide application prospects, and provides more choices for efficient targeting and identification of silk fibroin and silk fibroin materials .
Description
Technical field
The present invention relates to a kind of preparation methods of fibroin fluorescence probe, belong to field of biotechnology.
Background technique
Large biological molecule such as proteins and peptides have biocompatibility, functionality and diversity, are nature biotechnologies
Important component and the mankind are used for nature remodeling, the necessary raw material improved the quality of living.Therefore, to albumen in material
Matter progress qualitative and quantitative analysis has and its important role, Chang Yaoyong have the biological method of height sensitivity.To egg
The conventional method of white matter content identification and quantification has biuret method, phenol reagent process method and ultraviolet absorption method etc., but these are traditional
The shortcomings that there are sensitivity is low for method, non-specific measurement.With the progress of science and technology, mass spectrum especially flight time mass spectrum
(MALDI-TOF) important technology of identification spectrum protein is had become.Mainly have when MALDI-TOF detects protein
2 steps: ion repeller and delay are extracted, to reach the measurement to protein content in sample.This technology can be complete
The protein of pairs of extremely low content is detected, and the detection sensitivity of protein is improved.
Fibroin albumen is a kind of natural macromolecule, due to its unique mechanical property and good biocompatibility, drop
Solution property and stability, easy to process and modification, and essence is the design feature of protein, becomes pharmacy, chemical industry, food addition
Noticeable one kind biomaterial in the fields such as agent and organizational project.Since fibroin albumen has biological degradability, biofacies
The excellent performances such as capacitive, biodegradability and machinability can be prepared into the material of variform, such as cellular, membranaceous, fine
Shape and tubulose etc. are tieed up, is widely paid close attention to and is studied in scientific research.Fibroin albumen is mainly by glycine, alanine, silk
18 kinds of amino acid compositions such as propylhomoserin, tyrosine.Although the above method or technology can detect protein, but still cannot
Fibroin albumen can not be carried out especially under the interference containing other oroteins by carrying out specificity identification to fibroin albumen
It is qualitative and quantitative.
Fibroin molecular weight reaches as high as 350KDa, and the fibroin albumen of low molecular weight also has 20kDa, and exists in this section
A very big molecular weight span, therefore fibroin albumen primary structure and multilevel structure are complicated；Especially when fibroin albumen with it is other
After albumen mixing, commonsense method is difficult to carry out fibroin qualitative and quantitative.Therefore it develops a kind of special for fibroin albumen progress
The probe that the opposite sex identifies has very big challenge.In conclusion there are no a kind of efficient, simple method occur up to now
To fibroin albumen or the specific identification of silk fibroin material progress and quantitatively.
Summary of the invention
Fluorescent probe technique is important research content in image technology, passes through electrostatic stress, chemical reaction, hydrogen bond action etc.
Method by the object (such as biomolecule, tissue, material) of quasi- detection specific wavelength excitation under generate fluorescence and by naked eyes or
The process of person's instrument detection.
For deficiency present in conventional fabrication techniques, the invention proposes a kind of preparation method of fibroin fluorescence probe,
Centered on the fibroin affinity peptide that phage selection obtains, by being grafted with fluorescein, specific detection silk fibroin material is obtained
Fluorescence probe, the simple one-step synthesis of this method.
The technical scheme is that using following steps:
1) phage library is screened with silk fibroin material, obtains the bacteriophage specifically bound with fibroin albumen；
2) step 2) being obtained to carry out gene sequencing with fibroin albumen specific binding bacteriophage, sequencing is obtained
The corresponding amino acid sequence of gene is as fibroin affinity peptide primary sequence；
3) Peptide systhesis is carried out to the fibroin affinity peptide primary sequence that step (2) obtains by peptide synthesis technology, obtained
Fibroin is affine Gly-His-Lys；
4) the affine Gly-His-Lys of fibroin and fluorescein obtained step (3) combine, and obtain fibroin fluorescence probe.
The fibroin fluorescence probe that step (4) obtains is identified and applied in the embodiment of the present invention, fibroin fluorescence probe
Can the material to silk fibroin material or containing fibroin albumen carry out specifically bind and detected by fluorescence microscope.
The step 1) specifically:
A. it closes silk fibroin material: the buffer containing TBS and BSA, TBS and the respective concentration of BSA is added in silk fibroin material
For 0.1-10mg/ml；
B. screening of the silk fibroin material to phage library: phage display peptide is added in the buffer of fibroin membrane, room temperature shaking table
In with shake 0.5-8h under 150-220rpm revolving speed；
C. it washs: being washed fibroin membrane 1-10 times with cleaning solution；
D. it elutes: cleaning solution is removed, eluent room temperature is added and is incubated for 1-20 minutes, will be specifically bound with silk fibroin material
Bacteriophage elution get off；
E. it neutralizes: neutralizer is added, so that the phagocytosis body fluid after elution is in neutrality after mixing with neutralizer；
F. Phage amplification: the phagocytosis body fluid after neutralization is transferred in ER2738 bacterium, in the shaking table of 37 DEG C of temperature with
150-220rpm revolving speed shake 3-24h, obtains bacteriophage and ER2738 bacterium mixed liquor；
G. bacteriophage purifies: bacteriophage and ER2738 bacterium mixed liquor being centrifuged, the mixing of PEG and NaCl are then added to
It is purified in liquid, obtains the bacteriophage bacterium solution of affine silk fibroin material；
I. above-mentioned steps a~g process is one cycle, by step a~g by after 2-6 repetitive cycling, obtaining and silk
The phagocytosis body fluid of cellulosic material specific binding.
The embodiment of the present invention is further handled in the following ways, come in verification step (2) with silk fibroin material specificity knot
The compatibility of the bacteriophage of conjunction: the bacteriophage progress coated plate with silk fibroin material specific binding after screening will specifically be bitten
Thallus infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as blue in occurred on next day LB plate
Color spot point then shows that bacteriophage has high-affinity.There is blue spot to which explanation is not due to bacteriophage itself, but
By there is blue spot after screening and silk fibroin material specific binding.
Fibroin, material in the step a are prepared in the following ways: being first dissolved in silk protein powder organic molten
Fibroin solutions are formed in agent or water, fibroin solutions drop is air-dried on polyethylene board, ethanol postincubation is added to make silk
Albumen solidification, obtains the fibroin membrane for being not dissolved in water.
The silk fibroin material includes but is not limited to fibroin membrane, fibroin porous support, silk fibroin powder, fibroin fiber, fibroin
The silk fibroin materials such as microballoon, fibroin hydrogel, silk fabrics or the material doped with fibroin albumen.
Washing in the step 1) includes being washed with TBST, and phosphate buffer (PBS), simulated body fluid also can be selected
(SBF), the non-strong buffer such as physiological saline, Tris-HCl buffer, washing times are 1-10 times, and preferably number is 3-7 times.
Elution in the step 1) includes using Gly-HCl eluent, and acid, alkalinity or strong elution also can be selected
Liquid.
Neutralizer in the step 1) uses Tris-HCl neutralizer.
The fluorescein is selected from one of the following group or a variety of: fluorescein isothiocynate (FITC), rhodamine, blue are glimmering
Photoprotein, green fluorescent protein, yellow fluorescence protein, Alexa Fluor, Cy3, Cy5 fluorescein etc..
The fibroin is affine Gly-His-Lys and fluorescein combine be will by way of chemical covalent crosslinking or chelation
The two is attached.
Currently preferred method is fluorescein and fibroin affinity peptide chemical graft process.When fluorescein in alkaline solution with
When fibroin affinity peptide reacts, the sulphur carbon amine key of the amino and fluorescein of C-terminal or N-terminal is combined on fibroin affinity peptide, is formed
A kind of fluorescein-fibroin affinity peptide conjugate.Meanwhile the connection type of fluorescein and fibroin affinity peptide can also be chemical covalent
The modes such as crosslinking, chelation.
The fibroin fluorescence probe that the present invention is prepared is for detection and identification silk fibroin material or contains fibroin egg
White material.The fibroin fluorescence probe can be micro- to fibroin membrane, fibroin porous support, silk fibroin powder, fibroin fiber, fibroin
The silk fibroin materials such as ball, fibroin hydrogel, silk fabrics or material containing fibroin albumen carry out specific detection, are a kind of sensitive
Spend method that is high, quick, practical, conveniently, efficiently detecting silk fibroin material.
Probe of the present invention is made of affinity peptide and fluorescent dye comprising specifically binding with fibroin albumen.The parent
It is to screen to obtain by phage library with peptide, fluorescent dye is that commercialization is commercially available.
It is demonstrated experimentally that the bacteriophage that the present invention is screened has very high compatibility to silk fibroin material.This probe sensitivity
Height, detection speed is fast, and application prospect is extensive, and efficient targeting, the identification for fibroin albumen and silk fibroin material provide more selections.
Compared with prior art, the present invention has following prominent characteristics:
(1) high sensitivity: compared to traditional method, the method for this patent can be carried out fibroin albumen in microgram rank
Identification；
(2) specificity is high: there is the specificity in conjunction with silk fibroin material, and nonspecific proteins will not be detected；
(3) convenient: not need complicated sample making course and cumbersome instrumentation step；
(4) quickly: fibroin identification quickly, can be completed within half an hour；
(5) practical: specificity identification can be carried out to the material containing fibroin albumen and quantified, silk fibroin material will not be destroyed
The structural intergrity of itself；
Detailed description of the invention
Fig. 1 is that the fibroin fluorescence probe of Example 1 and Example 2 of the present invention preparation identifies that fibroin nanofiber and fibroin are more
The picture of hole bracket, control group are polythene strip.
Fig. 2 is the quantity histogram of the bacteriophage after 4 wheel screenings of embodiment 1.
Fig. 3 is in embodiment 3, fibroin FITC fluorescence probe and control group (FITC aqueous solution) concentration it is extremely low (2 micrograms/
Milliliter) in the case where to fibroin nanofiber carry out fluorescent staining figure.
Specific embodiment
Below by embodiment, the present invention is described in further detail, following embodiment be explanation of the invention and
The invention is not limited to following embodiments.
The embodiment of the present invention is as follows:
Embodiment 1:
1) phage peptide library produced using commercialized NEB obtains and fibroin albumen knot 5 wheel screening of fibroin membrane progress
The bacteriophage of conjunction, detailed process is as follows；
1.1) fibroin membrane is prepared:
A. fibroin aqueous solution, concentration 1mg/mL are obtained；
B. fibroin solutions drop is air-dried in polyethylene board, adds ethanol postincubation to make its solidification, obtains being not dissolved in water
Bombyx mori silk fibroin film.
1.2) polypeptide sequence of the screening in conjunction with fibroin albumen
A. it closes silk fibroin material: the buffer of TBS+BSA (0.5mg/ml) being added in bombyx mori silk fibroin film；
B. screening of the silk fibroin material to phage library: this is to each bacteriophage type, by commercialized M13-12 bacteriophage
Random 10 microlitres of display libraries are added in fibroin membrane, and 150rpm shakes 0.5h in room temperature shaking table；
C. it washs: being washed fibroin membrane 1 time with TBST cleaning solution；
D. it elutes: the TBST cleaning solution in polyethylene board orifice plate is removed as far as possible, Gly-HCl eluent, which is added, (joined
BSA), room temperature is incubated for 1 minute, will be got off with the bacteriophage elution that silk fibroin material is specifically bound.
E. it neutralizes: Tris-HCl neutralizer is added, be neutral after mixing；
F. bacteriophage elution liquid Phage amplification: is transferred in the ER2738 bacterium shaken overnight to (OD600 value is about
0.5) 150rpm, 3h, are shaken in 37 DEG C of shaking tables, obtained bacteriophage/ER2738 bacterium mixed liquor.
G. bacteriophage purifies: bacteriophage obtained above/ER2738 bacterium mixed liquor being centrifuged, it is mixed to be then added to PEG/NaCl
It closes and is purified in liquid, respective concentration is 20g/L and 40g/L in PEG and NaCl mixed liquor, obtains phagocytosis body fluid.
H: the above process is 1 circulation, repeats step a~g after 4 wheel circulations, the fibroin being enriched with is affine to be bitten
Thallus, and counted, the counting difference of four-wheel is as shown in Figure 2.
1.3) it verifies the compatibility of screened bacteriophage and silk fibroin material: the bacteriophage after screening is subjected to coated plate: will
Bacteriophage infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as occurred on next day LB plate
Blue spot then shows that bacteriophage is affinity.
2) gene sequencing is carried out to the bacteriophage that obtains of screening, releases the amino acid sequence of fibroin affinity peptide；
3) the fibroin affinity peptide of crude separation is obtained by Solid phase peptide synthssis technology, obtains fibroin using purification process
Affinity peptide powder；
4) different sulphur FITC (cyanic acid fluorescein) and DIEA (diisopropylethylamine), FTIC, DIEA and fibroin affinity peptide is added
It is reacted 24 hours in DMF (dimethylformamide) solvent being protected from light, concentration/mixing ratio of FTIC, DIEA and fibroin affinity peptide
Example is 5:1, can prepare fibroin FTIC probe.
5) by the fibroin FTIC probe in step 4) to fibroin nanofiber carry out specific detection, different materials it
Between, it can recognize that fibroin nano-fiber material, shown in middle graph as shown in figure 1, and control group (TCP plastic material) such as Fig. 1
In left hand view, do not issue fluorescence.It proves that fibroin FTIC probe can carry out specificity identification to silk fibroin material, has very
High silk fibroin material specificity.
Embodiment 2:
1) phage peptide library produced using commercialized NEB obtains and fibroin albumen knot 5 wheel screening of fibroin membrane progress
The bacteriophage of conjunction, detailed process is as follows；
1.1) fibroin membrane is prepared:
A. fibroin aqueous solution, concentration 10mg/mL are obtained；
B. fibroin solutions drop is air-dried in polyethylene board, adds ethanol postincubation to make its solidification, obtains being not dissolved in water
Bombyx mori silk fibroin film.
1.2) polypeptide sequence of the screening in conjunction with fibroin albumen
A. it closes silk fibroin material: the buffer of TBS+BSA (10mg/ml) being added in bombyx mori silk fibroin film；
B. screening of the silk fibroin material to phage library: this is to each bacteriophage type, by commercialized M13-12 bacteriophage
Random 10 microlitres of display libraries are added in fibroin membrane, and 220rpm shakes 0.5h in room temperature shaking table；
C. it washs: being washed fibroin membrane 10 times with TBST cleaning solution；
D. it elutes: the TBST cleaning solution in polyethylene board orifice plate is removed as far as possible, Gly-HCl eluent, which is added, (joined
BSA), room temperature is incubated for 1 minute, will be got off with the bacteriophage elution that silk fibroin material is specifically bound.
E. it neutralizes: Tris-HCl neutralizer is added, be neutral after mixing；
F. bacteriophage elution liquid Phage amplification: is transferred in the ER2738 bacterium shaken overnight to (OD600 value is about
0.5) 150rpm, 3h, are shaken in 37 DEG C of shaking tables, obtained bacteriophage/ER2738 bacterium mixed liquor.
G. bacteriophage purifies: bacteriophage obtained above/ER2738 bacterium mixed liquor being centrifuged, it is mixed to be then added to PEG/NaCl
It closes and is purified in liquid, respective concentration is 20g/L and 40g/L in PEG and NaCl mixed liquor, obtains phagocytosis body fluid.
H: the above process is 1 circulation, repeats step a~g after 4 wheel circulations, the fibroin being enriched with is affine to be bitten
Thallus, and counted.
1.3) it verifies the compatibility of screened bacteriophage and silk fibroin material: the bacteriophage after screening is subjected to coated plate: will
Bacteriophage infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as occurred on next day LB plate
Blue spot then shows that bacteriophage is affinity.
2) gene sequencing is carried out to the bacteriophage that obtains of screening, releases the amino acid sequence of fibroin affinity peptide；
3) the fibroin affinity peptide of crude separation is obtained by Solid phase peptide synthssis technology, obtains fibroin using purification process
Affinity peptide powder；
4) different sulphur FITC (cyanic acid fluorescein) and DIEA (diisopropylethylamine), FTIC, DIEA and fibroin affinity peptide is added
It is reacted 24 hours in DMF (dimethylformamide) solvent being protected from light, concentration/mixing ratio of FTIC, DIEA and fibroin affinity peptide
Example is 1:1, can prepare fibroin FTIC probe.
5) the fibroin FTIC probe in step 4) is subjected to specific detection to fibroin porous support, can recognize that fibroin
Porous support materials, shown in the right figure as shown in figure 1, and the left hand view of control group (polyethylene board material) as shown in figure 1, do not send out
Fluorescence out.
Embodiment 3:
1) phage peptide library produced using commercialized NEB obtains and fibroin albumen knot 5 wheel screening of fibroin membrane progress
The bacteriophage of conjunction, detailed process is as follows；
1.1) fibroin membrane is prepared:
A. fibroin aqueous solution, concentration 50mg/mL are obtained；
B. fibroin solutions drop is air-dried in polyethylene board, adds ethanol postincubation to make its solidification, obtains being not dissolved in water
Bombyx mori silk fibroin film.
1.2) polypeptide sequence of the screening in conjunction with fibroin albumen
A. it closes silk fibroin material: the buffer of TBS+BSA (1mg/ml) being added in bombyx mori silk fibroin film；
B. screening of the silk fibroin material to phage library: this is to each bacteriophage type, by commercialized M13-12 bacteriophage
Random 10 microlitres of display libraries are added in fibroin membrane, and 220rpm shakes 0.5h in room temperature shaking table；
C. it washs: being washed fibroin membrane 10 times with TBST cleaning solution；
D. it elutes: the TBST cleaning solution in polyethylene board orifice plate is removed as far as possible, Gly-HCl eluent, which is added, (joined
BSA), room temperature is incubated for 1 minute, will be got off with the bacteriophage elution that silk fibroin material is specifically bound.
E. it neutralizes: Tris-HCl neutralizer is added, be neutral after mixing；
F. bacteriophage elution liquid Phage amplification: is transferred in the ER2738 bacterium shaken overnight to (OD600 value is about
0.5) 220rpm, 3h, are shaken in 37 DEG C of shaking tables, obtained bacteriophage/ER2738 bacterium mixed liquor.
G. bacteriophage purifies: bacteriophage obtained above/ER2738 bacterium mixed liquor being centrifuged, it is mixed to be then added to PEG/NaCl
It closes and is purified in liquid, respective concentration is 20g/L and 40g/L in PEG and NaCl mixed liquor, obtains phagocytosis body fluid.
H: the above process is 1 circulation, repeats step a~g after 4 wheel circulations, the fibroin being enriched with is affine to be bitten
Thallus, and counted.
1.3) it verifies the compatibility of screened bacteriophage and silk fibroin material: the bacteriophage after screening is subjected to coated plate: will
Bacteriophage infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as occurred on next day LB plate
Blue spot then shows that bacteriophage is affinity.
2) gene sequencing is carried out to the bacteriophage that obtains of screening, releases the amino acid sequence of fibroin affinity peptide；
3) the fibroin affinity peptide of crude separation is obtained by Solid phase peptide synthssis technology, obtains fibroin using purification process
Affinity peptide powder；
4) different sulphur FITC (cyanic acid fluorescein) and DIEA (diisopropylethylamine), FTIC, DIEA and fibroin affinity peptide is added
It is reacted 24 hours in DMF (dimethylformamide) solvent being protected from light, concentration/mixing ratio of FTIC, DIEA and fibroin affinity peptide
Example is 1:3, can prepare fibroin FTIC probe.
5) the fibroin FTIC probe in step 4) is subjected to specific detection to fibroin nanofiber, in concentration extremely low (such as 2
Mcg/ml) in the case where, strong fluorescence is issued, therefore can identify fibroin nanofiber, such as the right part of flg institute in Fig. 3
Show.And fluorescence is not issued with the control group of concentration (FITC aqueous solution), such as the left hand view in Fig. 3, therefore it can not identify silk
Plain nanofiber.It proves that fibroin FTIC probe can carry out specificity identification to the other silk fibroin material of Gamma Magnitude, has very high
Sensitivity.
Embodiment 4:
1) 3 wheel screenings are carried out to fibroin membrane using the phage peptide library of commercialization NEB production to obtain in conjunction with fibroin albumen
Bacteriophage；
1.1) fibroin membrane is prepared:
A. fibroin aqueous solution, concentration 100mg/mL are obtained；
B. fibroin solutions drop is air-dried in polyethylene board, adds absolute alcohol processing to make its solidification, is not dissolved in
The bombyx mori silk fibroin film of water.
1.2) polypeptide sequence of the screening in conjunction with fibroin albumen
A. it closes silk fibroin material: the buffer of TBS+BSA (0.2mg/ml) being added in bombyx mori silk fibroin film；
B. screening of the silk fibroin material to phage library: this is to each bacteriophage type, by commercialized M13-12 bacteriophage
Random 10 microlitres of display libraries are added in fibroin membrane, and 160rpm shakes 8h in room temperature shaking table；
C. it washs: being washed fibroin membrane 10 times with TBST cleaning solution；
D. it elutes: the TBST cleaning solution in polyethylene board orifice plate is removed as far as possible, Gly-HCl eluent, which is added, (joined
BSA), room temperature is incubated for 20 minutes, will be got off with the bacteriophage elution that silk fibroin material is specifically bound.
E. it neutralizes: Tris-HCl neutralizer is added, be neutral after mixing；
F. bacteriophage elution liquid Phage amplification: is transferred in the ER2738 bacterium shaken overnight to (OD600 value is about
0.5) 220rpm, is shaken in 37 DEG C of shaking tables, for 24 hours, obtained bacteriophage/ER2738 bacterium mixed liquor.
G. bacteriophage purifies: bacteriophage obtained above/ER2738 bacterium mixed liquor being centrifuged, it is mixed to be then added to PEG/NaCl
It closes and is purified in liquid, respective concentration is 20g/L and 40g/L in PEG and NaCl mixed liquor, obtains phagocytosis body fluid.
H: the above process is 1 circulation, repeats step a~g after 6 wheel circulations, the fibroin being enriched with is affine to be bitten
Thallus.
1.3) it verifies the compatibility of screened bacteriophage and silk fibroin material: the bacteriophage after screening is subjected to coated plate: will
Bacteriophage infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as occurred on next day LB plate
Blue spot then shows that bacteriophage is affinity.
2) gene sequencing is carried out to the bacteriophage that obtains of screening, the amino acid sequence of fibroin affinity peptide can be released；
3) the fibroin affinity peptide of crude separation is obtained by Solid phase peptide synthssis technology, obtains fibroin using purification process
Affinity peptide powder；
4) fibroin affinity peptide is reacted in the carbonate buffer solution of PH9~9.5, rhodamine is marked in fibroin affinity peptide
On, fibroin rhodamine probe can be prepared.Fibroin rhodamine probe can carry out specificity to the other silk fibroin material of Gamma Magnitude
Identification has very high sensitivity.
Embodiment 5:
1) 3 wheel screenings are carried out to fibroin membrane using the phage peptide library of commercialization NEB production to obtain in conjunction with fibroin albumen
Bacteriophage；
1.1) fibroin membrane is prepared:
A. fibroin aqueous solution, concentration 100mg/mL are obtained；
B. fibroin solutions drop is air-dried in polyethylene board, adds absolute alcohol processing to make its solidification, is not dissolved in
The bombyx mori silk fibroin film of water.
1.2) polypeptide sequence of the screening in conjunction with fibroin albumen
A. it closes silk fibroin material: the buffer of TBS+BSA (0.2mg/ml) being added in bombyx mori silk fibroin film；
B. screening of the silk fibroin material to phage library: this is to each bacteriophage type, by commercialized M13-12 bacteriophage
Random 10 microlitres of display libraries are added in fibroin membrane, and 160rpm shakes 8h in room temperature shaking table；
C. it washs: being washed fibroin membrane 10 times with TBST cleaning solution；
D. it elutes: the TBST cleaning solution in polyethylene board orifice plate is removed as far as possible, Gly-HCl eluent, which is added, (joined
BSA), room temperature is incubated for 20 minutes, will be got off with the bacteriophage elution that silk fibroin material is specifically bound.
E. it neutralizes: Tris-HCl neutralizer is added, be neutral after mixing；
F. bacteriophage elution liquid Phage amplification: is transferred in the ER2738 bacterium shaken overnight to (OD600 value is about
0.5) 220rpm, is shaken in 37 DEG C of shaking tables, for 24 hours, obtained bacteriophage/ER2738 bacterium mixed liquor.
G. bacteriophage purifies: bacteriophage obtained above/ER2738 bacterium mixed liquor being centrifuged, it is mixed to be then added to PEG/NaCl
It closes and is purified in liquid, respective concentration is 20g/L and 40g/L in PEG and NaCl mixed liquor, obtains phagocytosis body fluid.
H: the above process is 1 circulation, repeats step a~g after 6 wheel circulations, the fibroin being enriched with is affine to be bitten
Thallus.
1.3) it verifies the compatibility of screened bacteriophage and silk fibroin material: the bacteriophage after screening is subjected to coated plate: will
Bacteriophage infects ER2738 bacterium again, and is uniformly coated on the solid LB plate containing Xgal/IPTG, as occurred on next day LB plate
Blue spot then shows that bacteriophage is affinity.
2) gene sequencing is carried out to the bacteriophage that obtains of screening, the amino acid sequence of fibroin affinity peptide can be released；
3) the fibroin affinity peptide of crude separation is obtained by Solid phase peptide synthssis technology, obtains fibroin using purification process
Affinity peptide powder；
4) fibroin affinity peptide is reacted in the carbonate buffer solution of PH9~9.5, rhodamine is marked in fibroin affinity peptide
On, fibroin rhodamine probe can be prepared；
5) the fibroin rhodamine probe of acquisition identifies micro silk fabric, fluorescent staining is the positive, and is compareed
Group (non-silk clothes) dyeing is feminine gender, it was demonstrated that probe of the invention can identify modern age or the ancient silk true and false.
Claims (5)
1. a kind of preparation method of fibroin fluorescence probe, it is characterized in that using following steps:
1) phage library is screened with silk fibroin material, obtains the bacteriophage specifically bound with fibroin albumen；
2) step 1) being obtained to carry out gene sequencing with fibroin albumen specific binding bacteriophage, the gene that sequencing is obtained
Corresponding amino acid sequence is as fibroin affinity peptide primary sequence；
3) Peptide systhesis is carried out to the fibroin affinity peptide primary sequence that step 2) obtains by peptide synthesis technology, obtains fibroin parent
And Gly-His-Lys；
4) fibroin obtained step 3) is affine Gly-His-Lys and fluorescent dye combination, obtain fibroin fluorescence probe；
The step 1) specifically:
A. it closes silk fibroin material: the buffer containing TBS and BSA is added in silk fibroin material；
B. screening of the silk fibroin material to phage library: phage display peptide is added in the buffer of fibroin membrane, in room temperature shaking table with
Shake 0.5-8h under 160rpm revolving speed；
C. it washs: being washed fibroin membrane 1-10 times with cleaning solution；
D. it elutes: cleaning solution is removed, eluent room temperature is added and is incubated for 1-20 minutes, will be bitten with what silk fibroin material was specifically bound
Thallus elutes；
E. it neutralizes: neutralizer is added, so that the phagocytosis body fluid after elution is in neutrality after mixing with neutralizer；
F. Phage amplification: the phagocytosis body fluid after neutralization is transferred in ER2738 bacterium, with 150- in the shaking table of 37 DEG C of temperature
220rpm revolving speed shake 3-24h, obtains bacteriophage and ER2738 bacterium mixed liquor；
G. bacteriophage purifies: bacteriophage and ER2738 bacterium mixed liquor being centrifuged, are then added in the mixed liquor of PEG and NaCl
It is purified, obtains the bacteriophage bacterium solution of affine silk fibroin material；
I. above-mentioned steps a~g process is that one cycle obtains and fibroin material by step a~g after 2-6 repetitive cycling
Expect the phagocytosis body fluid of specific binding；
Silk fibroin material in the step a is prepared in the following ways: first by silk protein powder be dissolved in organic solvent or
Fibroin solutions are formed in person's water, fibroin solutions drop is air-dried on polyethylene board, ethanol postincubation is added to make fibroin
Solidification, obtains the fibroin membrane for being not dissolved in water.
2. a kind of preparation method of fibroin fluorescence probe as described in claim 1, it is characterised in that: the step in the step 1)
The washing of rapid c includes using TBST cleaning solution, phosphate buffer, simulated body fluid, physiological saline, Tris-HCl buffer, washing
Number is 1-10 times；Elution in the step 1) includes using Gly-HCl eluent, acid, alkaline eluant；The step 1)
In neutralizer use Tris-HCl neutralizer.
3. a kind of preparation method of fibroin fluorescence probe as described in claim 1, it is characterised in that: the fluorescent dye is selected from
With one of the following group or a variety of: fluorescein isothiocynate, rhodamine, blue fluorescent protein, green fluorescent protein, yellow fluorescence
Albumen, Alexa Fluor, Cy3, Cy5.
4. a kind of preparation method of fibroin fluorescence probe as described in claim 1, it is characterised in that: the fibroin affinity peptide
It is to be connected the two by way of chemical graft process, chemical covalent crosslinking or chelation that powder and fluorescent dye, which combine,
It connects.
5. a kind of preparation method of fibroin fluorescence probe as described in claim 1, it is characterised in that: the silk being prepared
Plain fluorescence probe is used for detection and identification silk fibroin material or the material containing fibroin albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611234585.9A CN107057683B (en) | 2016-12-28 | 2016-12-28 | A kind of preparation method of fibroin fluorescence probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611234585.9A CN107057683B (en) | 2016-12-28 | 2016-12-28 | A kind of preparation method of fibroin fluorescence probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107057683A CN107057683A (en) | 2017-08-18 |
| CN107057683B true CN107057683B (en) | 2019-04-12 |
Family
ID=59623951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611234585.9A Active CN107057683B (en) | 2016-12-28 | 2016-12-28 | A kind of preparation method of fibroin fluorescence probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107057683B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107418559B (en) * | 2017-07-20 | 2019-07-02 | 广州博徕斯生物科技股份有限公司 | A kind of bacteriophage electrochemical luminescence signals amplification probe and its preparation method and application |
| CN108761086A (en) * | 2018-06-11 | 2018-11-06 | 浙江大学 | A kind of sericin visible detection method |
| CN109030827A (en) * | 2018-06-11 | 2018-12-18 | 浙江大学 | A kind of sericin quantitative detecting method |
| CN110305202B (en) * | 2019-07-03 | 2021-06-08 | 浙江大学 | A method for improving the biomineralization ability of silk fibroin |
| CN114767876B (en) * | 2022-03-30 | 2024-04-19 | 浙江大学 | A composite porous microsphere carrying bacteriophage and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552727A (en) * | 2003-12-19 | 2004-12-08 | 复旦大学附属中山医院 | A liver cancer tumor blood vessel specific targeting polypeptide |
| CN102977189A (en) * | 2012-12-10 | 2013-03-20 | 刘敏 | Polypeptide combined with FAP (Fibroblast Activation Protein) |
| CN104447989A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Silkworm fibroin antibody preparing method |
| CN104483478A (en) * | 2014-12-31 | 2015-04-01 | 浙江理工大学 | Detection method for ancient silk fabrics |
| CN105424943A (en) * | 2015-11-12 | 2016-03-23 | 浙江理工大学 | Detecting method for distinguishing silk types |
| CN106188241A (en) * | 2016-07-20 | 2016-12-07 | 长春理工大学 | A kind of fluorescent probe detecting bladder chalone C and construction method |
-
2016
- 2016-12-28 CN CN201611234585.9A patent/CN107057683B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552727A (en) * | 2003-12-19 | 2004-12-08 | 复旦大学附属中山医院 | A liver cancer tumor blood vessel specific targeting polypeptide |
| CN102977189A (en) * | 2012-12-10 | 2013-03-20 | 刘敏 | Polypeptide combined with FAP (Fibroblast Activation Protein) |
| CN104447989A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Silkworm fibroin antibody preparing method |
| CN104483478A (en) * | 2014-12-31 | 2015-04-01 | 浙江理工大学 | Detection method for ancient silk fabrics |
| CN105424943A (en) * | 2015-11-12 | 2016-03-23 | 浙江理工大学 | Detecting method for distinguishing silk types |
| CN106188241A (en) * | 2016-07-20 | 2016-12-07 | 长春理工大学 | A kind of fluorescent probe detecting bladder chalone C and construction method |
Non-Patent Citations (1)
| Title |
|---|
| Selection of silk-binding peptides by phage display;Yoko Nomura等;《Biotechnol Lett》;20110123;第33卷;1069-1073 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107057683A (en) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107057683B (en) | A kind of preparation method of fibroin fluorescence probe | |
| Feiner‐Gracia et al. | Super‐resolution microscopy unveils dynamic heterogeneities in nanoparticle protein corona | |
| Zhang et al. | A nano-bio interfacial protein corona on silica nanoparticle | |
| CA2560216C (en) | Methods for reducing the range in concentrations of analyte species in a sample | |
| Muzard et al. | M13 Bacteriophage‐Activated Superparamagnetic Beads for Affinity Separation | |
| CN106636068B (en) | A kind of screening technique of fibroin affinity peptide | |
| CN103270049A (en) | Spider silk fusion protein structures for binding to an organic target | |
| Wu et al. | Double phage displayed peptides co-targeting-based biosensor with signal enhancement activity for colorimetric detection of Staphylococcus aureus | |
| Tang et al. | An epitope imprinted polymer with affinity for kininogen fragments prepared by metal coordination interaction for cancer biomarker analysis | |
| CN102680705A (en) | Method for quantitatively detecting allergen alpha-lactalbumin based on quantum dot fluorescence | |
| Cho et al. | Antibody-free and selective detection of okadaic acid using an affinity peptide-based indirect assay | |
| CN106188241B (en) | A kind of fluorescence probe and construction method detecting cystatin C | |
| Krause et al. | IgE epitope profiling for allergy diagnosis and therapy–parallel analysis of a multitude of potential linear epitopes using a high throughput screening platform | |
| Cusano et al. | Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum | |
| CN110684079A (en) | A kind of specific binding peptide of polycaprolactone nanoparticle, its screening method and application | |
| Zhu et al. | Preparation of poly (caffeic acid)-coated epitope molecularly imprinted polymers and investigation of adsorption performance toward ovalbumin | |
| AU2001282035B2 (en) | Peptides capable of functioning as mimotopes for estradiol analytes | |
| Ma et al. | Quantification of silk protein using phage nanofibers with high binding specificity | |
| CN117224490A (en) | TCM microsphere suitable for traditional Chinese medicine complex system target fishing and preparation method and application thereof | |
| Park et al. | Discovering melamine-specific bioreceptors via phage display, constructing its validation method based on the quenching on nanocomplex, and applying screened bioreceptor to the electrochemical assay of melamine | |
| KR102701904B1 (en) | Peptides specifically binding to filamentous fungus spore | |
| Gregersen et al. | Peptide‐Stabilized, Fluorescent Silver Nanoclusters: Solid‐Phase Synthesis and Screening | |
| CN103252217A (en) | Novel lipopolysaccharides (LPS) specific adsorption blood purification material and preparation method thereof | |
| WO2014138028A1 (en) | Liquid array platform for multiplexed analysis of molecule-protein interactions | |
| US20230393140A1 (en) | Binding assays involving a plurality of synthetic compounds, targets, and counter targets |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |