CN107389641A - A kind of method based on immune vestige method detection ancient times argillization silk goods - Google Patents

A kind of method based on immune vestige method detection ancient times argillization silk goods Download PDF

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CN107389641A
CN107389641A CN201710645247.2A CN201710645247A CN107389641A CN 107389641 A CN107389641 A CN 107389641A CN 201710645247 A CN201710645247 A CN 201710645247A CN 107389641 A CN107389641 A CN 107389641A
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silk
argillization
carbon point
solution
fluorescent carbon
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CN107389641B (en
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胡智文
陈茹茹
李津
梁军龙
尚亚廷
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Zhejiang Sci Tech University ZSTU
Zhejiang University of Science and Technology ZUST
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    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

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Abstract

The present invention relates to historical relic detection field, discloses a kind of method based on immune vestige method detection ancient times argillization silk goods, and the present invention is first prepared for the silk secondary antibody of fluorescent carbon point mark, progressively uses ionic liquid and PM afterwards13Modern silk and ancient times argillization silk fabric cultural relics sample are hydrolyzed alkali protease, after obtaining protein extract, through dialysing, purifying, carry out SDS PAGE gel electrophoresises, obtained protein band is transferred on pvdf membrane, after the secondary antibody marked through silk primary antibody and fluorescent carbon point is incubated, immunofluorescence band can be observed in gel imaging system, differentiates the species of Ancient Silk Textile.Chemical levels are few in the present invention, and reaction is gentle, environmentally friendly；When being detected to Ancient Silk Textile, there is the characteristics of few amount of samples, directly perceived, quick and high sensitivity.

Description

A kind of method based on immune vestige method detection ancient times argillization silk goods
Technical field
The present invention relates to historical relic detection field, more particularly to it is a kind of based on immune vestige method detection ancient times argillization silk goods Method.
Background technology
China is the silk area of origin that the world recognizes extensively, and the invention of silk has act in the history culture of the Chinese nation The status of sufficient weight.For thousands of years, silk is always ancient Chinese symbol, and long magnificent Chinese culture by silk it Go to the world on road.What China silk mainly utilized is silk, and silk is natural protein fibre, is to pass through peptide bond by amino acid Act on the natural macromolecular material being formed by connecting.Silk fabrics is imbedded in underground, through excess temperature, humidity, soil acid-base property, thin The influence of bacterium, mould etc., protein fibre are easily aging, degraded.
In ancient times in silk remnants, it can be seen that the silk remnants form of expression from archaeological excavation over the years and be broadly divided into Four types：Silk goods are in kind, are ashed silk goods, mineralising silk goods and argillization silk goods.Especially argillization silk goods, definite Say be textile impression, the textile material of its original is probably silk, it is also possible to other yarn fabrics, for sake of convenience, lead to Argillization silk goods are often referred to as before textile material is not identified.Its textile material of this kind of remnants has been degraded, and it is degraded Product or remnants are merely able to see the shape or pattern of fabric among soil, or through rain drop erosion in soil surface.Therefore such as What extracts effective information, to historical relic discriminating and silk using modern advanced natural science means from ancient times argillization silk goods Origin have important value.
In research at this stage, the research meanses to Ancient Silk Textile still use infrared spectrum, drawn mostly both at home and abroad The technologies such as graceful spectrum, X-ray diffraction, these sensitivities are low, larger by impurity effect, are not suitable for entering micro historical relic sample How row detection, find a kind of means of more science to identify that Ancient Silk Textile is always the problem of historical relic's protection circle.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides one kind based on immune vestige method detection ancient times argillization silk goods Method.When being detected using the inventive method to ancient times argillization silk goods, there is the characteristics of directly perceived, accurate, high sensitivity.
The present invention concrete technical scheme be：A kind of method based on immune vestige method detection ancient times argillization silk goods, bag Include following steps：
1）The preparation of fluorescent carbon point：Fructose is dissolved in deionized water and the in the mixed solvent of absolute ethyl alcohol, in hydrothermal reaction kettle Interior reaction, fluorescent carbon point solution is made, after through dialysis purification, freeze-drying, obtain fluorescent carbon point powder.
2）Fluorescent carbon point marks the preparation of silk fibroin secondary antibody：Fluorescent carbon point is coupled using carbodiimides mediated method With silk fibroin secondary antibody, fluorescent carbon point mark silk secondary antibody is made.
3）Modern silk is weighed as control sample, with 1:95-105 bath raio is containing 0.4-0.6wt% Na2PO4With 0.8-1.2wt%C17H3525-35min is boiled in COONa mixed solution, carries out degumming process, common degumming is twice；Degumming it Afterwards, silk is washed by rubbing with the hands more than 4 times with deionized water, is put into oven drying, obtain modern natural silk fiber；Then by modern silkworm Silk fibroin fiber is with 1:45-55 bath raio is immersed in [AMIM] Cl ionic liquids, heating response, cooling；Add PM13- alkalescence eggs White enzyme powder constant-temperature incubation 5-7h, carries out enzyme deactivation, obtains fibroin albumen/ionic liquid solution A.
In scouring processes, with the hydrolysis of silk gum, the concentration of amino acid gradually increases in solution, and pH is reduced, degumming effect Rate weakens, so alkali need to be added to maintain the stabilization of degumming liquid pH value, but alkali concn crosses conference damage fibroin, so using C17H35COONa is as buffer.
4）The soil sample with ancient times argillization silk goods is weighed, after mortar grinder, is added by solid-to-liquid ratio 25-35g/60mL Into [AMIM] Cl ionic liquids, heating response, cooling；Add PM13- basic protein enzyme powder constant-temperature incubation 5-7h, is gone out Enzyme, obtain ionic liquid suspension；By suspension high speed refrigerated centrifuge, take supernatant liquor obtain egg fibroin it is white/ionic liquid is molten Liquid B.
5）Respectively to fibroin it is white/ionic liquid solution A, B in add absolute ethyl alcohol, soak repeatedly, separate out albumen, filter out After albumen, deionized water dissolving is added, obtains pure fibroin albumen extract solution A, B.
Using being dialysed under cryogenic to the extract solution of modern silk and ancient times argillization silk goods, it is possible to reduce silk Fibroin loses, and ensures precision.
6）By fibroin albumen extract solution A, B respectively under the conditions of 1-5 DEG C with bag filter dialysis 40-50h, it is miscellaneous to remove small molecule Matter；It is about 150-250 μ L to be concentrated in vacuo to final volume, respectively obtains fibroin albumen concentrate A, B.
7）PAGE gel electrophoresis：40-60 μ L fibroin albumen concentrate A, B are measured respectively, it is dilute with the buffer solutions of CB 9.6 Release to 4-6mL, 4-6min is heated under the conditions of 98-102 DEG C, carry out denaturation treatment；To after room temperature after cooling, by two kinds of samples and What protein Marker additions prepared exempts to contaminate glue（Upper strata is concentration glue, and lower floor is separation gel）In well, in a cell Fill it up with electrolyte, after the completion of electrophoresis, electrophoretic image can be observed in imaging systems.
Gel electrophoresis is carried out using dye glue is exempted from, the use of chemical reagent can be reduced, while save the time.
8）Electrotransfer：Glue after electrophoresis is laid in and turned through methanol and electricity on the treated pvdf membrane of buffer solution, is clipped in filter Transhipment interlayer is assembled among paper and sponge, is put into wet type membrane-transferring device, constant current transferring film.
The pvdf membrane used is first soaked with methanol, it is therefore an objective to activate the group on schedule above pvdf membrane, make it be easier with Electronegative protein combines.
9）Film is imaged：After the completion of electrotransfer, pvdf membrane is taken out, is put into gel imaging system and is imaged, observe protein band Transfer case.
Film is imaged, observes the transfer case of protein band, it is convenient to grasp experiment progress, Optimal Experimental step.
10）Closing：Pvdf membrane is taken out, film 4-6min is washed with 10-20mL TBS liquid, is located at ambient temperature with confining liquid Manage 50-70min.
11）Immune processing：With antibody diluent with 950-1050:1 volume ratio dilution primary antibody, it is dilute to be immersed in primary antibody by film Release in liquid, shaking table is incubated 1 ~ 2 h under room temperature condition；Film is washed with TBST liquid after taking-up film, immerses the fluorescence of 8-12mL dilutions afterwards In the secondary antibody liquid of carbon point mark 1-2 h are incubated under room temperature condition.
12）Luminous colour developing：Directly the film after immune processing is imaged in gel imaging system, using fluorescent carbon point in purple Launch the performance of fluorescence under outer light irradiation, obtain Western blotting image.If two samples all obtain fluorescent bands, illustrate ancient times argillization Silk goods belong to silk；If only modern silk has fluorescent bands, illustrate that ancient times argillization silk goods are not belonging to silk.
Preferably, step 1）In, the preparation method of fluorescent carbon point is specially：By 8-12mL deionized waters and 8-12mL without Water-ethanol mixes, and adds 0.35-0.45g fructose, stirring is to being completely dissolved；Mixed solution is poured into hydrothermal reaction kettle and sealed, 2 ~ 3 h of heating in 155-165 DEG C of baking oven are placed in, treat that solution is changed into light yellow, reaction is completed, and is naturally cooled to room temperature, is obtained glimmering Light carbon dots solution；The bag filter that obtained solution molecular cut off is 1000 is dialysed into 20-28 h to remove other small molecules Impurity；Finally the carbon dots solution of purifying is freeze-dried, can obtain fluorescent carbon point powder.
Fluorescent carbon point surface has many defect energy bands, when being excited light irradiation, because its electronics and hole are measured Sub- confinement, continuous band structure become the discrete energy level structure with molecular characterization, and radiation restructuring can be occurred by being excited rear exciton And launch fluorescence.
Preferably, step 2）In, the preparation method of fluorescent carbon point mark silk secondary antibody is specific as follows：By fluorescent carbon point, Carbodiimides and secondary antibody are added in 9-11mmol/L PBS, after stirring 2 ~ 3 h at ambient temperature, are delayed with PBS Fliud flushing carries out repeatedly washing centrifugation to solution, obtains the silk fibroin secondary antibody of fluorescent carbon point mark.
Fluorescent carbon point is after coupling, the enhancing of its fluorescence capability.Contain abundant group such as carboxyl, hydroxyl in fluorescent carbon point surface Base, amino etc., they to a certain extent can be with the exciton of trapped radiation fluorescence, therefore fluorescence capability strengthens after coupling, together When can also avoid fluorescent carbon point particle aggregation phenomenon.
Preferably, step 3）, step 4）In, the preparation of the PM13- alkali proteases：To 95-105mL 0.1M The pectination PEG of 0.10-0.20g alkali proteases and 3.5-4.0g MW15000 is added in sodium borate buffer solution, at 1-5 DEG C Under the conditions of be slowly stirred 0.5-1.5h, under conditions of additional 45-55mL 1mmol/L HCl ultrafiltration remove unreacted PM13, In triplicate, filtrate is freeze-dried, obtains PM13- basic protein enzyme powder.
The present invention first carries out preliminarily solubilised using ionic liquid to sample, reuses PM13- alkali protease is carried out to fibroin Further dissolving.Ionic liquid is the green solvent of a kind of great application prospect of rising in recent years, is a kind of intensive polar solvent, Hydrogen bond can be formed with the amino in fibroin albumen macromolecular and carboxyl, so as to destroy the hydrogen bond inside silk fibroin molecular, so as to Realize the dissolving to protein.Biology enzyme is a kind of nontoxic and environment-friendly catalyst, possesses efficient specificity； And dosage is few, pollution is reduced, is economized on resources；Hydrolysising condition（Temperature, pH）Gently, it is small to amino acid destruction, meanwhile, through PM13Repair The hydrolysis ability and stability for the alkali protease adornd all have strengthened, and can improve the dissolution rate to albumen in sample and extraction Rate.
The present invention uses PM13Alkali protease is modified, to keep stabilization of the alkali protease in ionic liquid Property and activity.Alkali protease is also a kind of protein in itself, and ionic liquid has certain destruction, PM to its structure13 Place is a kind of comb copolymer, can be covered in alkali protease surface, prevent alkali protease from being destroyed by solion, simultaneously Compatibility by PM13 chains to IL, the stability of enzyme can be not only improved, ensure activity of the enzyme in ionic liquid, and make Enzyme is uniformly dispersed in IL.
Preferably, step 6）In, the molecular cut off of bag filter is 1000.
Preferably, step 7）In, electrophoresis concrete operations are：Albumen enters separation gel after first constant pressure 80V, 8-12min, adjusts Whole constant voltage is 120V, and 15-25min rear electrophoresis are completed, and take out gel, observe electrophoretic image.
Preferably, step 8）In, in electrotransfer operation, the pvdf membrane used first soaks 50-70s, then electricity consumption with methanol Turn buffer solution immersion；Filter paper used, sponge need electricity consumption to turn buffer solution infiltration, according to sponge-filter paper-running gel- The order assembling transhipment interlayer of pvdf membrane-filter paper-sponge, constant current transferring film is the h of transferring film 1 ~ 2 under 100mA constant currents；This operation Using wet method transferring film, heat release in transfer process, membrane-transferring device need to be placed in ice bath.
Preferably, step 10）In, after being incubated primary antibody and secondary antibody, wash film three times with TBST liquid on shaking table at room temperature, often Secondary 4-6min.To elute uncombined primary antibody and fluorescence secondary antibody.
Preferably, step 10）In, the confining liquid is 5wt% skimmed milk powers.
Preferably, step 11）In, the antibody diluent is TBST liquid.
It is compared with the prior art, the beneficial effects of the invention are as follows：
（1）The present invention use source of the fructose as fluorescent carbon point, makes full use of resource, energy-conserving and environment-protective, green harmless.
（2）The present invention as fluorescence imaging material, shows strong fluorescent stability, resistance to photobleaching, hair from fluorescent carbon point The ejected wave length excellent fluorescence property such as controllable, is compared with fluorometric reagent with quantum dot, also with toxicity is low, good biocompatibility With particle diameter it is controllable the features such as, can substitute conventional fluorescent dyestuff and quantum dot and applied to the research of life science..
（3）The present invention adds C in scouring processes17H35COONa buffers, degumming efficiency is improved, while reduced to silk The injury of cellulose fiber.
（4）The present invention is progressively handled fibroin albumen, carried using the effect of the dual dissolution of ionic liquid and biology enzyme The high solubility of fibroin albumen.Meanwhile and ionic liquid is " green solvent ", environmentally friendly, group can design, and be easy to Recovery, can be recycled.Biology enzyme is nontoxic, with environment-friendly, while dosage is few, economizes on resources；It is specific high, act on bar Part is gentle, and the destruction to fibroin albumen is small.
（5）Inventive samples dosage is few, can intuitively, it is accurate, detect fibroin albumen high sensitivity, especially for Addle ancient times argillization silk goods that are serious, examining difficulty.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1：
1）Fluorescent carbon point（CDs）Preparation：10mL deionized water and absolute ethyl alcohol is measured respectively（Volume ratio 1:1）, mix molten Solution adds 0.40 g fructose, stirring is to being completely dissolved in beaker.Mixed solution is poured into hydrothermal reaction kettle and sealed, is placed in 2 h are heated in 160 DEG C of baking ovens, treat that solution is changed into light yellow, reaction is completed, and is naturally cooled to room temperature, is obtained fluorescent carbon point solution； The solution bag filter that will be obtained（Molecular cut off 1000）24h dialyse to remove other small molecular weight impurities；Finally to purifying Carbon dots solution is freeze-dried, and can obtain fluorescent carbon point powder.
2）Fluorescent carbon point marks the preparation of silk fibroin secondary antibody：Fluorescent carbon point, carbodiimides and secondary antibody are added By 1:20:20 mass ratio is added in 10mmol/L PBS, after stirring 2 h at ambient temperature, uses PBS Repeatedly washing centrifugation is carried out to solution, you can obtain the silk fibroin secondary antibody of fluorescent carbon point mark.
3）0.3g modern times silk is weighed as control sample, with 1:100 bath raio is containing 0.5% Na2PO4With 1% C17H3530min is boiled in COONa mixed solution, carries out degumming process, common degumming is twice；After degumming, by silk spend from Sub- water is washed by rubbing with the hands more than 4 times, is put into oven drying, obtains modern natural silk fiber.By modern natural silk fiber, with 1:50 Bath raio is immersed in [AMIM] Cl ionic liquids, heating response certain time, cooling；Add PM13- basic protein enzyme powder constant temperature is incubated 6h is educated, enzyme deactivation is carried out, obtains fibroin albumen/ionic liquid solution A.
Wherein, the PM13The preparation method of-alkali protease is：Add into 100mL 0.1M sodium borate buffer solution Enter 0.15g alkali proteases and 3.75g MW15000 pectination PEG, 1h, then additional 50mL are slowly stirred under the conditions of 4 DEG C Ultrafiltration removes unreacted PM under conditions of 1mmol/L HCl13, in triplicate, filtrate is freeze-dried, obtains PM13- Basic protein enzyme powder.
4）The soil sample that 30g carries ancient times argillization silk goods is weighed, after mortar grinder, adds 60mL [AMIM] Cl ions In liquid, according to 3）Middle step operation, obtain ionic liquid suspension.Suspension is subjected to high speed refrigerated centrifuge, supernatant liquor That is fibroin albumen/ionic liquid solution B.
5）Absolute ethyl alcohol is added in fibroin albumen/ionic liquid solution A, B, separates out albumen, filters out albumen, addition is gone Ionized water dissolves, and obtains pure fibroin albumen extract solution A, B.
6）Obtain two kinds of extract solutions are used into bag filter under the conditions of 4 DEG C（Molecular cut off 1000）Dialyse 48 h, removes Small molecular weight impurity；It is about 200 μ L using instrument concentration silk fibroin water solution to final volume is concentrated in vacuo.
7）PAGE gel electrophoresis：50 μ L fibroin albumen concentrate A, B are measured respectively, are diluted to the buffer solutions of CB 9.6 5mL, 5min is heated under the conditions of 100 DEG C, carry out denaturation treatment；To after room temperature after cooling, by two kinds of samples and protein What Marker additions prepared exempts to contaminate glue（Upper strata is concentration glue, and lower floor is separation gel）In well, electricity is filled it up with a cell Liquid is solved, after the completion of electrophoresis, electrophoretic image can be observed in imaging systems.Wherein, electrophoresis concrete operations are：First constant pressure 80V, Albumen enters separation gel after 10min, and adjustment constant voltage is 120V, and 20min rear electrophoresis are completed.
8）Electrotransfer：Glue after electrophoresis is laid in and turned through methanol and electricity on the treated pvdf membrane of buffer solution, is clipped in filter Transhipment interlayer is assembled among paper and sponge, filter paper used, sponge need electricity consumption to turn buffer solution infiltration, according to sponge-filter The order assembling transhipment interlayer of paper-running gel-pvdf membrane-filter paper-sponge, is put into wet type membrane-transferring device, in 100mA Membrane-transferring device, need to be placed in ice bath by the h of transferring film 1 under constant current, heat release in transfer process.
9）Film is imaged：After the completion of electrotransfer, pvdf membrane is taken out with tweezers, is put into gel imaging system and is imaged, observes egg Informal voucher band transfer case.
10）Closing：Pvdf membrane is taken out, film 5min is washed with 15mL TBS liquid, uses confining liquid（5% skimmed milk power）In room temperature bar 1h is handled under part.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:1000 dilution proportion primary antibody, primary antibody is immersed in by film In dilution, shaking table is incubated 1 h under room temperature condition；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time five Minute, 10mL is immersed afterwards with 1:In the secondary antibody liquid of the fluorescent carbon point mark of 1000 dilution proportion, 1.5 are incubated under room temperature condition H, after taking out film, wash film three times with TBST liquid on shaking table at room temperature, five minutes every time.
12）Luminous colour developing：Directly the film after immune processing is imaged in gel imaging system, existed using fluorescent carbon point Launch the performance of fluorescence under ultraviolet light, obtain Western blotting image.If two samples all obtain fluorescent bands, illustrate ancient times mud Change silk goods and belong to silk；If only modern silk has fluorescent bands, illustrate that ancient times argillization silk goods are not belonging to silk.
Embodiment 2：
1）Fluorescent carbon point（CDs）Preparation：10mL deionized water and absolute ethyl alcohol is measured respectively（Volume ratio 1:1）, mix molten Solution adds 0.40 g fructose, stirring is to being completely dissolved in beaker.Mixed solution is poured into hydrothermal reaction kettle and sealed, is placed in 2.5 h are heated in 160 DEG C of baking ovens, treat that solution is changed into light yellow, reaction is completed, and naturally cools to room temperature, it is molten to obtain fluorescent carbon point Liquid；The solution bag filter that will be obtained（Molecular cut off 1000）24 h dialyse to remove other small molecular weight impurities；Finally to pure The carbon dots solution of change is freeze-dried, and can obtain fluorescent carbon point powder.
2）Fluorescent carbon point marks the preparation of silk fibroin secondary antibody：Fluorescent carbon point, carbodiimides and secondary antibody are added By 1:20:20 mass ratio is added in 10mmol/L PBS, after stirring 2.5 h at ambient temperature, is buffered with PBS Liquid carries out repeatedly washing centrifugation to solution, you can obtains the silk fibroin secondary antibody of fluorescent carbon point mark.
3）0.3g modern times silk is weighed as control sample, with 1:100 bath raio is containing 0.5% Na2PO4With 1% C17H3530min is boiled in COONa mixed solution, carries out degumming process, common degumming is twice；After degumming, by silk spend from Sub- water is washed by rubbing with the hands more than 4 times, is put into oven drying, obtains natural silk fiber.Modern natural silk fiber, with 1:50 bath raio leaching Enter in [AMIM] Cl ionic liquids, heating response certain time, cool down；Add PM13- basic protein enzyme powder constant-temperature incubation 6h, Enzyme deactivation is carried out, obtains fibroin albumen/ionic liquid solution A.
Wherein, the PM13The preparation method of-alkali protease is：Add into 100mL 0.1M sodium borate buffer solution Enter 0.15g alkali proteases and 3.75g MW15000 pectination PEG, 1h, then additional 50mL are slowly stirred under the conditions of 4 DEG C Ultrafiltration removes unreacted PM under conditions of 1mmol/L HCl13, in triplicate, filtrate is freeze-dried, obtains PM13- Basic protein enzyme powder.
4）The soil sample that 30g carries ancient times argillization silk goods is weighed, after mortar grinder, adds 60mL [AMIM] Cl ions In liquid, according to 3）Middle step operation, obtain ionic liquid suspension.Suspension is subjected to high speed refrigerated centrifuge, supernatant liquor That is fibroin albumen/ionic liquid solution B.
5）Absolute ethyl alcohol is added in fibroin albumen/ionic liquid solution A, B, separates out albumen, filters out albumen, addition is gone Ionized water dissolves, and obtains pure fibroin albumen extract solution A, B.
6）Obtain two kinds of extract solutions are used into bag filter under the conditions of 4 DEG C（Molecular cut off 1000）Dialyse 48 h, removes Small molecular weight impurity；It is about 200 μ L using instrument concentration silk fibroin water solution to final volume is concentrated in vacuo.
7）PAGE gel electrophoresis：50 μ L fibroin albumen concentrate A, B are measured respectively, are diluted to the buffer solutions of CB 9.6 5mL, 5min is heated under the conditions of 100 DEG C, carry out denaturation treatment；To after room temperature after cooling, by two kinds of samples and protein What Marker additions prepared exempts to contaminate glue（Upper strata is concentration glue, and lower floor is separation gel）In well, electricity is filled it up with a cell Liquid is solved, after the completion of electrophoresis, electrophoretic image can be observed in imaging systems.Wherein, electrophoresis concrete operations are：First constant pressure 80V, Albumen enters separation gel after 10min, and adjustment constant voltage is 120V, and 20min rear electrophoresis are completed.
8）Electrotransfer：Glue after electrophoresis is laid in and turned through methanol and electricity on the treated pvdf membrane of buffer solution, is clipped in filter Transhipment interlayer is assembled among paper and sponge, filter paper used, sponge need electricity consumption to turn buffer solution infiltration, according to sponge-filter The order assembling transhipment interlayer of paper-running gel-pvdf membrane-filter paper-sponge, is put into wet type membrane-transferring device, in 100mA Membrane-transferring device, need to be placed in ice bath by the h of transferring film 1.5 under constant current, heat release in transfer process.
9）Film is imaged：After the completion of electrotransfer, pvdf membrane is taken out with tweezers, is put into gel imaging system and is imaged, observes egg Informal voucher band transfer case.
10）Closing：Pvdf membrane is taken out, film 5min is washed with 15mL TBS liquid, uses confining liquid（5% skimmed milk power）In room temperature bar 1h is handled under part.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:1000 dilution proportion primary antibody, primary antibody is immersed in by film In dilution, shaking table is incubated 1.5 h under room temperature condition；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time Five minutes, 10mL was immersed afterwards with 1:In the secondary antibody liquid of the fluorescent carbon point mark of 2000 dilution proportion, it is incubated under room temperature condition 1.5 h, after taking out film, wash film three times with TBST liquid on shaking table at room temperature, five minutes every time.
12）Luminous colour developing：Directly the film after immune processing is imaged in gel imaging system, existed using fluorescent carbon point Launch the performance of fluorescence under ultraviolet light, obtain Western blotting image.If two samples all obtain fluorescent bands, illustrate ancient times mud Change silk goods and belong to silk；If only modern silk has fluorescent bands, illustrate that ancient times argillization silk goods are not belonging to silk.
Embodiment 3：
1）Fluorescent carbon point（CDs）Preparation：10mL deionized water and absolute ethyl alcohol is measured respectively（Volume ratio 1:1）, mix molten Solution adds 0.40 g fructose, stirring is to being completely dissolved in beaker.Mixed solution is poured into hydrothermal reaction kettle and sealed, is placed in 3 h are heated in 160 DEG C of baking ovens, treat that solution is changed into light yellow, reaction is completed, and is naturally cooled to room temperature, is obtained fluorescent carbon point solution； The solution bag filter that will be obtained（Molecular cut off 1000）24 h dialyse to remove other small molecular weight impurities；Finally to purifying Carbon dots solution is freeze-dried, and can obtain fluorescent carbon point powder.
2）Fluorescent carbon point marks the preparation of silk fibroin secondary antibody：Fluorescent carbon point, carbodiimides and secondary antibody are added By 1:20:20 mass ratio is added in 10mmol/L PBS, after stirring 2 ~ 3 h at ambient temperature, is buffered with PBS Liquid carries out repeatedly washing centrifugation to solution, you can obtains the silk fibroin secondary antibody of fluorescent carbon point mark.
3）0.3g modern times silk is weighed as control sample, with 1:100 bath raio is containing 0.5% Na2PO4With 1% C17H3530min is boiled in COONa mixed solution, carries out degumming process, common degumming is twice；After degumming, by silk spend from Sub- water is washed by rubbing with the hands more than 4 times, is put into oven drying, obtains natural silk fiber.Modern natural silk fiber, with 1:50 bath raio leaching Enter in [AMIM] Cl ionic liquids, heating response certain time, cool down；Add PM13- basic protein enzyme powder constant-temperature incubation 6h, Enzyme deactivation is carried out, obtains fibroin albumen/ionic liquid solution A.
Wherein, the PM13The preparation method of-alkali protease is：Add into 100mL 0.1M sodium borate buffer solution Enter 0.15g alkali proteases and 3.75g MW15000 pectination PEG, 1h, then additional 50mL are slowly stirred under the conditions of 4 DEG C Ultrafiltration removes unreacted PM under conditions of 1mmol/L HCl13, in triplicate, filtrate is freeze-dried, obtains PM13- Basic protein enzyme powder.
4）The soil sample that 30g carries ancient times argillization silk goods is weighed, after mortar grinder, adds 60mL [AMIM] Cl ions In liquid, according to 3）Middle step operation, obtain ionic liquid suspension.Suspension is subjected to high speed refrigerated centrifuge, supernatant liquor That is fibroin albumen/ionic liquid solution B.
5）Absolute ethyl alcohol is added in fibroin albumen/ionic liquid solution A, B, separates out albumen, filters out albumen, addition is gone Ionized water dissolves, and obtains pure fibroin albumen extract solution A, B.
6）Obtain two kinds of extract solutions are used into bag filter under the conditions of 4 DEG C（Molecular cut off 1000）Dialyse 48 h, removes Small molecular weight impurity；It is about 200 μ L using instrument concentration silk fibroin water solution to final volume is concentrated in vacuo.
7）PAGE gel electrophoresis：50 μ L fibroin albumen concentrate A, B are measured respectively, are diluted to the buffer solutions of CB 9.6 5mL, 5min is heated under the conditions of 100 DEG C, carry out denaturation treatment；To after room temperature after cooling, by two kinds of samples and protein What Marker additions prepared exempts to contaminate glue（Upper strata is concentration glue, and lower floor is separation gel）In well, electricity is filled it up with a cell Liquid is solved, after the completion of electrophoresis, electrophoretic image can be observed in imaging systems.Wherein, electrophoresis concrete operations are：First constant pressure 80V, Albumen enters separation gel after 10min, and adjustment constant voltage is 120V, and 20min rear electrophoresis are completed.
8）Electrotransfer：Glue after electrophoresis is laid in and turned through methanol and electricity on the treated pvdf membrane of buffer solution, is clipped in filter Transhipment interlayer is assembled among paper and sponge, filter paper used, sponge need electricity consumption to turn buffer solution infiltration, according to sponge-filter The order assembling transhipment interlayer of paper-running gel-pvdf membrane-filter paper-sponge, is put into wet type membrane-transferring device, in 100mA Membrane-transferring device, need to be placed in ice bath by the h of transferring film 2 under constant current, heat release in transfer process.
9）Film is imaged：After the completion of electrotransfer, pvdf membrane is taken out with tweezers, is put into gel imaging system and is imaged, observes egg Informal voucher band transfer case.
10）Closing：Pvdf membrane is taken out, film 5min is washed with 15mL TBS liquid, uses confining liquid（5% skimmed milk power）In room temperature bar 1h is handled under part.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:1000 dilution proportion primary antibody, primary antibody is immersed in by film In dilution, shaking table is incubated 2 h under room temperature condition；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time five Minute, 10mL is immersed afterwards with 1:In the secondary antibody liquid of the fluorescent carbon point mark of 3000 dilution proportion, 1.5 are incubated under room temperature condition H, after taking out film, wash film three times with TBST liquid on shaking table at room temperature, five minutes every time.
12）Luminous colour developing：Directly the film after immune processing is imaged in gel imaging system, existed using fluorescent carbon point Launch the performance of fluorescence under ultraviolet light, obtain Western blotting image.If two samples all obtain fluorescent bands, illustrate ancient times mud Change silk goods and belong to silk；If only modern silk has fluorescent bands, illustrate that ancient times argillization silk goods are not belonging to silk.
Raw materials used in the present invention, equipment, it is the conventional raw material, equipment of this area unless otherwise noted；In the present invention Method therefor, it is the conventional method of this area unless otherwise noted.
It is described above, only it is presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, it is every according to the present invention Any simple modification, change and the equivalent transformation that technical spirit is made to above example, still fall within the technology of the present invention side The protection domain of case.

Claims (10)

  1. A kind of 1. method based on immune vestige method detection ancient times argillization silk goods, it is characterised in that comprise the following steps：
    1）The preparation of fluorescent carbon point：Fructose is dissolved in deionized water and the in the mixed solvent of absolute ethyl alcohol, in hydrothermal reaction kettle Interior reaction, fluorescent carbon point solution is made, after through dialysis purification, freeze-drying, obtain fluorescent carbon point powder；
    2）Fluorescent carbon point marks the preparation of silk fibroin secondary antibody：Fluorescent carbon point and silkworm are coupled using carbodiimides mediated method Silk fibroin albumen secondary antibody, fluorescent carbon point mark silk secondary antibody is made；
    3）Modern silk is weighed as control sample, with 1:95-105 bath raio is containing 0.4-0.6wt% Na2PO4And 0.8- 1.2wt%C17H3525-35min is boiled in COONa mixed solution, carries out degumming process, common degumming is twice；, will after degumming Silk is washed by rubbing with the hands more than 4 times with deionized water, is put into oven drying, obtains modern natural silk fiber；Then by modern silk silk Cellulose fiber is with 1:45-55 bath raio is immersed in [AMIM] Cl ionic liquids, heating response, cooling；Add PM13- alkali protease Powder constant-temperature incubation 5-7h, enzyme deactivation is carried out, obtains fibroin albumen/ionic liquid solution A；
    4）The soil sample with ancient times argillization silk goods is weighed, after mortar grinder, is added to by solid-to-liquid ratio 25-35g/60mL In [AMIM] Cl ionic liquids, heating response, cooling；Add PM13- basic protein enzyme powder constant-temperature incubation 5-7h, enzyme deactivation is carried out, Obtain ionic liquid suspension；By suspension high speed refrigerated centrifuge, take supernatant liquor obtain egg fibroin it is white/ionic liquid solution B；
    5）Respectively to fibroin it is white/ionic liquid solution A, B in add absolute ethyl alcohol, soak repeatedly, separate out albumen, filter out albumen Afterwards, deionized water dissolving is added, obtains pure fibroin albumen extract solution A, B；
    6）By fibroin albumen extract solution A, B respectively under the conditions of 1-5 DEG C with bag filter dialysis 40-50h, small molecular weight impurity is removed； It is about 150-250 μ L to be concentrated in vacuo to final volume, respectively obtains fibroin albumen concentrate A, B；
    7）PAGE gel electrophoresis：40-60 μ L fibroin albumen concentrate A, B are measured respectively, are diluted to the buffer solutions of CB 9.6 4-6mL, 4-6min is heated under the conditions of 98-102 DEG C, carry out denaturation treatment；To after room temperature after cooling, by two kinds of samples and albumen Matter Marker add prepare exempt to contaminate in glue well, fill it up with electrolyte in a cell, after the completion of electrophoresis, can be in imaging Electrophoretic image is observed in system；
    8）Electrotransfer：Glue after electrophoresis is laid in and turned through methanol and electricity on the treated pvdf membrane of buffer solution, be clipped in filter paper and Transhipment interlayer is assembled among sponge, is put into wet type membrane-transferring device, constant current transferring film；
    9）Film is imaged：After the completion of electrotransfer, pvdf membrane is taken out, is put into gel imaging system and is imaged, observation protein band transfer Situation；
    10）Closing：Pvdf membrane is taken out, film 4-6min is washed with 10-20mL TBS liquid, 50- is handled at ambient temperature with confining liquid 70min；
    11）Immune processing：With antibody diluent with 950-1050:1 volume ratio dilution primary antibody, primary antibody dilution is immersed in by film In, shaking table is incubated 1 ~ 2 h under room temperature condition；Film is washed with TBST liquid after taking-up film, immerses the fluorescent carbon point of 8-12mL dilutions afterwards 1-2 h are incubated in the secondary antibody liquid of mark under room temperature condition；
    12）Luminous colour developing：Directly the film after immune processing is imaged in gel imaging system, using fluorescent carbon point in ultraviolet light The performance of the lower transmitting fluorescence of irradiation, obtains Western blotting image；If two samples all obtain fluorescent bands, illustrate ancient times argillization silk weaving Product belong to silk；If only modern silk has fluorescent bands, illustrate that ancient times argillization silk goods are not belonging to silk.
  2. A kind of 2. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 1）In, the preparation method of fluorescent carbon point is specially：8-12mL deionized waters and 8-12mL absolute ethyl alcohols are mixed, added 0.35-0.45g fructose, stirring is to being completely dissolved；Mixed solution is poured into hydrothermal reaction kettle and sealed, is placed in 155-165 DEG C of baking 2 ~ 3 h are heated in case, treat that solution is changed into light yellow, reaction is completed, and is naturally cooled to room temperature, is obtained fluorescent carbon point solution；Will To solution molecular cut off be 1000 bag filter dialyse 20-28 h to remove other small molecular weight impurities；Finally to purifying Carbon dots solution be freeze-dried, can obtain fluorescent carbon point powder.
  3. A kind of 3. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 2）In, the preparation method of fluorescent carbon point mark silk secondary antibody is specific as follows：Fluorescent carbon point, carbodiimides and secondary antibody are added Enter into 9-11mmol/L PBS, after stirring 2 ~ 3 h at ambient temperature, solution is carried out repeatedly with PBS Washing centrifugation, obtains the silk fibroin secondary antibody of fluorescent carbon point mark.
  4. A kind of 4. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 3）, step 4）In, the PM13The preparation of-alkali protease：Add into 95-105mL 0.1M sodium borate buffer solution Enter 0.10-0.20g alkali proteases and 3.5-4.0g MW15000 pectination PEG, 0.5- is slowly stirred under the conditions of 1-5 DEG C 1.5h, the unreacted PM of ultrafiltration removing under conditions of additional 45-55mL 1mmol/L HCl13, in triplicate, filtrate is carried out Freeze-drying, obtains PM13- basic protein enzyme powder.
  5. A kind of 5. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 6）In, the molecular cut off of bag filter is 1000.
  6. A kind of 6. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 7）In, electrophoresis concrete operations are：Albumen enters separation gel after first constant pressure 80V, 8-12min, and adjustment constant voltage is 120V, 15-25min rear electrophoresis are completed, and take out gel, observe electrophoretic image.
  7. A kind of 7. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 8）In, in electrotransfer operation, the pvdf membrane used first soaks 50-70s with methanol, then electricity consumption turns buffer solution immersion；It is used Filter paper, sponge need electricity consumption turn buffer solution infiltration, according to sponge-filter paper-running gel-pvdf membrane-filter paper-sponge Order assembling transhipment interlayer, constant current transferring film is the h of transferring film 1 ~ 2 under 100mA constant currents；This operation uses wet method transferring film, shifts Heat release in journey, membrane-transferring device need to be placed in ice bath.
  8. A kind of 8. method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, it is characterised in that Step 10）In, after being incubated primary antibody and secondary antibody, wash film three times with TBST liquid on shaking table at room temperature, each 4-6min.
  9. 9. a kind of method based on immune vestige method detection ancient times argillization silk goods as described in claim 1 or 8, its feature exist In step 10）In, the confining liquid is 5wt% skimmed milk powers.
  10. 10. a kind of method based on immune vestige method detection ancient times argillization silk goods as claimed in claim 1, its feature exist In step 11）In, the antibody diluent is TBST liquid.
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CN108387435A (en) * 2018-01-30 2018-08-10 中国计量大学 A kind of trace fibroin albumen enrichment method
CN108828236A (en) * 2018-08-23 2018-11-16 浙江理工大学 A method of ancient times woolen knitwear relic is detected based on Western blot
CN109060927A (en) * 2018-08-30 2018-12-21 浙江理工大学 A method of the detected through gel electrophoresis ancient times woolen knitwear dyed using carbon quantum dot
CN109187699A (en) * 2018-09-05 2019-01-11 浙江理工大学 A method of detection Ancient Silk Textile
CN109187699B (en) * 2018-09-05 2020-06-23 浙江理工大学 Method for detecting ancient silk fabric
CN109187949B (en) * 2018-09-07 2021-06-08 浙江理工大学 Method for identifying types of remnant wool of ancient wool fabrics based on immunoblotting method
CN109187949A (en) * 2018-09-07 2019-01-11 浙江理工大学 A method of ancient times woolen knitwear relic sort of wool is identified based on Western blot
CN109283332A (en) * 2018-11-11 2019-01-29 浙江理工大学 A method of based on fibroin microscratch mark in Western Bolt measurement ancient site
CN109283332B (en) * 2018-11-11 2021-07-13 浙江理工大学 Method for determining fibroin micro-traces in ancient site based on Western Blot
CN110333341A (en) * 2019-07-04 2019-10-15 浙江理工大学 A method of silk fabric cultural relics sample is identified based on protein biochip technology
CN110441526A (en) * 2019-07-10 2019-11-12 浙江理工大学 A method of wool textile historical relic sample is identified based on protein biochip technology
CN110412297A (en) * 2019-08-26 2019-11-05 浙江理工大学 A method of sample fidelity is simulated based on Western blot silk fabric cultural relics
CN111781371A (en) * 2020-06-18 2020-10-16 中国计量大学 Method for rapidly detecting trace silk fibroin on archaeological site based on magnetic bead carrier double-antibody sandwich ELISA
CN111781371B (en) * 2020-06-18 2023-01-03 中国计量大学 Method for rapidly detecting trace silk fibroin on archaeological site based on magnetic bead carrier double-antibody sandwich ELISA
CN113030488A (en) * 2021-04-01 2021-06-25 浙江理工大学 Method for detecting silk fibroin by using immunomagnetic surface enhanced Raman substrate
CN113030488B (en) * 2021-04-01 2022-04-01 浙江理工大学 Method for detecting silk fibroin by using immunomagnetic surface enhanced Raman substrate

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