CN105295080B - A kind of preparation method of function fibroin membrane beneficial to cell adherence - Google Patents
A kind of preparation method of function fibroin membrane beneficial to cell adherence Download PDFInfo
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- CN105295080B CN105295080B CN201510705912.3A CN201510705912A CN105295080B CN 105295080 B CN105295080 B CN 105295080B CN 201510705912 A CN201510705912 A CN 201510705912A CN 105295080 B CN105295080 B CN 105295080B
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Abstract
The invention discloses a kind of preparation method of the function fibroin membrane beneficial to cell adherence, step is：It will carry (RGD)4Polypeptide or (RGD)8The prokaryotic system expression vector transfection Escherichia coli cell of polypeptide gene, Fiber differentiation a few hours；Centrifuge cell bacterium solution, is collected and broken bacterium cell, the albumen supernatant purifying after crushing, digestion one-step method are collected, are lyophilized, obtaining (RGD)4Polypeptide powder or (RGD)8Polypeptide powder；After silkworm silk or silk cocoon are carried out into degumming, dissolving, dialysis, filtering and concentrating, it is cast in the vessel of certain area and air-dries, obtain bombyx mori silk fibroin film；By bombyx mori silk fibroin film immersion in the cross-linking agent solution of excess, add (RGD)4Polypeptide or (RGD)8Polypeptide, 4 DEG C of reaction overnights, produces function fibroin membrane.The function fibroin membrane that the present invention prepares has excellent cell adherence performance, belongs to bio-medical material, without cytotoxicity, has the recognition site of CAP, is advantageous to cell adherence, growth, promotes defective tissue healing.
Description
Technical field
The present invention relates to a kind of preparation method of the silk fibroin material applied to biomedical sector, and in particular to one kind is beneficial to
The preparation method of the function fibroin membrane of cell adherence.
Background technology
RGD sequence is made up of arginine, glycine and aspartic acid, is widely present in various kinds of cell epimatrix, collagen
Albumen, FTN, laminin, vitronectin, elastin laminin etc. extracellular matrix, can be with a variety of integrins
Specific binding, contributes to cell adherence, promotes attached cell adhesion growth.Fibroin membrane through RGD chemical modifications and other change
Property material such as high molecular polymer can improve adhesion, diffusion and propagation (such as J Biomed Mater of the cell on material
Res A, 2003,67 (2), 559-570；Biomaterials, 2002,23：4315-4323 etc.).RGD peptide can also be competitive
Suppress the various attachment proteinses of such as fibrin and hematoblastic combination, and adhesion (Letters of the blood platelet on material
In Peptide Science, 2002,9：101-109), so as to preventing the formation of thrombus.
The RGD peptide of existing sale is from the peptide of chemical synthesis, including chain type peptide and cyclic peptide.With genetic engineering
The fast development of technology, recombination expression turn into a kind of important technology prepared by Functional Polypeptides.There is research to point out a kind of containing for restructuring
RGD silk-fibroin have good cell adherence and multiplication capacity (chemical journal, 2006,64：1273-1278).There is report to claim
Adhesiveness and differentiation capability of the osteocyte on the oligopeptides containing RGD of recombination expression are better than the coated culture plate of lysine
(Biotechnol Lett, 2007,29：359-363).And for example it is undifferentiated to point out that restructuring RGD spider silk fibroins can be supported for research
Mouse bone-forming cell differentiation (Biomaterials, 2008,29：2556-2563)；By RGD recombinant spider silks and polyvinyl alcohol
High polymer material combines, can support rat embryo fibroblast cell growth (Chinese Reconstructive surgery magazine, 2009,23：
747-750) etc..
Silk is a kind of good natural biologic material, and that studies both at home and abroad is main based on bombyx mori silk fibroin, and on open country
Silk such as tussah silk or wild silk yarn are used for research report seldom (the Journal of Wuhan University of of biomaterial
Technology:Materials Scinece, 2011,26 (6)：1044-1048；Biomed Mater, 2006,1：181-
187), due to can not be domestic, yield be very low.It will be appreciated, however, that divide in the amino acid sequence of tussah silk peptide or giant silkworm fibroin
Be furnished with the unit repeated fragment of the largely tripeptides containing RGD, this unit sequence containing RGD come from natural wild silkworm fibroin and
The function of repetitive sequence without research report, is not more applied also.
The content of the invention
RGD functional polypeptides fragment but silk yield are contained based on natural tussore silk fibroin (tussah silk peptide or giant silkworm fibroin)
Extremely low present situation, the present invention is intended to provide a kind of functional polypeptide (- RGD-) for coming from organism synthesis4(- RGD-)8And its
The preparation method of modified bombyx mori silk fibroin film, wherein-RGD- amino acid sequence is come from natural tussore silk fibroin
One section, the function fibroin membrane prepared belongs to bio-medical material, without cytotoxicity, the identification position with CAP
Point, be advantageous to cell adherence, growth, promote defective tissue healing.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
A kind of preparation method of function fibroin membrane beneficial to cell adherence, comprises the following steps：
Step 1) wherein contains RGD tri- according to the analysis to tussah silk peptide or giant silkworm fibroin amino acid sequence, design one
The unit peptide fragment GSGAGGRGDGGYGDGSS of peptide sequence, is named as (- RGD-)1Polypeptide gene；
Step 2) is using technique for gene engineering to (- the RGD-)1Polypeptide gene carries out 4 times of clones, and design construction is taken
With (- RGD-)4The prokaryotic system expression vector of polypeptide gene, (- the RGD-)4The amino acid sequence of polypeptide is such as
SEQ.ID.NO.1；
Or, using technique for gene engineering to (- the RGD-)1Polypeptide gene carries out 8 times of clones, and design construction carries
(-RGD-)8The prokaryotic system expression vector of polypeptide gene, (- the RGD-)8The amino acid sequence of polypeptide such as SEQ.ID.NO.2；
Step 3) is carried design construction is good (- RGD-)4Polypeptide or (- RGD-)8The prokaryotic system of polypeptide gene
Expression vector (Bio-Medical Materials and Engineering, 2014,24：2057-2064) transfection Escherichia coli
(BL21) cell, with Luria-Bertani culture mediums through isopropyl-β-D-thiogalactoside Fiber differentiation a few hours；
Step 4) centrifuge cell bacterium solution, collects bacterium cell and broken bacterium cell, and the albumen supernatant after then crushing adds
Enter into the affinity chromatography of glutathione transferase one-step method purifying, digestion, collection, lyophilized, final acquisition (- RGD-)4Polypeptide
Powder or (- RGD-)8Polypeptide powder, the certain density aqueous solution is configured during use；
After silkworm silk or silk cocoon are carried out degumming, dissolving by step 5), it is poured into bag filter, is dialysed with deionized water,
Then the silk fibroin protein solution of filtering and concentrating to 3~8%, it is cast in the vessel of certain area and air-dries, obtain domestic silkworm silk
Plain film；
Step 6) in the cross-linking agent solution of excess, adds obtained above-mentioned bombyx mori silk fibroin film immersion (- RGD-)4It is more
Peptide or (- RGD-)8Polypeptide, 4 DEG C of reaction overnights, produces function fibroin membrane.
Compared with prior art, the invention has the advantages that：
RGD functional polypeptides fragment but silk yield are contained based on natural tussore silk fibroin (tussah silk peptide or giant silkworm fibroin)
Extremely low present situation, the present invention is intended to provide a kind of preparation method of function fibroin membrane beneficial to cell adherence.Pass through this method
The function fibroin membrane prepared has excellent cell adherence performance, belongs to bio-medical material, without cytotoxicity, has thin
The recognition site of born of the same parents' attachment proteins, be advantageous to cell adherence, growth, promote defective tissue healing.Its reason is due to the present invention
The polypeptide of offer comes from one section (- RGD-) of the functional sequence containing RGD in natural wild silkworm fibroin1Polypeptide and its repetition sequence
Row, not only peptide chain side base contains substantial amounts of hydrophilic radical, is advantageous to cell growth and organization healing；And the peptide of each molecule
Chain contains the recognition site RGD of multiple CAPs, the same polypeptide for being grafted a molecule, (- RGD-)4Polypeptide and (-
RGD-)8Polypeptide can at most have 4 and 8 RGD sites respectively, can advantageously promote the adhesion of cell and sprawl.Meanwhile
Polypeptide provided by the invention comes from organism expressing, and nontoxic nonirritant, molecular weight is single, can be covalent with silk fibroin protein
With reference to, stably assign the more preferable cell adherence function of bombyx mori silk fibroin film.In addition, the acquisition of RGD containing peptides is (pure in the present invention
Change, digestion) it is that a step is completed in GST affinity columns, reduce the destruction to expression product.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, described in detail below with presently preferred embodiments of the present invention as after.
Embodiment
Below in conjunction with embodiment, to describe the present invention in detail.
A kind of preparation method of function fibroin membrane beneficial to cell adherence, can be implemented by following examples.Preparing
Before the function fibroin membrane, first wherein contained according to the analysis to tussah silk peptide or giant silkworm fibroin amino acid sequence, design one
The unit peptide fragment GSGAGGRGDGGYGDGSS of RGD tripeptide sequences, is named as (- RGD-)1Polypeptide.Then genetic engineering skill is used
Art is to (- the RGD-)1Polypeptide gene carries out 4 times of clones, and design construction carries (- RGD-)4The prokaryotic system of polypeptide gene
Expression vector, (- the RGD-)4The amino acid sequence of polypeptide such as SEQ.ID.NO.1；Or using technique for gene engineering to it is described (-
RGD-)1Polypeptide gene carries out 8 times of clones, and design construction carries (- RGD-)8The prokaryotic system expression vector of polypeptide gene,
(- the RGD-)8The amino acid sequence of polypeptide such as SEQ.ID.NO.2.
Embodiment one：
1st, will carry (- RGD-)4Prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, apply
The Luria-Bertani solid mediums containing ampicillin are overlying on, are inverted into 37 DEG C of biochemical cultivation case culture 14~16
Hour.The single bacterium colony of picking is put into the 4mL fresh Luria-Bertani fluid nutrient mediums containing ampicillin, and insertion is shaken
Swing speed 200r/min 37 DEG C of air table shaken cultivation 8~10 hours.The bacterium solution of culture is pressed 1:100 ratio is new in 1L
Amplification cultivation in the fresh Luria-Bertani fluid nutrient mediums containing ampicillin.When bacterium solution density reaches OD600=0.3~2.1
When add 0~1.0mM isopropyl-β-D-thiogalactoside Fiber differentiation 1~8 hour, 4 DEG C are collected by centrifugation bacterium cell.
2nd, the bacterium cell of collection is resuspended with the combination buffer of glutathione transferase (GST) affinity chromatography and mixed, put
In ultrasonic disruption cell on ice, release protein.After the completion of broken 12000r/min, 4 DEG C of centrifugation 10min collect containing (-
RGD-)4The supernatant of polypeptide.Expression contains (- RGD-)4The albumen of peptide sequence is the fusion protein GST- containing GST labels
(-RGD-)4。
3rd, containing GST- (- RGD-)4Supernatant perfusion GST affinity columns, elute nonspecific proteins.Then by every
Milligram fusion protein adds 20 DEG C of 5~10u fibrin ferments and placed 16 hours.It is last slowly flow out and collect efflux obtain (-
RGD-)4Polypeptide solution.
4th, by (- RGD-)4Polypeptide solution addition desalting column purifies all salt components in solution, (- the RGD-) of collection4It is more
The peptide aqueous solution immediately with liquid nitrogen freezing, be subsequently placed in freeze drier and be dried to (- RGD-)4Polypeptide powder.
5th, silkworm raw silk is in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, by the family that a certain amount of degumming is clean
Silk element is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), in 72 DEG C of water-baths
It is stirred well to fibroin fully to dissolve, silk fibroin solution is encased in bag filter, is dialysed in 4 DEG C of deionized waters 3 days, every 2 hours
Change a deionized water, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, evaporated
Certain moisture is removed, obtains the silk fibroin water solution that concentration is 4~10%.
6th, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water.In ice
Under the conditions of bath, appropriate MES is added into every hole and is handled 1 hour, then adds excessive EDC and NHS mixed liquor, ice bath bar
Part is handled 1 hour.Xiang Kongzhong adds 0.01 μM of (- RGD-)4Polypeptide 1mL, reaction overnight at 4 DEG C, is finally rushed with deionized water
Wash clean, unreacted polypeptide and crosslinking agent are removed, cell culture is used for after irradiated sterilizing.
7th, 2 × 10 are inoculated with into hole5Individual L929 cells, adhesion experiment result：It is grafted (- RGD-)42 on the fibroin membrane of polypeptide
The cell adherence rate of hour is more than 90%, and unused (- RGD-)4The cell adherence rate of peptide modified fibroin membrane is 87%,
Significant difference be present.When being transplanted for internal defective tissue, material, which can adhere to normal cell faster and be advantageous to tissue, to be repaiied
It is multiple.
Embodiment two：
1st, will carry (- RGD-)8Prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, apply
The Luria-Bertani solid mediums containing ampicillin are overlying on, are inverted into 37 DEG C of biochemical cultivation case culture 14~16
Hour.The single bacterium colony of picking is put into the 4mL fresh Luria-Bertani fluid nutrient mediums containing ampicillin, and insertion is shaken
Swing speed 200r/min 37 DEG C of air table shaken cultivation 8~10 hours.The bacterium solution of culture is pressed 1:100 ratio is new in 1L
Amplification cultivation in the fresh Luria-Bertani fluid nutrient mediums containing ampicillin.When bacterium solution density reaches OD600=0.3~2.1
When add 0~1.0mM isopropyl-β-D-thiogalactoside Fiber differentiation 1~8 hour, 4 DEG C are collected by centrifugation bacterium cell.
2nd, the bacterium cell of collection is resuspended with the combination buffer of glutathione transferase (GST) affinity chromatography and mixed, put
In ultrasonic disruption cell on ice, release protein.After the completion of broken 12000r/min, 4 DEG C of centrifugation 10min collect containing (-
RGD-)8The supernatant of polypeptide.Expression contains (- RGD-)8The albumen of peptide sequence is the fusion protein GST- containing GST labels
(-RGD-)8。
3rd, containing GST- (- RGD-)8Supernatant perfusion GST affinity columns, elute nonspecific proteins.Then by every
Milligram fusion protein adds 20 DEG C of 5~10u fibrin ferments and placed 16 hours.It is last slowly flow out and collect efflux obtain (-
RGD-)8Polypeptide solution.
4th, by (- RGD-)8Polypeptide solution addition desalting column purifies all salt components in solution, (- the RGD-) of collection8It is more
The peptide aqueous solution immediately with liquid nitrogen freezing, be subsequently placed in freeze drier and be dried to (- RGD-)8Polypeptide powder.
5th, by silkworm raw silk in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, a certain amount of degumming is clean
Bombyx mori silk fibroin is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), 72 DEG C of water-baths
In be stirred well to fibroin and fully dissolve, silk fibroin solution is encased in bag filter, dialysed 3 days in 4 DEG C of deionized waters, every 2 is small
Deionized water of Shi Genghuan, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, steamed
Hair removes certain moisture, obtains the silk fibroin water solution that concentration is 4~10%.
6th, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water.In ice
Under the conditions of bath, appropriate MES is added into every hole and is handled 1 hour, then adds excessive EDC and NHS mixed liquor, ice bath bar
Part is handled 1 hour.Xiang Kongzhong adds 0.01 μM of (- RGD-)8Polypeptide 1mL, reaction overnight at 4 DEG C, is finally rushed with deionized water
Wash clean, unreacted polypeptide and crosslinking agent are removed, cell culture is used for after irradiated sterilizing.
7th, 2 × 10 are inoculated with into hole5Individual L929 cells, adhesion experiment result：It is grafted (- RGD-)82 on the fibroin membrane of polypeptide
The cell adherence rate of hour is more than 92.5%, and unused (- RGD-)8The cell adherence rate of peptide modified fibroin membrane is
87%, significant difference.When being transplanted for internal defective tissue, material, which can adhere to normal cell faster and be advantageous to tissue, to be repaiied
It is multiple.
Embodiment three：
1st, will carry (- RGD-)4Prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, apply
The Luria-Bertani solid mediums containing ampicillin are overlying on, are inverted into 37 DEG C of biochemical cultivation case culture 14~16
Hour.The single bacterium colony of picking is put into the 4mL fresh Luria-Bertani fluid nutrient mediums containing ampicillin, and insertion is shaken
Swing speed 200r/min 37 DEG C of air table shaken cultivation 8~10 hours.The bacterium solution of culture is pressed 1:100 ratio is new in 1L
Amplification cultivation in the fresh Luria-Bertani fluid nutrient mediums containing ampicillin.When bacterium solution density reaches OD600=0.3~2.1
When add 0~1.0mM isopropyl-β-D-thiogalactoside Fiber differentiation 1~8 hour, 4 DEG C are collected by centrifugation bacterium cell.
2nd, the bacterium cell of collection is resuspended with the combination buffer of glutathione transferase (GST) affinity chromatography and mixed, put
In ultrasonic disruption cell on ice, release protein.After the completion of broken 1200r/min, 4 DEG C of centrifugation 10min collect containing (-
RGD-)4The supernatant of polypeptide.Expression contains (- RGD-)4The albumen of peptide sequence is the fusion protein GST- containing GST labels
(-RGD-)4。
3rd, containing GST- (- RGD-)4Supernatant perfusion GST affinity columns, elute nonspecific proteins.Then by every
Milligram fusion protein adds 20 DEG C of 5~10u fibrin ferments and placed 16 hours.It is last slowly flow out and collect efflux obtain (-
RGD-)4Polypeptide solution.
4th, by (- RGD-)4Polypeptide solution addition desalting column purifies all salt components in solution, (- the RGD-) of collection4It is more
The peptide aqueous solution immediately with liquid nitrogen freezing, be subsequently placed in freeze drier and be dried to (- RGD-)4Polypeptide powder.
5th, by silkworm raw silk in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, a certain amount of degumming is clean
Bombyx mori silk fibroin is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), 72 DEG C of water-baths
In be stirred well to fibroin and fully dissolve, silk fibroin solution is encased in bag filter, dialysed 3 days in 4 DEG C of deionized waters, every 2 is small
Deionized water of Shi Genghuan, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, steamed
Hair removes certain moisture, obtains the silk fibroin water solution that concentration is 4~10%.
6th, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water.In ice
Under the conditions of bath, appropriate MES is added into every hole and is handled 1 hour, then adds excessive EDC and NHS mixed liquor, ice bath bar
Part is handled 1 hour.Xiang Kongzhong adds 0.005 μM of (- RGD-)4Polypeptide 1mL, reaction overnight at 4 DEG C, is finally rushed with deionized water
Wash clean, unreacted polypeptide and crosslinking agent are removed, cell culture is used for after irradiated sterilizing.
7th, 2.5~10 × 10 are inoculated with into hole4Individual L929 cells, cell proliferation experiment result：After culture 3 days, grafting (-
RGD-)45.7 times of cell quantity when cell quantity is is inoculated with the fibroin membrane of polypeptide, than non-grafted (- RGD-)4The fibroin of polypeptide
Cell quantity increase about 19% on film.
Example IV：
1st, will carry (- RGD-)4Prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, apply
The Luria-Bertani solid mediums containing ampicillin are overlying on, are inverted into 37 DEG C of biochemical cultivation case culture 14~16
Hour.The single bacterium colony of picking is put into the 4mL fresh Luria-Bertani fluid nutrient mediums containing ampicillin, and insertion is shaken
Swing speed 200r/min 37 DEG C of air table shaken cultivation 8~10 hours.The bacterium solution of culture is pressed 1:100 ratio is new in 1L
Amplification cultivation in the fresh Luria-Bertani fluid nutrient mediums containing ampicillin.When bacterium solution density reaches OD600=0.3~2.1
When add 0~1.0mM isopropyl-β-D-thiogalactoside Fiber differentiation 1~8 hour, 4 DEG C are collected by centrifugation bacterium cell.
2nd, the bacterium cell of collection is resuspended with the combination buffer of glutathione transferase (GST) affinity chromatography and mixed, put
In ultrasonic disruption cell on ice, protein is discharged.After the completion of broken 12000r/min, 4 DEG C of centrifugation 10min collect containing (-
RGD-)4The supernatant of polypeptide.Expression contains (- RGD-)4The albumen of peptide sequence is the fusion protein GST- containing GST labels
(-RGD-)4。
3rd, containing GST- (- RGD-)4Supernatant perfusion GST affinity columns, elute nonspecific proteins.Then by every
Milligram fusion protein adds 20 DEG C of 5~10u fibrin ferments and placed 16 hours.It is last slowly flow out and collect efflux obtain (-
RGD-)4Polypeptide solution.
4th, by (- RGD-)4Polypeptide solution addition desalting column purifies all salt components in solution, (- the RGD-) of collection4It is more
The peptide aqueous solution immediately with liquid nitrogen freezing, be subsequently placed in freeze drier and be dried to (- RGD-)4Polypeptide powder.
5th, by silkworm raw silk in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, a certain amount of degumming is clean
Bombyx mori silk fibroin is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), 72 DEG C of water-baths
In be stirred well to fibroin and fully dissolve, silk fibroin solution is encased in bag filter, dialysed 3 days in 4 DEG C of deionized waters, every 2 is small
Deionized water of Shi Genghuan, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, steamed
Hair removes certain moisture, obtains the silk fibroin water solution that concentration is 4~10%.
6th, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water.In ice
Under the conditions of bath, appropriate MES is added into every hole and is handled 1 hour, then adds excessive EDC and NHS mixed liquor, ice bath bar
Part is handled 1 hour.Xiang Kongzhong adds 0.015 μM of (- RGD-)4Polypeptide 1mL, reaction overnight at 4 DEG C, is finally rushed with deionized water
Wash clean, unreacted polypeptide and crosslinking agent are removed, cell culture is used for after irradiated sterilizing.
7th, 2.5~10 × 10 are inoculated with into hole4Individual L929 cells, cell proliferation experiment result：After culture 3 days, grafting (-
RGD-)47.1 times of cell quantity when cell quantity is is inoculated with the fibroin membrane of polypeptide, than non-grafted (- RGD-)4The fibroin of polypeptide
Cell quantity increase by 48% or so on film.
Embodiment five：
1st, will carry (- RGD-)8Prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, apply
The Luria-Bertani solid mediums containing ampicillin are overlying on, are inverted into 37 DEG C of biochemical cultivation case culture 14~16
Hour.The single bacterium colony of picking is put into the 4mL fresh Luria-Bertani fluid nutrient mediums containing ampicillin, and insertion is shaken
Swing speed 200r/min 37 DEG C of air table shaken cultivation 8~10 hours.The bacterium solution of culture is pressed 1:100 ratio is new in 1L
Amplification cultivation in the fresh Luria-Bertani fluid nutrient mediums containing ampicillin.When bacterium solution density reaches OD600=0.3~2.1
When add 0~1.0mM isopropyl-β-D-thiogalactoside Fiber differentiation 1~8 hour, 4 DEG C are collected by centrifugation bacterium cell.
2nd, the bacterium cell of collection is resuspended with the combination buffer of glutathione transferase (GST) affinity chromatography and mixed, put
In ultrasonic disruption cell on ice, protein is discharged.After the completion of broken 12000r/min, 4 DEG C of centrifugation 10min collect containing (-
RGD-)8The supernatant of polypeptide.Expression contains (- RGD-)8The albumen of peptide sequence is the fusion protein GST- containing GST labels
(-RGD-)8。
3rd, containing GST- (- RGD-)8Supernatant perfusion GST affinity columns, elute nonspecific proteins.Then by every
Milligram fusion protein adds 20 DEG C of 5~10u fibrin ferments and placed 16 hours.It is last slowly flow out and collect efflux obtain (-
RGD-)8Polypeptide solution.
4th, by (- RGD-)8Polypeptide solution addition desalting column purifies all salt components in solution, (- the RGD-) of collection8It is more
The peptide aqueous solution immediately with liquid nitrogen freezing, be subsequently placed in freeze drier and be dried to (- RGD-)8Polypeptide powder.
5th, by silkworm raw silk in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, a certain amount of degumming is clean
Bombyx mori silk fibroin is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), 72 DEG C of water-baths
In be stirred well to fibroin and fully dissolve, silk fibroin solution is encased in bag filter, dialysed 3 days in 4 DEG C of deionized waters, every 2 is small
Deionized water of Shi Genghuan, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, steamed
Hair removes certain moisture, obtains the silk fibroin water solution that concentration is 4~10%.
6th, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water.In ice
Under the conditions of bath, appropriate MES is added into every hole and is handled 1 hour, then adds excessive EDC and NHS mixed liquor, ice bath bar
Part is handled 1 hour.Xiang Kongzhong adds 0.015 μM of (- RGD-)8Polypeptide 1mL, reaction overnight at 4 DEG C, is finally rushed with deionized water
Wash clean, unreacted polypeptide and crosslinking agent are removed, cell culture is used for after irradiated sterilizing.
7th, 2.5~10 × 10 are inoculated with into hole4Individual L929 cells, cell proliferation experiment result：After culture 3 days, grafting (-
RGD-)87.9 times of cell quantity when cell quantity is is inoculated with the fibroin membrane of polypeptide, than non-grafted (- RGD-)8Polypeptide fibroin membrane
Upper cell quantity increase by 65%.
Embodiment six：
1st, by silkworm raw silk in the Na that concentration is 0.06%2CO3Degumming is boiled in solution, a certain amount of degumming is clean
Bombyx mori silk fibroin is with 1:10 bath raio is put into ternary solution calcium chloride ethanol water (mol ratio 1:2:8), 72 DEG C of water-baths
In be stirred well to fibroin and fully dissolve, silk fibroin solution is encased in bag filter, dialysed 3 days in 4 DEG C of deionized waters, every 2 is small
Deionized water of Shi Genghuan, you can obtain the silk fibroin protein aqueous solution.Silk fibroin solution after dialysis is placed under fan, steamed
Hair removes certain moisture, obtains the silk fibroin water solution that concentration is 4~10%.
2nd, silk fibroin water solution is added in 24 orifice plates, per the μ l of hole 300~1000, is put under ventilating kitchen and air-dries, system
Into one layer of fibroin membrane for being layered on culture plate bottom, then rinsed well with after 70% Ethanol Treatment 30min with deionized water, through spoke
According to being used for cell culture after sterilizing.
3rd, 2 × 10 are inoculated with into hole5Individual L929 cells, the cell adherence rate that adhesion experiment result obtains 2 hours are 87%.To
2.5~10 × 10 are inoculated with hole4Individual L929 cells, after cell culture three days, on fibroin membrane cell quantity for inoculation when cell number
4.8 times of amount.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (1)
1. the preparation method of a kind of function fibroin membrane beneficial to cell adherence, it is characterised in that comprise the following steps：
Step 1) wherein contains RGD tripeptides sequences according to the analysis to tussah silk peptide or giant silkworm fibroin amino acid sequence, design one
The unit peptide fragment GSGAGGRGDGGYGDGSS of row, is named as (- RGD-)1Polypeptide gene；
Step 2) is using technique for gene engineering to (- the RGD-)1Polypeptide gene carry out 4 times clone, and design construction carry (-
RGD-)4The prokaryotic system expression vector of polypeptide gene, (- the RGD-)4The amino acid sequence of polypeptide is SEQ.ID.NO.1；
Or, using technique for gene engineering to (- the RGD-)1Polypeptide gene carry out 8 times clone, and design construction carry (-
RGD-)8The prokaryotic system expression vector of polypeptide gene, (- the RGD-)8The amino acid sequence of polypeptide is SEQ.ID.NO.2；
Step 3) is carried design construction is good (- RGD-)4Polypeptide gene or (- RGD-)8The prokaryotic system of polypeptide gene
Expression vector transfection Escherichia coli cell, induced and trained through isopropyl-β-D-thiogalactoside with Luria-Bertani culture mediums
Support a few hours；
Step 4) centrifuge cell bacterium solution, collects bacterium cell and broken bacterium cell, and the albumen supernatant after then crushing is added to
One-step method purifying, digestion, collection, lyophilized, final acquisition (- RGD-) in the affinity chromatography of glutathione transferase4Polypeptide powder
Or (- RGD-)8Polypeptide powder, the certain density aqueous solution is configured during use；
After silkworm silk or silk cocoon are carried out degumming, dissolving by step 5), it is poured into bag filter, is dialysed with deionized water, then
The silk fibroin protein solution of filtering and concentrating to 3~8%, it is cast in the vessel of certain area and air-dries, obtain bombyx mori silk fibroin film；
Step 6) in the cross-linking agent solution of excess, adds obtained above-mentioned bombyx mori silk fibroin film immersion (- RGD-)4Polypeptide or
(-RGD-)8Polypeptide, 4 DEG C of reaction overnights, produces function fibroin membrane.
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| CN101011596A (en) * | 2007-02-08 | 2007-08-08 | 浙江理工大学 | Process for preparing antibiotic-peptide modified-fibroin film material |
| CN104099372A (en) * | 2014-07-23 | 2014-10-15 | 苏州大学 | Cationic silk fibroin/gene compound, and preparation method and application thereof |
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| CN101011596A (en) * | 2007-02-08 | 2007-08-08 | 浙江理工大学 | Process for preparing antibiotic-peptide modified-fibroin film material |
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