CN105295080A - Method for preparing functional silk fibroin protein membrane beneficial to cell adhesion - Google Patents
Method for preparing functional silk fibroin protein membrane beneficial to cell adhesion Download PDFInfo
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- CN105295080A CN105295080A CN201510705912.3A CN201510705912A CN105295080A CN 105295080 A CN105295080 A CN 105295080A CN 201510705912 A CN201510705912 A CN 201510705912A CN 105295080 A CN105295080 A CN 105295080A
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Abstract
The invention discloses a method for preparing a functional silk fibroin protein membrane beneficial to cell adhesion. The method includes the steps that a pronucleus system expression vector carried with (-RGD-)4 polypeptide or (-RGD-)8 polypeptide genes conducts transfection on escherichia coli cells, and induction culture is carried out for several hours; cell bacterial liquid is centrifuged, bacterial cells are collected and crushed, crushed protein supernate is subjected to purification, enzyme digestion one-step method collection and freeze-drying, and (-RGD-)4 polypeptide powder or (-RGD-)8 polypeptide powder is obtained; silkworm silk or silkworm cocoons are poured into a container of certain area to be air-dried after being subjected to degumming, dissolving, dialysis, filtration and concentration, and accordingly a silkworm silk fibroin protein membrane is obtained; the silkworm silk fibroin protein membrane is dipped in an excessive cross-linking agent solution, then (-RGD-)4 polypeptide or (-RGD-)8 polypeptide is added, an overnight reaction is carried out at the temperature of 4 DEG C, and accordingly the functional silk fibroin protein membrane is obtained. The functional silk fibroin protein membrane prepared through the method has excellent cell adhesion performance, belongs to biomedical materials, does not have cytotoxicity, is provided with cell adhesion protein recognition sites, and is beneficial to cell adhesion and growth and capable of promoting defect tissue healing.
Description
Technical field
The present invention relates to a kind of preparation method being applied to the silk fibroin material of biomedical sector, be specifically related to a kind of preparation method being beneficial to the function fibroin membrane of cell adhesion.
Background technology
RGD sequence is made up of arginine, glycine and aspartic acid, extensively be present in various kinds of cell epimatrix, collagen protein, fibronectin, ln, vitronectin, elastin etc. extracellular matrix, can with multiple integrin specific binding, contribute to cell adhesion, impel attached cell to adhere to growth.Through the fibroin membrane of RGD chemically modified and other material modified as high molecular polymer etc. can improve the adhesion of cell on material, diffusion and propagation (as JBiomedMaterResA, 2003,67 (2), 559-570; Biomaterials, 2002,23:4315-4323 etc.).RGD peptide can also competitive inhibition as the various attachment proteins of scleroproein etc. and hematoblastic combination, and the adhesion of thrombocyte on material (LettersinPeptideScience, 2002,9:101-109), thus the formation stoping thrombus.
The RGD peptide of existing sale is all the peptide coming from chemosynthesis, comprises chain type peptide and cyclic peptide.Along with the fast development of genetic engineering technique, the recombinant expressed a kind of important technology having become functional peptides and prepared.A kind of silk-protein containing RGD of restructuring has good cell adhesion and multiplication capacity (chemical journal, 2006,64:1273-1278) to have research to point out.Report is had to claim osteocyte at the recombinant expressed culture plate (BiotechnolLett, 2007,29:359-363) being better than Methionin bag quilt containing the adhesivity on the oligopeptides of RGD and differentiation capability.And for example research points out that restructuring RGD spider silk fibroin can support the differentiation (Biomaterials, 2008,29:2556-2563) of undifferentiated mouse bone-forming cell; RGD recombinant spider silk and high-molecular polyvinyl alcohol material are combined, growth of rat embryo fibroblast cell (Chinese Reconstructive surgery magazine, 2009,23:747-750) etc. can be supported.
Silk is a kind of good natural biologic material, domestic and international research main based on bombyx mori silk fibroin, and report seldom (JournalofWuhanUniversityofTechnology:MaterialsScinece about the research that wild silk yarn is used for biomaterial as tussah silk or wild silk yarn, 2011,26 (6): 1044-1048; BiomedMater, 2006,1:181-187), due to can not be domestic, output be very low.But it should be noted that, a large amount of unit repeated fragments containing RGD tripeptides is distributed with in the aminoacid sequence of tussah silk peptide or giant silkworm fibroin, this coming from also does not study report containing the unit sequence of RGD and the function of tumor-necrosis factor glycoproteins in natural wild silkworm fibroin, does not more apply.
Summary of the invention
Based on natural tussore silk fibroin (tussah silk peptide or giant silkworm fibroin) containing RGD functional polypeptide fragment but the extremely low present situation of silk yield, the present invention aims to provide a kind of functional polypeptide (-RGD-) coming from organism synthesis
4(-RGD-)
8and the preparation method of the bombyx mori silk fibroin film of modification, wherein the aminoacid sequence of-RGD-is a section of coming from natural tussore silk fibroin, the function fibroin membrane prepared belongs to bio-medical material, there is no cytotoxicity, there is the recognition site of cell adhesion protein, be conducive to cell adhesion, growth, the healing of promotion defective tissue.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
Be beneficial to a preparation method for the function fibroin membrane of cell adhesion, comprise the steps:
Step 1) according to the analysis to tussah silk peptide or giant silkworm fibroin aminoacid sequence, design a unit peptide section GSGAGGRGDGGYGDGSS wherein containing RGD tripeptide sequence, called after (-RGD-)
1polypeptide gene;
Step 2) adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 4 times of clones, and design construction carries (-RGD-)
4the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
4the aminoacid sequence of polypeptide is as SEQ.ID.NO.1;
Or, adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 8 times of clones, and design construction carries (-RGD-)
8the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
8the aminoacid sequence of polypeptide is as SEQ.ID.NO.2;
Step 3) by good the carrying (-RGD-) of design construction
4polypeptide or (-RGD-)
8prokaryotic system expression vector (the Bio-MedicalMaterialsandEngineering of polypeptide gene, 2014,24:2057-2064) transfection Escherichia coli (BL21) cell, with Luria-Bertani substratum through isopropyl-β-D-thiogalactoside(IPTG) inducing culture a few hours;
Step 4) eccentric cell bacterium liquid, collect mycetocyte broken mycetocyte, then the albumen supernatant liquor after fragmentation joined single stage method purifying in the affinity chromatography of Thiadiazolidine isomerase, enzyme is cut, collect, freeze-drying, finally obtain (-RGD-)
4polypeptide powder or (-RGD-)
8polypeptide powder, configures the certain density aqueous solution during use;
Step 5) silkworm silk or silk cocoon carried out come unstuck, dissolve after, be poured in dialysis tubing, with deionized water dialysis, then the silk fibroin protein solution of filtering and concentrating to 3 ~ 8%, is cast in the vessel of certain area air-dry, obtains bombyx mori silk fibroin film;
Step 6) by obtained above-mentioned bombyx mori silk fibroin film immersion in excessive cross-linking agent solution, then to add (-RGD-)
4polypeptide or (-RGD-)
8polypeptide, 4 DEG C of reaction overnight, obtain function fibroin membrane.
Compared with prior art, the present invention has following beneficial effect:
Based on natural tussore silk fibroin (tussah silk peptide or giant silkworm fibroin) containing RGD functional polypeptide fragment but the extremely low present situation of silk yield, the present invention aims to provide a kind of preparation method being beneficial to the function fibroin membrane of cell adhesion.By the function fibroin membrane prepared of the method, there is excellent cell adhesion performance, belong to bio-medical material, there is no cytotoxicity, there is the recognition site of cell adhesion protein, be conducive to cell adhesion, growth, the healing of promotion defective tissue.Its reason is because polypeptide provided by the invention comes from a section (-RGD-) containing the functional sequence of RGD in natural wild silkworm fibroin
1polypeptide and tumor-necrosis factor glycoproteins thereof, not only peptide chain side base contains a large amount of hydrophilic radicals, is conducive to Growth of Cells and organization healing; And the peptide chain of every a part contains the recognition site RGD of multiple cell adhesion protein, the polypeptide of same grafting a part, (-RGD-)
4polypeptide and (-RGD-)
8polypeptide can have at most 4 and 8 RGD sites respectively, can promote the adhesion of cell better and sprawl.Meanwhile, polypeptide provided by the invention comes from organism expressing, nontoxic nonirritant, and molecular weight is single, can with silk fibroin protein covalent attachment, stably give bombyx mori silk fibroin film better cell adhesion function.In addition, in the present invention, the acquisition (purifying, enzyme are cut) of RGD containing peptides is that a step completes in GST affinity column, decreases the destruction to expression product.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, be described in detail as follows below with preferred embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail.
Be beneficial to a preparation method for the function fibroin membrane of cell adhesion, can be implemented by following embodiment.Before this function fibroin membrane of preparation, first according to the analysis to tussah silk peptide or giant silkworm fibroin aminoacid sequence, design a unit peptide section GSGAGGRGDGGYGDGSS wherein containing RGD tripeptide sequence, called after (-RGD-)
1polypeptide.Then adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 4 times of clones, and design construction carries (-RGD-)
4the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
4the aminoacid sequence of polypeptide is as SEQ.ID.NO.1; Or adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 8 times of clones, and design construction carries (-RGD-)
8the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
8the aminoacid sequence of polypeptide is as SEQ.ID.NO.2.
Embodiment one:
1, will carry (-RGD-)
4prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, is coated on the Luria-Bertani solid medium containing penbritin, and the biochemical cultivation case being inverted into 37 DEG C is cultivated 14 ~ 16 hours.The single bacterium colony of picking puts into the fresh Luria-Bertani liquid nutrient medium containing penbritin of 4mL, air table 37 DEG C of shaking culture of insertion hunting speed 200r/min 8 ~ 10 hours.The bacterium liquid cultivated is contained amplification cultivation in the Luria-Bertani liquid nutrient medium of penbritin in the ratio of 1:100 in 1L is fresh.When the liquid-tight degree of bacterium reaches OD
600the isopropyl-β-D-thiogalactoside(IPTG) inducing culture 1 ~ 8 hour of 0 ~ 1.0mM is added, 4 DEG C of collected by centrifugation mycetocytes when=0.3 ~ 2.1.
2, by the resuspended mixing of binding buffer liquid of mycetocyte Thiadiazolidine isomerase (GST) affinity chromatography of collection, ultrasonic disruption cell, release protein is on ice placed in.After fragmentation completes, 12000r/min, 4 DEG C of centrifugal 10min collect containing (-RGD-)
4the supernatant liquor of polypeptide.That expresses contains (-RGD-)
4the albumen of peptide sequence is the fusion rotein GST-(-RGD-) containing GST label
4.
3, containing GST-(-RGD-)
4supernatant liquor perfusion GST affinity column, wash-out nonspecific proteins.Then add 5 ~ 10u zymoplasm 20 DEG C by every milligram of fusion rotein to place 16 hours.Finally slowly flow out and collect effluent liquid and namely obtain (-RGD-)
4polypeptide solution.
4, by (-RGD-)
4polypeptide solution adds desalting column and purifies all salt components in solution, (-the RGD-) of collection
4polypeptid solution immediately with liquid nitrogen freezing, be then placed in freeze drier and be dried to (-RGD-)
4polypeptide powder.
5, silkworm raw silk is the Na of 0.06% in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
6, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, clean with deionized water rinsing after then using 70% Ethanol Treatment 30min.Under condition of ice bath, in every hole, add appropriate MES process 1 hour, then add the mixed solution of excessive EDC and NHS, condition of ice bath process 1 hour.Xiang Kongzhong adds (-the RGD-) of 0.01 μM
4polypeptide 1mL, reaction overnight at 4 DEG C, finally clean with deionized water rinsing, remove unreacted polypeptide and linking agent, for cell cultures after irradiation sterilization.
7, Xiang Kongzhong inoculation 2 × 10
5individual L929 cell, adhesion experiment result: grafting (-RGD-)
4the cell adhesion rate of fibroin membrane upper 2 hour of polypeptide is more than 90%, and does not use (-RGD-)
4the cell adhesion rate of peptide modified fibroin membrane is 87%, there is significant difference.When transplanting for defective tissue in body, material can adhere to normal cell faster and be conducive to tissue repair.
Embodiment two:
1, will carry (-RGD-)
8prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, is coated on the Luria-Bertani solid medium containing penbritin, and the biochemical cultivation case being inverted into 37 DEG C is cultivated 14 ~ 16 hours.The single bacterium colony of picking puts into the fresh Luria-Bertani liquid nutrient medium containing penbritin of 4mL, air table 37 DEG C of shaking culture of insertion hunting speed 200r/min 8 ~ 10 hours.The bacterium liquid cultivated is contained amplification cultivation in the Luria-Bertani liquid nutrient medium of penbritin in the ratio of 1:100 in 1L is fresh.When the liquid-tight degree of bacterium reaches OD
600the isopropyl-β-D-thiogalactoside(IPTG) inducing culture 1 ~ 8 hour of 0 ~ 1.0mM is added, 4 DEG C of collected by centrifugation mycetocytes when=0.3 ~ 2.1.
2, by the resuspended mixing of binding buffer liquid of mycetocyte Thiadiazolidine isomerase (GST) affinity chromatography of collection, ultrasonic disruption cell, release protein is on ice placed in.After fragmentation completes, 12000r/min, 4 DEG C of centrifugal 10min collect containing (-RGD-)
8the supernatant liquor of polypeptide.That expresses contains (-RGD-)
8the albumen of peptide sequence is the fusion rotein GST-(-RGD-) containing GST label
8.
3, containing GST-(-RGD-)
8supernatant liquor perfusion GST affinity column, wash-out nonspecific proteins.Then add 5 ~ 10u zymoplasm 20 DEG C by every milligram of fusion rotein to place 16 hours.Finally slowly flow out and collect effluent liquid and namely obtain (-RGD-)
8polypeptide solution.
4, by (-RGD-)
8polypeptide solution adds desalting column and purifies all salt components in solution, (-the RGD-) of collection
8polypeptid solution immediately with liquid nitrogen freezing, be then placed in freeze drier and be dried to (-RGD-)
8polypeptide powder.
5, be the Na of 0.06% by silkworm raw silk in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
6, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, clean with deionized water rinsing after then using 70% Ethanol Treatment 30min.Under condition of ice bath, in every hole, add appropriate MES process 1 hour, then add the mixed solution of excessive EDC and NHS, condition of ice bath process 1 hour.Xiang Kongzhong adds (-the RGD-) of 0.01 μM
8polypeptide 1mL, reaction overnight at 4 DEG C, finally clean with deionized water rinsing, remove unreacted polypeptide and linking agent, for cell cultures after irradiation sterilization.
7, Xiang Kongzhong inoculation 2 × 10
5individual L929 cell, adhesion experiment result: grafting (-RGD-)
8the cell adhesion rate of fibroin membrane upper 2 hour of polypeptide is more than 92.5%, and does not use (-RGD-)
8the cell adhesion rate of peptide modified fibroin membrane is 87%, significant difference.When transplanting for defective tissue in body, material can adhere to normal cell faster and be conducive to tissue repair.
Embodiment three:
1, will carry (-RGD-)
4prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, is coated on the Luria-Bertani solid medium containing penbritin, and the biochemical cultivation case being inverted into 37 DEG C is cultivated 14 ~ 16 hours.The single bacterium colony of picking puts into the fresh Luria-Bertani liquid nutrient medium containing penbritin of 4mL, air table 37 DEG C of shaking culture of insertion hunting speed 200r/min 8 ~ 10 hours.The bacterium liquid cultivated is contained amplification cultivation in the Luria-Bertani liquid nutrient medium of penbritin in the ratio of 1:100 in 1L is fresh.When the liquid-tight degree of bacterium reaches OD
600the isopropyl-β-D-thiogalactoside(IPTG) inducing culture 1 ~ 8 hour of 0 ~ 1.0mM is added, 4 DEG C of collected by centrifugation mycetocytes when=0.3 ~ 2.1.
2, by the resuspended mixing of binding buffer liquid of mycetocyte Thiadiazolidine isomerase (GST) affinity chromatography of collection, ultrasonic disruption cell, release protein is on ice placed in.After fragmentation completes, 1200r/min, 4 DEG C of centrifugal 10min collect containing (-RGD-)
4the supernatant liquor of polypeptide.That expresses contains (-RGD-)
4the albumen of peptide sequence is the fusion rotein GST-(-RGD-) containing GST label
4.
3, containing GST-(-RGD-)
4supernatant liquor perfusion GST affinity column, wash-out nonspecific proteins.Then add 5 ~ 10u zymoplasm 20 DEG C by every milligram of fusion rotein to place 16 hours.Finally slowly flow out and collect effluent liquid and namely obtain (-RGD-)
4polypeptide solution.
4, by (-RGD-)
4polypeptide solution adds desalting column and purifies all salt components in solution, (-the RGD-) of collection
4polypeptid solution immediately with liquid nitrogen freezing, be then placed in freeze drier and be dried to (-RGD-)
4polypeptide powder.
5, be the Na of 0.06% by silkworm raw silk in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
6, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, clean with deionized water rinsing after then using 70% Ethanol Treatment 30min.Under condition of ice bath, in every hole, add appropriate MES process 1 hour, then add the mixed solution of excessive EDC and NHS, condition of ice bath process 1 hour.Xiang Kongzhong adds (-the RGD-) of 0.005 μM
4polypeptide 1mL, reaction overnight at 4 DEG C, finally clean with deionized water rinsing, remove unreacted polypeptide and linking agent, for cell cultures after irradiation sterilization.
7, Xiang Kongzhong inoculation 2.5 ~ 10 × 10
4individual L929 cell, cell proliferation experiment result: cultivate after 3 days, grafting (-RGD-)
4when on the fibroin membrane of polypeptide, cell quantity is inoculation 5.7 times of cell quantity, than non-grafting (-RGD-)
4cell quantity increase about 19% on the fibroin membrane of polypeptide.
Embodiment four:
1, will carry (-RGD-)
4prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, is coated on the Luria-Bertani solid medium containing penbritin, and the biochemical cultivation case being inverted into 37 DEG C is cultivated 14 ~ 16 hours.The single bacterium colony of picking puts into the fresh Luria-Bertani liquid nutrient medium containing penbritin of 4mL, air table 37 DEG C of shaking culture of insertion hunting speed 200r/min 8 ~ 10 hours.The bacterium liquid cultivated is contained amplification cultivation in the Luria-Bertani liquid nutrient medium of penbritin in the ratio of 1:100 in 1L is fresh.When the liquid-tight degree of bacterium reaches OD
600the isopropyl-β-D-thiogalactoside(IPTG) inducing culture 1 ~ 8 hour of 0 ~ 1.0mM is added, 4 DEG C of collected by centrifugation mycetocytes when=0.3 ~ 2.1.
2, by the resuspended mixing of binding buffer liquid of mycetocyte Thiadiazolidine isomerase (GST) affinity chromatography of collection, ultrasonic disruption cell is on ice placed in, release protein.After fragmentation completes, 12000r/min, 4 DEG C of centrifugal 10min collect containing (-RGD-)
4the supernatant liquor of polypeptide.That expresses contains (-RGD-)
4the albumen of peptide sequence is the fusion rotein GST-(-RGD-) containing GST label
4.
3, containing GST-(-RGD-)
4supernatant liquor perfusion GST affinity column, wash-out nonspecific proteins.Then add 5 ~ 10u zymoplasm 20 DEG C by every milligram of fusion rotein to place 16 hours.Finally slowly flow out and collect effluent liquid and namely obtain (-RGD-)
4polypeptide solution.
4, by (-RGD-)
4polypeptide solution adds desalting column and purifies all salt components in solution, (-the RGD-) of collection
4polypeptid solution immediately with liquid nitrogen freezing, be then placed in freeze drier and be dried to (-RGD-)
4polypeptide powder.
5, be the Na of 0.06% by silkworm raw silk in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
6, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, clean with deionized water rinsing after then using 70% Ethanol Treatment 30min.Under condition of ice bath, in every hole, add appropriate MES process 1 hour, then add the mixed solution of excessive EDC and NHS, condition of ice bath process 1 hour.Xiang Kongzhong adds (-the RGD-) of 0.015 μM
4polypeptide 1mL, reaction overnight at 4 DEG C, finally clean with deionized water rinsing, remove unreacted polypeptide and linking agent, for cell cultures after irradiation sterilization.
7, Xiang Kongzhong inoculation 2.5 ~ 10 × 10
4individual L929 cell, cell proliferation experiment result: cultivate after 3 days, grafting (-RGD-)
4when on the fibroin membrane of polypeptide, cell quantity is inoculation 7.1 times of cell quantity, than non-grafting (-RGD-)
4on the fibroin membrane of polypeptide, cell quantity increases about 48%.
Embodiment five:
1, will carry (-RGD-)
8prokaryotic system expression vector transfection Escherichia coli (BL21) cell of polypeptide gene, is coated on the Luria-Bertani solid medium containing penbritin, and the biochemical cultivation case being inverted into 37 DEG C is cultivated 14 ~ 16 hours.The single bacterium colony of picking puts into the fresh Luria-Bertani liquid nutrient medium containing penbritin of 4mL, air table 37 DEG C of shaking culture of insertion hunting speed 200r/min 8 ~ 10 hours.The bacterium liquid cultivated is contained amplification cultivation in the Luria-Bertani liquid nutrient medium of penbritin in the ratio of 1:100 in 1L is fresh.When the liquid-tight degree of bacterium reaches OD
600the isopropyl-β-D-thiogalactoside(IPTG) inducing culture 1 ~ 8 hour of 0 ~ 1.0mM is added, 4 DEG C of collected by centrifugation mycetocytes when=0.3 ~ 2.1.
2, by the resuspended mixing of binding buffer liquid of mycetocyte Thiadiazolidine isomerase (GST) affinity chromatography of collection, ultrasonic disruption cell is on ice placed in, release protein.After fragmentation completes, 12000r/min, 4 DEG C of centrifugal 10min collect containing (-RGD-)
8the supernatant liquor of polypeptide.That expresses contains (-RGD-)
8the albumen of peptide sequence is the fusion rotein GST-(-RGD-) containing GST label
8.
3, containing GST-(-RGD-)
8supernatant liquor perfusion GST affinity column, wash-out nonspecific proteins.Then add 5 ~ 10u zymoplasm 20 DEG C by every milligram of fusion rotein to place 16 hours.Finally slowly flow out and collect effluent liquid and namely obtain (-RGD-)
8polypeptide solution.
4, by (-RGD-)
8polypeptide solution adds desalting column and purifies all salt components in solution, (-the RGD-) of collection
8polypeptid solution immediately with liquid nitrogen freezing, be then placed in freeze drier and be dried to (-RGD-)
8polypeptide powder.
5, be the Na of 0.06% by silkworm raw silk in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
6, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, clean with deionized water rinsing after then using 70% Ethanol Treatment 30min.Under condition of ice bath, in every hole, add appropriate MES process 1 hour, then add the mixed solution of excessive EDC and NHS, condition of ice bath process 1 hour.Xiang Kongzhong adds (-the RGD-) of 0.015 μM
8polypeptide 1mL, reaction overnight at 4 DEG C, finally clean with deionized water rinsing, remove unreacted polypeptide and linking agent, for cell cultures after irradiation sterilization.
7, Xiang Kongzhong inoculation 2.5 ~ 10 × 10
4individual L929 cell, cell proliferation experiment result: cultivate after 3 days, grafting (-RGD-)
8when on the fibroin membrane of polypeptide, cell quantity is inoculation 7.9 times of cell quantity, than non-grafting (-RGD-)
8on polypeptide fibroin membrane, cell quantity increases by 65%.
Embodiment six:
1, be the Na of 0.06% by silkworm raw silk in concentration
2cO
3boil in solution and come unstuck, a certain amount of clean bombyx mori silk fibroin that comes unstuck is put into ternary solution calcium chloride ethanol water (mol ratio is for 1:2:8) with the bath raio of 1:10, be stirred well to fibroin in 72 DEG C of water-baths fully to dissolve, silk fibroin solution is encased in dialysis tubing, dialyse 3 days in 4 DEG C of deionized waters, within every 2 hours, change a deionized water, the silk fibroin protein aqueous solution can be obtained.Under silk fibroin solution after dialysis is placed in fan, the moisture that evaporative removal is certain, obtaining concentration is the silk fibroin water solution of 4 ~ 10%.
2, silk fibroin water solution is joined in 24 orifice plates, every hole 300 ~ 1000 μ l, air-dry under putting into ventilating kitchen, make one deck fibroin membrane be layered on bottom culture plate, then clean with deionized water rinsing after using 70% Ethanol Treatment 30min, for cell cultures after irradiation sterilization.
3, Xiang Kongzhong inoculation 2 × 10
5individual L929 cell, the cell adhesion rate that adhesion experiment result obtains 2 hours is 87%.Xiang Kongzhong inoculation 2.5 ~ 10 × 10
4individual L929 cell, cell cultures is after three days, 4.8 times of cell quantity when cell quantity is inoculation on fibroin membrane.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. be beneficial to a preparation method for the function fibroin membrane of cell adhesion, it is characterized in that, comprise the steps:
Step 1) according to the analysis to tussah silk peptide or giant silkworm fibroin aminoacid sequence, design a unit peptide section GSGAGGRGDGGYGDGSS wherein containing RGD tripeptide sequence, called after (-RGD-)
1polypeptide gene;
Step 2) adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 4 times of clones, and design construction carries (-RGD-)
4the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
4the aminoacid sequence of polypeptide is as SEQ.ID.NO.1;
Or, adopt genetic engineering technique to described (-RGD-)
1polypeptide gene carries out 8 times of clones, and design construction carries (-RGD-)
8the prokaryotic system expression vector of polypeptide gene, described (-RGD-)
8the aminoacid sequence of polypeptide is as SEQ.ID.NO.2;
Step 3) by good the carrying (-RGD-) of design construction
4polypeptide gene or (-RGD-)
8the prokaryotic system expression vector transfection Escherichia coli cell of polypeptide gene, with Luria-Bertani substratum through isopropyl-β-D-thiogalactoside(IPTG) inducing culture a few hours;
Step 4) eccentric cell bacterium liquid, collect mycetocyte broken mycetocyte, then the albumen supernatant liquor after fragmentation joined single stage method purifying in the affinity chromatography of Thiadiazolidine isomerase, enzyme is cut, collect, freeze-drying, finally obtain (-RGD-)
4polypeptide powder or (-RGD-)
8polypeptide powder, configures the certain density aqueous solution during use;
Step 5) silkworm silk or silk cocoon carried out come unstuck, dissolve after, be poured in dialysis tubing, with deionized water dialysis, then the silk fibroin protein solution of filtering and concentrating to 3 ~ 8%, is cast in the vessel of certain area air-dry, obtains bombyx mori silk fibroin film;
Step 6) by obtained above-mentioned bombyx mori silk fibroin film immersion in excessive cross-linking agent solution, then to add (-RGD-)
4polypeptide or (-RGD-)
8polypeptide, 4 DEG C of reaction overnight, obtain function fibroin membrane.
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| CN110003518A (en) * | 2019-04-16 | 2019-07-12 | 苏州大学 | A kind of activity silk fibroin porous material or active fibroin protein film and preparation method thereof |
| CN110041557A (en) * | 2019-04-16 | 2019-07-23 | 苏州大学 | A kind of function fibroin porous material or function fibroin membrane and preparation method thereof |
| CN110066418A (en) * | 2019-04-16 | 2019-07-30 | 苏州大学 | A kind of activity fibroin porous material or active fibroin membrane and preparation method thereof |
| CN111499728A (en) * | 2020-04-17 | 2020-08-07 | 安徽中盛溯源生物科技有限公司 | Method for rapidly preparing human glass fibronectin and application |
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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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| CN1807460A (en) * | 2006-02-13 | 2006-07-26 | 浙江理工大学 | Recombinant fusion protein of fibroin and RGD, and its biological synthesis method |
| 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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| CN110003518A (en) * | 2019-04-16 | 2019-07-12 | 苏州大学 | A kind of activity silk fibroin porous material or active fibroin protein film and preparation method thereof |
| CN110041557A (en) * | 2019-04-16 | 2019-07-23 | 苏州大学 | A kind of function fibroin porous material or function fibroin membrane and preparation method thereof |
| CN110066418A (en) * | 2019-04-16 | 2019-07-30 | 苏州大学 | A kind of activity fibroin porous material or active fibroin membrane and preparation method thereof |
| CN110066418B (en) * | 2019-04-16 | 2021-08-31 | 苏州大学 | Active silk fibroin porous material or active silk fibroin membrane and preparation method thereof |
| CN110003518B (en) * | 2019-04-16 | 2021-08-31 | 苏州大学 | Active silk fibroin porous material or active silk fibroin film and preparation method thereof |
| CN110041557B (en) * | 2019-04-16 | 2021-08-31 | 苏州大学 | Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof |
| CN111499728A (en) * | 2020-04-17 | 2020-08-07 | 安徽中盛溯源生物科技有限公司 | Method for rapidly preparing human glass fibronectin and application |
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