CN109717969A - A kind of gene recombination spider silk fibroin tooth inlay and preparation method thereof - Google Patents
A kind of gene recombination spider silk fibroin tooth inlay and preparation method thereof Download PDFInfo
- Publication number
- CN109717969A CN109717969A CN201711030980.XA CN201711030980A CN109717969A CN 109717969 A CN109717969 A CN 109717969A CN 201711030980 A CN201711030980 A CN 201711030980A CN 109717969 A CN109717969 A CN 109717969A
- Authority
- CN
- China
- Prior art keywords
- spider silk
- silk fibroin
- gene recombination
- solution
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The present invention provides a kind of gene recombination spider silk fibroin tooth inlay and preparation method thereof, and preparation, the freeze-drying of gene recombination spider silk fibroin solution including gene recombination spider silk fibroin solution, gene recombination spider silk fibroin be dissolved in hexafluoroisopropanol or water, the preparation of gene recombination spider silk fibroin bar, machining.Enhance particle by the parameters such as control gene recombination spider silk fibroin solution preparation parameter, dry solidification and doping solid phase, it can control the mechanical property of gene recombination spider silk fibroin tooth inlay, it obtains and skeleton mechanics parameter, the matched excellent performance tooth inlay of dental articulation, and the tooth inlay that the present invention obtains is compatible with human body, will not generate inflammatory problems.
Description
Technical field
The present invention relates to biological medicine field of material technology, embedding more particularly to a kind of gene recombination spider silk fibroin tooth
Body and preparation method thereof.
Background technique
Tooth inlay is inside a kind of insertion tooth body, to restore the form of defect of teeth and the dummy of function.According to
It is different to make inlay material, alloy inlay, resin inlay and porcelain inlay can be divided into.Alloy inlay is prepared by metal alloy,
There are good ductility and mechanical performance, is the production ideal repair materials of backteeth inlay；Resin inlay is added by compound resin
Work forms, and ingredient and polymerization methods are more brilliant than the resin routinely filled, and material wears away small, easily repairing to tooth, is a kind of good
Good aesthstic inlay material；Then there are mainly three types of processing methods for porcelain inlay: having the porcelain directly made on fire proofed wood die embedding
Body, the porcelain inlay for having CAD/CAM to be ground out have the castable ceramic inlay for making to cast out on model after wax pattern embedding, they have Zhuo
Performance attractive in appearance more.The tooth inlay material of these three current main-streams have the shortcomings that one it is common, that is, it is incompatible with human body,
It is easy to generate rejection with human body, generates inflammation.
Spider silk fibroin is a kind of special fibrin, it not only has very high intensity, elasticity, flexibility, elongation
Degree and tensile strength, and the advantages that be also equipped with slim and graceful, bio-compatible, biodegradable, but its yield is too low.Based on gene work
Journey technology can be obtained and gene recombination spider silk fibroin similar in natural spider silk property of protein by engineering bacterium expression system.
Itself has good mechanical performance and physicochemical property, and has fabulous biocompatibility with human body, degrades it in human body
After be amino acid and polypeptide, effect harmless to the human body, so gene recombination spider silk fibroin is widely used in biomedicine
Research field.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of gene recombination spider silk fibroins
Tooth inlay and preparation method thereof, it is incompatible with human body for solving prior art Tooth inlay, it is easy to generate row with human body
Reprimand reaction, leads to the problem of inflammation.
In order to achieve the above objects and other related objects, the present invention provides a kind of gene recombination spider silk fibroin tooth inlay
Preparation method, the preparation method includes at least:
1) engineering bacteria through everfermentation and expressing gene recombinant spider silk proteins is dissolved in certain density buffer solution
In, it is then crushed the engineering bacteria, and be centrifuged, collects the first supernatant liquor；
2) first supernatant liquor is kept the temperature, and continues to be centrifuged, collect the second supernatant liquor；
3) nickel layer is added in second supernatant liquor, is washed away using the first cleaning solution and is adsorbed on the nickel layer
The foreign protein on column is analysed, the second cleaning solution is recycled to elute the target gene recombinant spider silk being adsorbed in the nickel layer
Albumen, and collect target gene recombinant spider silk proteins solution；
4) the target gene recombinant spider silk proteins solution of collection is fitted into bag filter and is dialysed, Zhi Houli
The heart collects third supernatant liquor, to obtain required gene recombination spider silk fibroin solution；
5) freeze drying process is utilized, the gene recombination spider silk fibroin solution that step 4) obtains is changed into anhydrous
Gene recombination spider silk fibroin；
6) the anhydrous gene recombination spider silk fibroin is dissolved in hexafluoroisopropanol or water, obtains genetic recombination spider
The hexafluoroisopropanol solution or aqueous solution of spider's thread protein；
7) the hexafluoroisopropanol solution or aqueous solution of the gene recombination spider silk fibroin are injected in specific mold,
And successively
Gene recombination spider silk fibroin bar is made by methyl alcohol process and air-dried technique, then by the genetic recombination spider
Silk-fibroin bar is cut into required tooth inlay.
Preferably, the buffer solution in the step 1) is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution,
Wherein, trishydroxymethylaminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, the concentration of sodium chloride is 0.1~
1000mmol/L, the concentration of imidazoles are 0.1~1000mmol/L.
Preferably, when being crushed the engineering bacteria in the step 1), cracking pressure is 600~2000bar, the engineering bacteria
Quality and volume of buffer solution ratio are 10g:1mL~10g:1000m L；The revolving speed of centrifuge separation is 1r/min~40000r/
Min, time are 1s~10h, and temperature when centrifugation is -3 DEG C~30 DEG C.
Preferably, in the step 2), first supernatant liquor is kept the temperature using water bath with thermostatic control, holding temperature model
Enclosing is 20~100 DEG C, and soaking time range is 0.1~100h.
Preferably, in the step 2), the revolving speed of centrifuge separation is 1r/min~40000r/min, and the time is 1s~10h,
Temperature when centrifugation is -3 DEG C~30 DEG C.
Preferably, in the step 3), nickel layer analysis is added in second supernatant liquor that volume is 10~1000ml
Column.
Preferably, in the step 3), first cleaning solution is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazoles
Solution, wherein trishydroxymethylaminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, and the concentration of sodium chloride is
0.1~1000mmol/L, imidazole concentration are 0.1~1000mmol/L, and the volume of first cleaning solution is 10~1000ml.
Preferably, in the step 3), second cleaning solution is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazoles
Solution, wherein the concentration of sodium chloride is 0.1~1000mmol/L；Trishydroxymethylaminomethane-hydrochloric acid pH value is 8, and concentration is
0.1~100mmol/L；Imidazole concentration is 0.1~1000mmol/L, and the volume of second cleaning solution is 10~1000ml.
Preferably, in the step 4), dialysis procedure are as follows: be first placed in gradient dialysis one in certain density phosphate buffer
It is transferred in ultrapure water and dialyses after fixing time；Bag filter specification be 10~10000000Da, dialysis procedure be stand dialysis or
Magnetic agitation dialysis, magnetic stirring speed are 1r/min~2000r/min.
Preferably, when carrying out gradient dialysis, the concentration of the phosphate buffer is 10~1000mmol/L, and replacement phosphoric acid is slow
The time interval of fliud flushing is 0.1~100h, and the volume of changed phosphate buffer is 1mL~1000L every time.
Preferably, in the step 4), the revolving speed of centrifuge separation is 1r/min~40000r/min, and the time is 1s~10h,
Temperature when centrifugation is -3 DEG C~30 DEG C.
Preferably, in the step 5), freeze drying process are as follows: first by gene recombination spider silk fibroin aqueous solution 0
It is freezed in~-80 DEG C of environment, then vacuum refrigeration is handled 1~500 hour in freeze drier, freeze drier work ginseng
Number are as follows: 0.001~1mBar of vacuum pressure, 0~-80 DEG C of temperature.
Preferably, in the step 5), solid phase, which is added, in the anhydrous gene recombination spider silk fibroin enhances particle, Gu
Mutually enhancing granular materials is ceramic powders, metal powder or polymer powder, and the solid phase enhancing particle and moisture-free basis are because of weight
Group spider silk fibroin mass ratio is 1:100~100:1.
Preferably, in the step 6), anhydrous gene recombination spider silk fibroin and hexafluoroisopropanol or water quality ratio are
1:1~1:10, temperature range are 5 DEG C~50 DEG C.
Preferably, after the solid phase enhancing particle is added, in the step 6), the anhydrous gene recombination spider silk fibroin
It is 1:1~1:10 with the mixture and hexafluoroisopropanol of solid phase enhancing particle or the mass ratio of water, temperature range is 5 DEG C~50
℃。
Preferably, in the step 7), methyl alcohol process method is methanol solution processing, the methanol solution treatment process
Are as follows: the mold of hexafluoroisopropanol solution or aqueous solution containing gene recombination spider silk fibroin is immersed in liquid methanol,
And every the methanol solution of replacement in 1~50 hour, changing liquid number is 1~50 time, until the solution in the mold is changed into
Gene recombination spider silk fibroin solid；Then the gene recombination spider silk fibroin solid is taken out and is continued from the mold
It is impregnated using methanol solution, and every the methanol solution of replacement in 1~50 hour, changing liquid number is 1~50 time.
Preferably, in the step 7), the air-dried technique is to make the genetic recombination spider silk protein under ventilated environment
Methanol volatilization in white solid, obtains the gene recombination spider silk fibroin bar, wherein ventilated environment temperature is 10~50
DEG C, the processing time is 1 day~200 days.
Also a kind of gene recombination spider silk fibroin tooth inlay of the present invention, is prepared by above-mentioned preparation method.
As described above, gene recombination spider silk fibroin tooth inlay and preparation method thereof of the invention, including genetic recombination
The preparation of spidroin solution, the freeze-drying of gene recombination spider silk fibroin solution, gene recombination spider silk fibroin are dissolved in
In hexafluoroisopropanol or water, gene recombination spider silk fibroin bar preparation, machining.By controlling genetic recombination spider silk
The parameters such as protein solution preparation parameter, dry solidification and doping solid phase enhance particle, can control genetic recombination spider silk protein
The mechanical property of white tooth inlay, acquisition and skeleton mechanics parameter, the matched excellent performance tooth inlay of dental articulation, and
And the tooth inlay that the present invention obtains is compatible with human body, will not generate inflammatory problems.
Detailed description of the invention
Fig. 1 is the preparation method flow chart of gene recombination spider silk fibroin tooth inlay of the present invention.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Please refer to attached drawing.It should be noted that only the invention is illustrated in a schematic way for diagram provided in the present embodiment
Basic conception, only shown in schema then with related component in the present invention rather than component count, shape when according to actual implementation
Shape and size are drawn, when actual implementation kenel, quantity and the ratio of each component can arbitrarily change for one kind, and its component cloth
Office's kenel may also be increasingly complex.
The present invention provides a kind of preparation method of gene recombination spider silk fibroin tooth inlay, and the preparation method is at least wrapped
It includes:
Step S1 is first carried out, the engineering bacteria through everfermentation and expressing gene recombinant spider silk proteins is dissolved in certain dense
In the buffer solution of degree, it is then crushed the engineering bacteria, and be centrifuged, collects the first supernatant liquor.
The engineering bacteria is usually the method for using genetic engineering, and foreign gene is made to obtain the mushroom cell strain of high efficient expression
System.Engineering bacteria is the novel microbial processed using modern biotechnology, is had multi-functional, efficient and adaptable
The features such as.
Using the engineering bacteria by high density fermentation and expressing gene recombinant spider silk proteins in this step, then will
The engineering bacteria is dissolved in certain density buffer solution.
As an example, the buffer solution is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution, wherein three
Hydroxymethyl aminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, the concentration of sodium chloride is 0.1~
1000mmol/L, the concentration of imidazoles are 0.1~1000mmol/L.More preferably, the trishydroxymethylaminomethane-concentration of hydrochloric acid is
10~80mmol/L, the concentration of sodium chloride are 50~500mmol/L, and the concentration of imidazoles is 100~800mmol/L.Certainly, in addition to
Trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution can choose other suitable solution also as buffer solution
The engineering bacteria is dissolved, it is unlimited herein.
As an example, cracking pressure is 600~2000bar, the engineering bacteria quality and buffering when being crushed the engineering bacteria
Liquor capacity ratio is 10g:1mL~10g:1000mL；The revolving speed of centrifuge separation be 1r/min~40000r/min, the time be 1s~
10h, temperature when centrifugation are -3 DEG C~30 DEG C.More preferably, cracking pressure is 800~1500bar, and the engineering bacteria quality is gentle
Rushing liquor capacity ratio is 10g:50mL~10g:500mL；The revolving speed of centrifuge separation is 100r/min~20000r/min, and the time is
1s~8h, temperature when centrifugation are -3 DEG C~25 DEG C
Then step S2 is executed, first supernatant liquor is kept the temperature, and continue to be centrifuged, the second upper layer is collected
Clear liquid.
As an example, can be kept the temperature using the first supernatant liquor described in water bath with thermostatic control, holding temperature range is 20~
100 DEG C, soaking time range is 0.1~100h.It is, of course, also possible to carry out message using other suitable message modes, herein
With no restrictions.More preferably, holding temperature range is 50~100 DEG C, and soaking time range is 10~100h.
As an example, the revolving speed being centrifuged after heat preservation is 1r/min~40000r/min, the time is 1s~10h,
Temperature when centrifugation is -3 DEG C~30 DEG C.More preferably, the revolving speed of centrifuge separation is 1000r/min~40000r/min, and the time is
1s~5h, temperature when centrifugation are -3 DEG C~10 DEG C.
Then step S3 is executed, nickel layer is added in second supernatant liquor, is washed away using the first cleaning solution
The foreign protein being adsorbed in the nickel layer recycles the second cleaning solution to elute the target being adsorbed in the nickel layer
Gene recombination spider silk fibroin, and collect target gene recombinant spider silk proteins solution.
The nickel layer tin column can adsorb whole albumen, including target gene recombinant spider silk proteins and remove target gene weight
Foreign protein except group spider silk fibroin.It is used for as an example, being added in the supernatant liquor that volume is 10~1000ml
The nickel layer of adhesion protein.
It as an example, the first cleaning solution type is unlimited, such as can be trishydroxymethylaminomethane-hydrochloric acid/chlorination
Sodium/imidazole solution, wherein trishydroxymethylaminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, sodium chloride
Concentration is 0.1~1000mmol/L, and imidazole concentration is 0.1~1000mmol/L, the volume of first cleaning solution is 10~
1000ml。
As an example, the type of second cleaning solution is unlimited, can for trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/
Imidazole solution, wherein the concentration of sodium chloride is 0.1~1000mmol/L；Trishydroxymethylaminomethane-hydrochloric acid pH value is 8, dense
Degree is 0.1~100mmol/L；Imidazole concentration is 0.1~1000mmol/L, the volume of second cleaning solution is 10~
1000ml。
It should be noted that first cleaning solution and the second cleaning solution can be identical solution, it is also possible to difference
Solution, according to identical solution, then according to demand selection various concentration come wash away respectively foreign protein and target gene recombination
Spider silk fibroin.
In the present embodiment, can choose trishydroxymethylaminomethane-hydrochloric acid concentration is 10mmol/L, the concentration of sodium chloride
For 100mmol/L, imidazole concentration is trishydroxymethylaminomethane-hydrochloric acid (PH=8)/sodium chloride/imidazole solution of 500mmol/L
Foreign protein is washed away, in addition the concentration of selective chlorination sodium is 50mmol/L；Trishydroxymethylaminomethane-concentration of hydrochloric acid is
50mmol/L；Trishydroxymethylaminomethane-hydrochloric acid (PH=8)/sodium chloride/imidazole solution that imidazole concentration is 200mmol/L comes
Wash away target gene recombinant spider silk proteins.The solution comprising target gene recombinant spider silk proteins is collected later.
Then step S4 is executed, the target gene recombinant spider silk proteins solution of collection is fitted into bag filter and is carried out
Dialysis, is centrifuged later, collects third supernatant liquor, to obtain required gene recombination spider silk fibroin solution；
As an example, dialysis procedure can be with are as follows: be first placed in gradient in certain density phosphate buffer and dialyse certain time
After be transferred in ultrapure water and dialyse；Bag filter specification is 10~10000000Da, and dialysis procedure is that standing dialysis or magnetic force stir
Dialysis is mixed, magnetic stirring speed is 1r/min~2000r/min.
As an example, the concentration of the phosphate buffer is 10~1000mmol/L when carrying out gradient dialysis, phosphoric acid is replaced
The time interval of buffer is 0.1~100h, and the volume of changed phosphate buffer is 1mL~1000L every time.
As an example, in the step 4), the revolving speed of centrifuge separation is 1r/min~40000r/min, the time be 1s~
10h, temperature when centrifugation are -3 DEG C~30 DEG C.
For example, the collected target gene recombinant spider silk proteins solution can be packed into the bag filter of 10000Da
In, it is placed in gradient in the phosphate buffer of 150mmol/L and stands dialysis, replacement in every 2 hours is primary, the phosphoric acid buffer replaced every time
Liquid product is 100L, is transferred in ultrapure water dialyses later.
As an example, dialysis finishes, it is centrifuged, the revolving speed of centrifuge separation is 1r/min~40000r/min, when
Between be 1s~10h, temperature when centrifugation is -3 DEG C~30 DEG C.More preferably, the revolving speed of centrifuge separation is 1r/min~10000r/
Min, time are 5h~10h, and temperature when centrifugation is 0~25 DEG C.
Then step S5 is executed, using freeze drying process, the gene recombination spider silk fibroin that step 4) is obtained
Solution is changed into anhydrous gene recombination spider silk fibroin.
As an example, in the step 5), freeze drying process are as follows: gene recombination spider silk fibroin aqueous solution exists first
It is freezed in 0~-80 DEG C of environment, then vacuum refrigeration is handled 1~500 hour in freeze drier, freeze drier work
Parameter are as follows: 0.001~1mBar of vacuum pressure, 0~-80 DEG C of temperature.
In this step, in order to further adjust gene recombination spider silk fibroin tooth inlay hardness and other mechanical property
Can, solid phase enhancing particle can be added in the anhydrous gene recombination spider silk fibroin, obtain gene recombination spider silk fibroin
Enhance particle mixed-powder with solid phase；Solid phase enhancing granular materials is ceramic powders, metal powder or polymer powder,
The solid phase enhancing particle and anhydrous gene recombination spider silk fibroin mass ratio are 1:100~100:1.
Step S6 is executed again, and the anhydrous gene recombination spider silk fibroin is dissolved in hexafluoroisopropanol or water, is obtained
The hexafluoroisopropanol solution or aqueous solution of gene recombination spider silk fibroin are obtained,.
As an example, the anhydrous gene recombination spider silk fibroin and hexafluoroisopropanol or water quality ratio are 1:1~1:
10, temperature range is 5 DEG C~50 DEG C.
If joined the solid phase enhancing particle in step S5, the anhydrous gene recombination spider silk fibroin and solid phase increase
The mixture and hexafluoroisopropanol of strong particle or the mass ratio of water are 1:1~1:10, and temperature range is 5 DEG C~50 DEG C.
For example, the anhydrous gene recombination spider silk fibroin and hexafluoroisopropanol or water quality ratio are 1:5, temperature range
It is 20 DEG C.If step joined the solid phase enhancing particle, the anhydrous gene recombination spider silk fibroin and solid phase enhance particle
Mixture and the mass ratio of hexafluoroisopropanol or water be 1:8, temperature range is 15 DEG C.
Step S7 is finally executed, the hexafluoroisopropanol solution or aqueous solution of the gene recombination spider silk fibroin are injected
In specific mold, and pass sequentially through methyl alcohol process and gene recombination spider silk fibroin bar is made in air-dried technique, then will be described
Gene recombination spider silk fibroin bar is cut into required tooth inlay.
As an example, the methyl alcohol process method is methanol solution processing, the methanol solution treatment process are as follows: will contain
The hexafluoroisopropanol solution of gene recombination spider silk fibroin or the mold of aqueous solution are immersed in liquid methanol, and every 1~
The methanol solution of replacement in 50 hours, changing liquid number is 1~50 time, until the solution in the mold is changed into genetic recombination spider
Spider's thread protein solid；Then the gene recombination spider silk fibroin solid is taken out from the mold and to continue to use methanol molten
Liquid impregnates, and every the methanol solution of replacement in 1~50 hour, changing liquid number is 1~50 time.
As an example, the air-dried technique refers to gene recombination spider silk fibroin solution after methyl alcohol process solidifies, logical
So that the methanol in the gene recombination spider silk fibroin solid is volatilized under wind environment, obtains the gene recombination spider silk fibroin stick
Material, wherein ventilated environment temperature is 10~50 DEG C, and the processing time is 1 day~200 days.
For example, the hexafluoroisopropanol solution of the gene recombination spider silk fibroin is injected in specific mold, by mold
It is immersed in liquid methanol, and every the methanol solution of replacement in 10 hours, changes liquid 10 times, until the solution in the mold turns
Become gene recombination spider silk fibroin solid；Then the gene recombination spider silk fibroin solid is taken out simultaneously from the mold
Methanol solution immersion is continued to use, and every the methanol solution of replacement in 5 hours, is changed liquid 15 times.Later in 25 DEG C of ventilated environments
Lower processing 10 days, makes the methanol in the gene recombination spider silk fibroin solid volatilize, obtains the genetic recombination spider silk protein
White bar,
Finally according to specific dental restortion clinical requirement, gene recombination spider silk fibroin bar is cut into required
Tooth inlay.
Tooth inlay obtained can be pure gene recombination spider silk fibroin and constitute, is also possible to genetic recombination spider
Silk-fibroin and solid phase enhancing particulate composite are constituted into, wherein pure gene recombination spider silk fibroin bar be moisture-free basis because
Recombinant spider silk proteins are prepared, and gene recombination spider silk fibroin is to pass through with solid phase enhancing particulate composite tooth inlay
Gene recombination spider silk fibroin is prepared with solid phase enhancing particle mixed-powder.
In conclusion the present invention provides a kind of gene recombination spider silk fibroin tooth inlay and preparation method thereof, including base
Because of the preparation of recombinant spider silk proteins solution, the freeze-drying of gene recombination spider silk fibroin solution, gene recombination spider silk fibroin
It is dissolved in hexafluoroisopropanol or water, the preparation of gene recombination spider silk fibroin bar, machining.By controlling genetic recombination
The parameters such as spidroin solution preparation parameter, dry solidification and doping solid phase enhance particle, can control genetic recombination spider
The mechanical property of spider's thread protein tooth inlay obtains and skeleton mechanics parameter, the matched excellent performance tooth of dental articulation
Inlay, and the tooth inlay that the present invention obtains is compatible with human body, will not generate inflammatory problems.
So the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (18)
1. a kind of preparation method of gene recombination spider silk fibroin tooth inlay, which is characterized in that the preparation method is at least wrapped
It includes:
1) engineering bacteria through everfermentation and expressing gene recombinant spider silk proteins is dissolved in certain density buffer solution, so
After be crushed the engineering bacteria, and be centrifuged, collect the first supernatant liquor；
2) first supernatant liquor is kept the temperature, and continues to be centrifuged, collect the second supernatant liquor；
3) nickel layer is added in second supernatant liquor, is washed away using the first cleaning solution and is adsorbed on the nickel layer
On foreign protein, recycle the second cleaning solution to elute the target gene recombinant spider silk egg that is adsorbed in the nickel layer
It is white, and collect target gene recombinant spider silk proteins solution；
4) the target gene recombinant spider silk proteins solution of collection is fitted into bag filter and is dialysed, is centrifuged later, received
Collect third supernatant liquor, to obtain required gene recombination spider silk fibroin solution；
5) utilize freeze drying process, by step 4) obtain the gene recombination spider silk fibroin solution be changed into moisture-free basis because
Recombinant spider silk proteins；
6) the anhydrous gene recombination spider silk fibroin is dissolved in hexafluoroisopropanol or water, obtains genetic recombination spider silk
The hexafluoroisopropanol solution or aqueous solution of albumen；
7) the hexafluoroisopropanol solution or aqueous solution of the gene recombination spider silk fibroin are injected in specific mold, and according to
It is secondary that gene recombination spider silk fibroin bar is made by methyl alcohol process and air-dried technique, then by the gene recombination spider silk fibroin
Bar is cut into required tooth inlay.
2. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
Buffer solution in step 1) is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution, wherein trihydroxy methyl amino first
Alkane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, and the concentration of sodium chloride is 0.1~1000mmol/L, imidazoles it is dense
Degree is 0.1~1000mmol/L.
3. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
When being crushed the engineering bacteria in step 1), cracking pressure is 600~2000bar, the engineering bacteria quality and volume of buffer solution
Than for 10g:1mL~10g:1000m L；The revolving speed of centrifuge separation is 1r/min~40000r/min, and the time is 1s~10h, from
The temperature when heart is -3 DEG C~30 DEG C.
4. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 2), first supernatant liquor is kept the temperature using water bath with thermostatic control, holding temperature range is 20~100 DEG C, heat preservation
Time range is 0.1~100h.
5. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 2), the revolving speed of centrifuge separation is 1r/min~40000r/min, and the time is 1s~10h, and temperature when centrifugation is -3 DEG C
~30 DEG C.
6. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 3), nickel layer is added in second supernatant liquor that volume is 10~1000ml.
7. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 3), first cleaning solution is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution, wherein trihydroxy methyl
Aminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L, and the concentration of sodium chloride is 0.1~1000mmol/L, miaow
Azoles concentration is 0.1~1000mmol/L, and the volume of first cleaning solution is 10~1000ml.
8. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 3), second cleaning solution is trishydroxymethylaminomethane-hydrochloric acid/sodium chloride/imidazole solution, wherein sodium chloride
Concentration is 0.1~1000mmol/L；Trishydroxymethylaminomethane-hydrochloric acid pH value is 8, and concentration is 0.1~100mmol/L；Miaow
Azoles concentration is 0.1~1000mmol/L, and the volume of second cleaning solution is 10~1000ml.
9. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: described
In step 4), dialysis procedure are as follows: be first placed in certain density phosphate buffer gradient dialysis be transferred to after a certain period of time it is ultrapure
It dialyses in water；Bag filter specification is 10~10000000Da, and dialysis procedure is that standing dialysis or magnetic agitation dialysis, magnetic force stir
Mixing speed is 1r/min~2000r/min.
10. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 9, it is characterised in that: into
When row gradient is dialysed, the concentration of the phosphate buffer is 10~1000mmol/L, and the time interval for replacing phosphate buffer is
0.1~100h, the volume of changed phosphate buffer is 1mL~1000L every time.
11. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 4), the revolving speed of centrifuge separation is 1r/min~40000r/min, and the time is 1s~10h, and temperature when centrifugation is -3
DEG C~30 DEG C.
12. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 5), freeze drying process are as follows: first that gene recombination spider silk fibroin aqueous solution is cold in 0~-80 DEG C of environment
Freeze, then vacuum refrigeration is handled 1~500 hour in freeze drier, freeze drier running parameter are as follows: vacuum pressure
0.001~1mBar, 0~-80 DEG C of temperature.
13. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 5), solid phase is added in the anhydrous gene recombination spider silk fibroin enhances particle, and the solid phase enhances granular material
Material is ceramic powders, metal powder or polymer powder, and the solid phase enhances particle and anhydrous gene recombination spider silk fibroin
Mass ratio is 1:100~100:1.
14. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 6), anhydrous gene recombination spider silk fibroin and hexafluoroisopropanol or water quality ratio are 1:1~1:10, temperature range
It is 5 DEG C~50 DEG C.
15. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 13, it is characterised in that: add
After entering the solid phase enhancing particle, in the step 6), the anhydrous gene recombination spider silk fibroin and solid phase enhance particle
The mass ratio of mixture and hexafluoroisopropanol or water is 1:1~1:10, and temperature range is 5 DEG C~50 DEG C.
16. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 7), methyl alcohol process method is methanol solution processing, the methanol solution treatment process are as follows: will contain genetic recombination spider
The hexafluoroisopropanol solution of spider's thread protein or the mold of aqueous solution are immersed in liquid methanol, and are replaced every 1~50 hour
Methanol solution, changing liquid number is 1~50 time, until the solution in the mold is changed into gene recombination spider silk fibroin and consolidates
Body；Then the gene recombination spider silk fibroin solid is taken out to from the mold and is continued to use methanol solution immersion, and
Every the methanol solution of replacement in 1~50 hour, changing liquid number is 1~50 time.
17. the preparation method of gene recombination spider silk fibroin tooth inlay according to claim 1, it is characterised in that: institute
It states in step 7), the air-dried technique is to wave the methanol in the gene recombination spider silk fibroin solid under ventilated environment
Hair, obtain the gene recombination spider silk fibroin bar, wherein ventilated environment temperature be 10~50 DEG C, processing the time be 1 day~
200 days.
18. a kind of gene recombination spider silk fibroin tooth inlay is obtained by the preparation method preparation of any one of claim 1~17
?.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711030980.XA CN109717969B (en) | 2017-10-30 | 2017-10-30 | Gene recombination spider silk protein tooth inlay and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711030980.XA CN109717969B (en) | 2017-10-30 | 2017-10-30 | Gene recombination spider silk protein tooth inlay and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109717969A true CN109717969A (en) | 2019-05-07 |
| CN109717969B CN109717969B (en) | 2021-11-02 |
Family
ID=66291201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711030980.XA Active CN109717969B (en) | 2017-10-30 | 2017-10-30 | Gene recombination spider silk protein tooth inlay and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109717969B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6620917B1 (en) * | 2000-01-20 | 2003-09-16 | The United States Of America As Represented By The Secretary Of The Army | Method for the purification and aqueous fiber spinning of spider silks and other structural proteins |
| CN1919358A (en) * | 2006-09-15 | 2007-02-28 | 浙江理工大学 | Method for preparing fibroin/calcium phosphate composite material regulated and controlled by silicon |
| CN102264401A (en) * | 2009-01-16 | 2011-11-30 | 圆光大学校产学协力团 | Concrete scaffold made of bone powder and fibrin glue |
| CN106667595A (en) * | 2015-11-11 | 2017-05-17 | 中国科学院上海微***与信息技术研究所 | Silk protein dental implant and preparation method thereof |
-
2017
- 2017-10-30 CN CN201711030980.XA patent/CN109717969B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6620917B1 (en) * | 2000-01-20 | 2003-09-16 | The United States Of America As Represented By The Secretary Of The Army | Method for the purification and aqueous fiber spinning of spider silks and other structural proteins |
| CN1919358A (en) * | 2006-09-15 | 2007-02-28 | 浙江理工大学 | Method for preparing fibroin/calcium phosphate composite material regulated and controlled by silicon |
| CN102264401A (en) * | 2009-01-16 | 2011-11-30 | 圆光大学校产学协力团 | Concrete scaffold made of bone powder and fibrin glue |
| CN106667595A (en) * | 2015-11-11 | 2017-05-17 | 中国科学院上海微***与信息技术研究所 | Silk protein dental implant and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 刘华: "蛛丝蛋白工程菌pNS2/BL21（DE3）发酵工艺和纯化方法的优化及表达产物的组织相容性研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 杨志明: "《组织工程基础与临床》", 30 April 2004 * |
| 涂桂云: "基因重组蜘蛛丝蛋白牙齿嵌体的制备方法", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109717969B (en) | 2021-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105025940B (en) | Cell transplantation eucaryotic cell structure body, bioaffinity polymer block and their manufacture method | |
| Liao et al. | Bone regeneration using adipose-derived stem cells in injectable thermo-gelling hydrogel scaffold containing platelet-rich plasma and biphasic calcium phosphate | |
| US20130059382A1 (en) | Biomedical materials for tissue engineering | |
| WO2002034885A1 (en) | Sericin-containing material, process for producing the same and method of using the same | |
| CN108926745B (en) | A kind of preparation method and its compound system of nanoporous micro rack | |
| CN107185039B (en) | Porous metal bone implant material and preparation method and application thereof | |
| CN101773684A (en) | Preparation method of fibroin/hydroxyapatite porous scaffold | |
| CN105832682A (en) | Method for preparing honeycomb silk fibroin porous microsphere sustained drug release vector | |
| CN106244659A (en) | The method of preparation poultry Cartilage collagen polypeptide | |
| CN108752466A (en) | A kind of chelated calcium preparation method of tuna bone collagen peptide | |
| CN106667595B (en) | Fibroin dental implant and preparation method thereof | |
| CN101928744B (en) | Process for extracting active collagen peptide from salmon trout waste | |
| CN107523600A (en) | Promote the preparation method and purposes of the bone collagen polypeptide of human osteoblast cell's propagation | |
| CN103468771A (en) | Method for extracting collagens from bovine achilles tendon | |
| CN105327399A (en) | Construction method of artificial blood vessel | |
| CN102886075A (en) | Human hard tissue repair material and preparation method thereof | |
| CN102127166A (en) | Method for separating purified ferritins from biological tissues or engineering bacteria | |
| Li et al. | Cathelicidin LL37 promotes osteogenic differentiation in vitro and bone regeneration in vivo | |
| CN109717969A (en) | A kind of gene recombination spider silk fibroin tooth inlay and preparation method thereof | |
| CN104558105A (en) | Antioxidative peptide of metapenaeus affinis as well as separation and extraction method and application of antioxidative peptide | |
| CN106075594A (en) | A kind of Thermal inactive nano-fiber tubular scaffold and preparation method thereof | |
| Yan et al. | Octopus-inspired gelatin-methacrylate scaffolds loaded with hBMSC-derived exosomes promote wound healing by regulating macrophage polarization | |
| CN102600504A (en) | Preparation method of mulberry silk tissue engineering scaffold | |
| WO2019045105A1 (en) | Abnormal cardiac rhythm myocardial model and method for producing same, agent for forming abnormal cardiac rhythm myocardial model, and method for evaluating drug efficacy of heart disease therapeutic | |
| CN101921318A (en) | Method for preparing antifreeze polypeptide through enzymolysis of pigskin collagen protein by utilizing alkali protease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |