CN106596820A - Method for analyzing influence of carbon-nanotube-reinforced silk on fibroin amino acid sequences based on proteomics - Google Patents
Method for analyzing influence of carbon-nanotube-reinforced silk on fibroin amino acid sequences based on proteomics Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses a method for analyzing influence of carbon-nanotube-reinforced silk on fibroin amino acid sequences based on proteomics. The method has the advantages that mulberry leaves sprayed with a carbon-nanotube-containing solution are supplied to larva silkworms to obtain reinforced silk, a contrast experiment involving the reinforced silk and common silk according to a proteomics method is conducted, alkaline degumming is employed and SDS anionic surfactants are added for improving degumming effects in an experiment process, and chymotrypsin is used for enzyme digestion in the solution to complete enzyme digestion separation; HPLC-MS/MS is used for mass spectrometric analysis, and the influence of the carbon-nanotube-reinforced silk on the fibroin amino acid sequences is judged according to obtained two groups of amino acid sequences; the method is high in sensitivity and accuracy, simple to operate and less in sample consumption.
Description
Technical field
The present invention relates to a kind of affect fibroin aminoacid sequence, it is more particularly to a kind of to be based on protein group
Learn analysis for carbon nanotubes and reinforce the method that silkworm silk affects on fibroin aminoacid sequence.
Background technology
Silkworm silk is one of ancient times Han nationality's civilization product, is that silk liquid concretionary continuous fiber is secreted when matured silkworm cocoons,
Also referred to as " natural silk ", it is one of animal fiber that the mankind utilize earliest as Pilus Caprae seu Oviss, and silkworm silk has gently, carefully, the advantages of soft,
But there is also low intensity, the shortcomings of external force resistance is destructive weak, in order to change this shortcoming of silkworm silk, many researchers do
Many researchs reinforcing silkworm silk, the silkworm silk crystal knot being known that by Raman spectrum and Electron Microscope images after reinforcing
Structure arrangement is more orderly, and various performances are significantly improved, but are whether that the change for being related to fibroin aminoacid sequence is difficult to do
Go out to judge.
The content of the invention
It is an object of the invention to overcome the shortcomings of that prior art is present, and provide a kind of quick, intuitively, it is easy based on
Proteome analysis CNT reinforces the method that silkworm silk affects on fibroin aminoacid sequence.
For achieving the above object, the invention provides following technical scheme：One kind is based on proteome analysis carbon nanometer
Pipe reinforces the method that silkworm silk affects on fibroin aminoacid sequence, and the method adopts following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) 0.5～2.5ml of aqueous solution sprayed on Folium Mori containing CNT, B groups silkworm larva institute are eaten in A group silkworm larvas
Food Folium Mori are not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 0.5～1.5% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice,
After 1h, solution and fibroin are separated, fibroin deionized water is rinsed and dried in 50～60 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,93～97 DEG C of 10～20min of steaming and decocting are heated to, silkworm silk solution is obtained, with retention molecule after cold filtration
The bag filter dialysis for 8000～14000 is measured, a water is changed every 2h or so in the 12h for starting that dialyses, one is changed every 4h after 12h
Secondary water, a water is changed after 24h every 8h, and silk fibroin protein solution, concentration are obtained after 72h；
E) take concentration after solution add mass fraction be 1.5～2% dithiothreitol, DTT solution 2～3ml alcohol in 40
At～50 DEG C keep 15～20min, be cooled to room temperature add mass fraction be 2.2～2.8% iodoacetamide solution 4～
5ml, keeps adding the chymotrypsin protein enzymatic solution of 50～80ul after 15～20min under dark surrounds, then dilute with formic acid solution
Release；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is contrasted with amino acid sequence database, than
Compared with A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups less, are divided with amino acid sequence database contrast difference
Analysis A groups, if aminoacid sequence has sequence of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Structure has an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
It is a kind of that the method that silkworm silk affects on fibroin aminoacid sequence is reinforced based on proteome analysis CNT,
Using following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) the aqueous solution 1.5ml sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Mulberry
Leaf is not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 1% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, after 1h,
Solution and fibroin are separated, fibroin deionized water is rinsed and dried in 55 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,95 DEG C of steaming and decocting 15min are heated to, silkworm silk solution is obtained, be with molecular cut off after cold filtration
11000 bag filter dialysis, a water is changed in the 12h for starting that dialyses every 2h or so, a water is changed every 4h after 12h, after 24h
A water is changed every 8h, silk fibroin protein solution, concentration are obtained after 72h；
E) take concentration after solution add mass fraction be 1.7% dithiothreitol, DTT solution 2.5ml alcohol at 45 DEG C
17min is kept, the iodoacetamide solution 4.5ml that room temperature adds mass fraction to be 2.5% is cooled to, is kept under dark surrounds
The chymotrypsin protein enzymatic solution of 65ul is added after 17min, is then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is contrasted with amino acid sequence database, than
Compared with A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups less, are divided with amino acid sequence database contrast difference
Analysis A groups, if aminoacid sequence has sequence of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Structure has an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
The invention has the beneficial effects as follows：It is reinforced to Folium Mori solution of the spray containing CNT with supplying silkworm larva
Silkworm silk compares experiment with common silkworm silk using proteomics method, subtraction degumming is adopted in experimentation and adds SDS cloudy
Ionic surface active agent improves degumming effect, carries out enzyme action in solution using chymase and completes enzyme action separation, adopts
HPLC-MS/MS carries out mass spectral analyses, judges that CNT reinforces silkworm silk to silkworm silk egg according to two groups of resulting aminoacid sequences
The impact of casamino acid sequence, the present invention reinforces silkworm silk to fibroin amino using proteomics method analysis for carbon nanotubes
The impact of acid sequence, on the one hand, sensitivity is high, simple to operate, on the other hand, accuracy is high, and sample usage amount is few.
Specific embodiment
The present invention is described in detail below in conjunction with specific embodiment：
It is a kind of that the method that silkworm silk affects on fibroin aminoacid sequence is reinforced based on proteome analysis CNT,
The method adopts following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) 0.5～2.5ml of aqueous solution sprayed on Folium Mori containing CNT, B groups silkworm larva institute are eaten in A group silkworm larvas
Food Folium Mori are not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 0.5～1.5% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice,
After 1h, solution and fibroin are separated, fibroin deionized water is rinsed and dried in 50～60 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,93～97 DEG C of 10～20min of steaming and decocting are heated to, silkworm silk solution is obtained, with retention molecule after cold filtration
The bag filter dialysis for 8000～14000 is measured, a water is changed every 2h or so in the 12h for starting that dialyses, one is changed every 4h after 12h
Secondary water, a water is changed after 24h every 8h, and silk fibroin protein solution, concentration are obtained after 72h；
E) take concentration after solution add mass fraction be 1.5～2% dithiothreitol, DTT solution 2～3ml alcohol in 40
At～50 DEG C keep 15～20min, be cooled to room temperature add mass fraction be 2.2～2.8% iodoacetamide solution 4～
5ml, keeps adding the chymotrypsin protein enzymatic solution of 50～80ul after 15～20min under dark surrounds, then dilute with formic acid solution
Release；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is entered with amino acid sequence database (seeing appendix 1)
Row contrast, compares A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups and amino acid sequence database contrast differences
Not less, A groups are analyzed, if aminoacid sequence has obvious sequence difference explanation silkworm silk reinforcing to silkworm silk egg with B groups in A groups
White sequential structure has an impact, if A groups aminoacid sequence is reinforced and aminoacid sequence knot with B groups no significant difference explanation silkworm silk
Structure is unrelated.
Embodiment 1：One kind reinforces silkworm silk to fibroin aminoacid sequence shadow based on proteome analysis CNT
Loud method, the method adopts following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) the aqueous solution 0.5ml sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Mulberry
Leaf is not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 0.5% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, 1h it
Afterwards, solution and fibroin are separated, fibroin deionized water is rinsed and dried in 50 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,93 DEG C of steaming and decocting 10min are heated to, silkworm silk solution is obtained, use molecular cut off to be 8000 after cold filtration
Bag filter dialysis, dialyse start 12h in changes a water every 2h or so, change a water every 4h after 12h, after 24h every
8h changes a water, and silk fibroin protein solution, concentration are obtained after 72h；
E) taking the solution after concentration adds the dithiothreitol, DTT solution 2ml alcohol that mass fraction is 1.5% to protect at 40 DEG C
15min is held, the iodoacetamide solution 4ml that room temperature adds mass fraction to be 2.2% is cooled to, under dark surrounds 15min is kept
The chymotrypsin protein enzymatic solution of 50ul is added afterwards, is then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is contrasted with amino acid sequence database, than
Compared with A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups and aminoacid sequence
Contrast difference less, analyzes A groups, if aminoacid sequence has obvious sequence difference explanation silkworm with B groups in A groups
Silk is reinforced the sequential structure to fibroin and is had an impact, if A groups aminoacid sequence is reinforced with B groups no significant difference explanation silkworm silk
It is unrelated with amino acid sequence structure.
Embodiment 2:One kind reinforces silkworm silk to fibroin aminoacid sequence shadow based on proteome analysis CNT
Loud method, the method adopts following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) the aqueous solution 1.5ml sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Mulberry
Leaf is not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 1% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, after 1h,
Solution and fibroin are separated, fibroin deionized water is rinsed and dried in 55 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,95 DEG C of steaming and decocting 15min are heated to, silkworm silk solution is obtained, be with molecular cut off after cold filtration
11000 bag filter dialysis, a water is changed in the 12h for starting that dialyses every 2h or so, a water is changed every 4h after 12h, after 24h
A water is changed every 8h, silk fibroin protein solution, concentration are obtained after 72h；
E) take concentration after solution add mass fraction be 1.7% dithiothreitol, DTT solution 2.5ml alcohol at 45 DEG C
17min is kept, the iodoacetamide solution 4.5ml that room temperature adds mass fraction to be 2.5% is cooled to, is kept under dark surrounds
The chymotrypsin protein enzymatic solution of 65ul is added after 17min, is then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is contrasted with amino acid sequence database, than
Compared with A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups less, are divided with amino acid sequence database contrast difference
Analysis A groups, if aminoacid sequence has sequence of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Structure has an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
Embodiment 3:One kind reinforces silkworm silk to fibroin aminoacid sequence shadow based on proteome analysis CNT
Loud method, the method adopts following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its etc.
Amount is randomly divided into A, two groups of B；
B) the aqueous solution 2.5ml sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Mulberry
Leaf is not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is 0.5% in mass fraction
Na2CO3Aqueous solution and mass fraction be 1.5% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, 1h it
Afterwards, solution and fibroin are separated, fibroin deionized water is rinsed and dried in 60 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:2 mixed solution
In, bath raio is 1：25,97 DEG C of steaming and decocting 20min are heated to, silkworm silk solution is obtained, be with molecular cut off after cold filtration
14000 bag filter dialysis, a water is changed in the 12h for starting that dialyses every 2h or so, a water is changed every 4h after 12h, after 24h
A water is changed every 8h, silk fibroin protein solution, concentration are obtained after 72h；
E) taking the solution after concentration adds the dithiothreitol, DTT solution 3ml alcohol that mass fraction is 2% to keep at 50 DEG C
20min, is cooled to the iodoacetamide solution 5ml that room temperature adds mass fraction to be 2.8%, keeps after 20min under dark surrounds
The chymotrypsin protein enzymatic solution of 80ul is added, is then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result is contrasted with amino acid sequence database, than
Compared with A, the fibroin aminoacid sequence in two groups of experimental results of B, B groups less, are divided with amino acid sequence database contrast difference
Analysis A groups, if aminoacid sequence has sequence of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Structure has an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment,
All technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
For those of ordinary skill, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Adnexa one
Bombyxmori Linnaeuss：Bombyx mori (5263aa)
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61 tkkmqrknkn hgilgknekm iktfvittds dgnesiveed vlmktlsdgt vaqsyvaada
121 gaysqsgpyv snsgysthqg ytsdfstsaa vgagagagaa agsgagagag ygaasgagag
181 agagagagyg tgagagagag ygagagagag agygagagag agagygagag agagagygag
241 agagagagyg agagagagag ygaasgagag agygqgvgsg aasgagagag agsaagsgag
301 agagtgagag ygagagagag agygaasgtg agygagagag yggasgagag agagagagag
361 agygtgagyg agagagagag agagygagag agygagygvg agagygagyg agagsgaasg
421 agsgagagsg agagsgagag sgagagsgag agsgagagsg agagsgagag sgtgagsgag
481 agygagagag ygagagsgaa sgagagsgag agsgagagsg agagsgagag sgagagygag
541 agagygagag agygagagvg ygagagsgaa sgagagsgag agsgagagsg agagsgagag
601 sgagagsgag agsgagagsg agagsgagvg ygagvgagyg agygagagag ygagagsgaa
661 sgagagagag agtgssgfgp yvanggysrs dgyeyawssd fgtgsgagag sgagagsgag
721 agsgagagsg agagsgagag ygagvgvgyg agygagagag ygagagsgaa sgagagsgag
781 agsgagagsg agagsgagag sgagagsgag agsgagagsg agagsgagag sgagvgsgag
841 agsgagagvg ygagagvgyg agagsgaasg agagsgagag sgagagsgag agsgagagsg
901 agagsgagag sgagagsgag agsgagvgyg agvgagygag ygagagagyg agagsgaasg
961 agagsgagag sgagagsgag agsgagagsg agagsgagag sgagagsgag agsgagagsg
1021 agagsgagag ygagagagyg agygagagag ygagagsgaa sgagsgagag sgagagagsg
1081 agagsgagag sgagagsgag agsgagagsg agagygagvg agygagygag agagygagag
1141 sgaasgagag sgagagsgag agsgagagsg agagsgagag sgagagsgag vgygagygag
1201 agagygagag sgaasgagag agagagtgss gfgpyvahgg ysgyeyawss esdfgtgsga
1261 gagsgagags gagagsgaga gsgagygagv gagygagyga gagagygaga gsgagsgaga
1321 gsgagagsga gagsgagags gagagsgaga gsgagagsga gagsgagagy gagygagaga
1381 gygagagsga gsgagagsga gagsgagags gagagsgaga gsgagagsga gagsgagagy
1441 gagvgagyga gygagagagy gagagsgags gagagsgaga gsgagagsga gvgsgagags
1501 gagagsgaga gsgagagyga gygagagagy gagagsgags gagagsgaga gsgagagsga
1561 gagsgagags gagagsgaga gsgagvgyga gvgagygagy gagagagyga gagsgaasga
1621 gagagagagt gssgfgpyva nggysgyeya wssesdfgtg sgagagsgag agsgagagsg
1681 agagsgagag ygagygagag agygagagsg agsgagagsg agagsgagag sgagagsgag
1741 agsgagagsg agagsgagsg sgagagsgag agsgagagyg agvgagygvg ygagagagyg
1801 agagsgaasg agagagagag tgssgfgpyv ahggysgyey awssesdfgt gsgagagsga
1861 gagsgagags gagagsgaga gsgagagsga gagygagvga gygaaygaga gagygagags
1921 gaasgagags gagagsgaga gsgagagsga gagsgagags gagagsgaga gsgagagsga
1981 gagsgagagy gagagagyga gagsgagsga gagsgagags gagagsgaga gsgagagsga
2041 gsgsgagags gagagsgaga gygagvgagy gagygagaga gygagagsga gsgagagsga
2101 gagygagaga gygagygaga gagygagagt gagsgagags gagagsgaga gsgagagsga
2161 gagsgagags gagsgsgaga gsgagagsga gagsgagags gagagsgaga gygagagagy
2221 gagygagaga gygagagsga gsgagagsga gagsgagags gagagygagy gagagsgaas
2281 gagagagaga gtgssgfgpy vahggysgye yawssesdfg tgsgagagsg agagagagag
2341 sgagagygag vgagygagyg agagagygag agsgtgsgag agsgagagyg agvgagygag
2401 agsgaafgag agagagsgag agsgagagsg agagsgagag sgagagygag ygagvgagyg
2461 agagsgaasg agagsgagag sgagagsgag agsgagagsg agagygagvg agygagygag
2521 agagygagag sgaasgagag sgagagagsg agagsgagag sgagagsgag sgagagsgag
2581 agsgagagyg agagsgaasg agagagagag tgssgfgpyv anggysgyey awssesdfgt
2641 gsgagagsga gagsgagags gagagsgaga gygagvgagy gagygagaga gygagagsga
2701 gsgagagsga gagsgagags gagagsgaga gsgagagsga gagygagags gaasgagags
2761 gagagsgaga gsgagagsga gagsgagags gagagygagv gagygvgyga gagagygaga
2821 gsgagsgaga gsgagagsga gagsgagags gagsgagags gagagsgaga gsgagsgaga
2881 gsgagagygv gygagagagy gagagsgags gagagsgaga gsgagagsga gsgagagsga
2941 gagsgagags gagagygagv gagygvgyga gagagygaga gsgagsgaga gsgagagsga
3001 gagsgagags gagagsgaga gsgagagsga gsgagagsga gagsgagags gagagsgags
3061 gagagsgaga gsgagagsga gagygagvga gygvgygagv gagygagags gaasgagags
3121 gagagagsga gagsgagags gagagsgaga gsgagagsga gagygagyga gvgagygaga
3181 gvgygagaga gygagagsga asgagagags gagagtgaga gsgagagyga gagsgaasga
3241 gagagagagt gssgfgpyva nggysgyeya wssesdfgtg sgagagsgag agsgagagsg
3301 agagsgagag ygagvgagyg agagsgagsg agagsgagag sgagagsgag agsgagagyg
3361 agagsgtgsg agagsgagag sgagagsgag agsgagagsg agagsgvgag ygvgygagag
3421 agygvgygag agagygagag sgtgsgagag sgagagsgag agsgagagsg agagsgagag
3481 sgagagygag vgagygvgyg agagagygag agsgagsgag agsgagagsg agagsgagag
3541 sgagsgagag sgagagsgag agsgagsgag agsgagagyg vgygagagag ygagagsgag
3601 sgagagsgag agsgagagsg agsgagagsg agagsgagag sgagagygag vgagygvgyg
3661 agagagygag agsgagsgag agsgagagsg agagsgagag sgagagsgag agsgagagsg
3721 agsgagagsg agagsgagag sgagagygag vgagygvgyg agagagygag agsgaasgag
3781 agagagagtg ssgfgpyvan ggysgyeyaw ssesdfgtgs gagagsgaga gsgagagyga
3841 gygagvgagy gagagvgyga gagagygaga gsgaasgaga gagagagsga gagsgagaga
3901 gsgagagyga gygigvgagy gagagvgyga gagagygaga gsgaasgaga gsgagagsga
3961 gagsgagags gagagsgaga gsgagagyga gygagvgagy gagagvgyga gagagygaga
4021 gsgaasgaga gagagagags gagagsgaga gsgagagsga gagsgagags gagagsgaga
4081 gsgagagsga gagygagvga gygagyggag agygagagsg aasgagagsg agagsgagag
4141 sgagagsgag agsgagagyg agagsgaasg agagagagag tgssgfgpyv nggysgyeya
4201 wssesdfgtg sgagagsgag agsgagagyg agvgagygag ygagagagyg agagsgaasg
4261 agagsgagag sgagagsgag agsgagsgag agsgagagsg agagsgagag sgagagsgag
4321 agygagvgag ygagygagag agygagagsg aasgagagsg agagagsgag agsgagagsg
4381 agagsgagag sgagagsgag sgagagsgag agygagygag vgagygagag vgygagagag
4441 ygagagsgaa sgagagsgsg agsgagagsg agagsgagag agsgagagsg agagsgagag
4501 ygagygagag sgaasgagag agagagtgss gfgpyvangg ysgyeyawss esdfgtgsga
4561 gagsgagags gagagygagv gagygagyga gagagygaga gsgagsgaga gsgagagsga
4621 gagsgagags gagagsgaga gsgagagsga gagygagyga gagagygaga gvgygagaga
4681 gygagagsga gsgagagsgs gagagsgsga gsgagagsga gagsgagags gagagsgaga
4741 gsgagagsga gagygagygi gvgagygaga gvgygagaga gygagagsga asgagagsga
4801 gagsgagags gagagsgaga gsgagagsga gagsgagags gagagsgaga gygagagvgy
4861 gagagsgaas gagagsgaga gsgagagsga gagsgagags gagagsgaga gsgagsgaga
4921 gsgagagyga gygagvgagy gagagygagy gvgagagyga gagsgagsga gagsgagags
4981 gagagsgaga gsgagagsga gsgagagyga gagagygaga gagygagags gaasgagaga
5041 gagsgagags gagagsgags gagagsgaga gygagagsga asgagagsga gagagagaga
5101 gsgagagsga gagygagags gaasgagaga gagtgssgfg pyvanggysr regyeyawss
5161 ksdfetgsga asgagagags gagagsgaga gsgagagsga gaggsvsyga grgygqgags
5221 aassvssass rsydysrrnv rkncgiprrq lvvkfralpc vnc
Claims (2)
1. it is a kind of that the method that silkworm silk affects on fibroin aminoacid sequence is reinforced based on proteome analysis CNT, its
It is characterised by adopting following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its equivalent with
Machine is divided into A, two groups of B；
B) 0.5～2.5ml of aqueous solution sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Mulberry
Leaf is not processed, and other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is in the Na that mass fraction is 0.5%2CO3
Aqueous solution and mass fraction be 0.5～1.5% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, 1h it
Afterwards, solution and fibroin are separated, fibroin deionized water is rinsed and dried in 50～60 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:In 2 mixed solution,
Bath raio is 1：25,93～97 DEG C of 10～20min of steaming and decocting are heated to, silkworm silk solution is obtained, molecular cut off is used after cold filtration
Bag filter for 8000～14000 is dialysed, and a water is changed every 2h or so in the 12h for starting that dialyses, and is changed once every 4h after 12h
Water, a water is changed after 24h every 8h, and silk fibroin protein solution, concentration are obtained after 72h；
E) take concentration after solution add mass fraction be 1.5～2% dithiothreitol, DTT solution 2～3ml alcohol in 40～50
15～20min is kept at DEG C, 4～5ml of iodoacetamide solution that room temperature adds mass fraction to be 2.2～2.8% is cooled to, it is black
Keep adding the chymotrypsin protein enzymatic solution of 50～80ul after 15～20min under dark situation, then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result are contrasted with amino acid sequence database, compare A,
Fibroin aminoacid sequence in two groups of experimental results of B, B groups less, analyze A with amino acid sequence database contrast difference
Group, if aminoacid sequence has sequential structure of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Have an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
2. it is a kind of that the method that silkworm silk affects on fibroin aminoacid sequence is reinforced based on proteome analysis CNT, its
It is characterised by adopting following steps：
A) choose a silkworm seed from same parent to be incubated under the same conditions, after treating that silkworm broken shell is grown up, by its equivalent with
Machine is divided into A, two groups of B；
B) the aqueous solution 1.5ml sprayed on Folium Mori containing CNT is eaten in A group silkworm larvas, B group silkworm larvas eat Folium Mori not
Process, other growth conditionss keep identical；
C) treat that larva spinning collects two groups of silkworm silks respectively with 1 after cocooing:100 bath raio is in the Na that mass fraction is 0.5%2CO3
Aqueous solution and mass fraction be 1% SDS mixed solutions in boiling boil 30min and carry out degumming, altogether degumming twice, after 1h, will be molten
Liquid and fibroin are separated, and fibroin deionized water is rinsed and dried in 55 DEG C of baking oven；
D) fibroin fiber that degumming is dried is put into the CaCl for preparing in advance2:H2O:Ethanol is 1:8:In 2 mixed solution,
Bath raio is 1：25, it is heated to 95 DEG C of steaming and decocting 15min, silkworm silk solution is obtained, with molecular cut off it is 11000 after cold filtration
Bag filter is dialysed, and a water is changed every 2h or so in the 12h for starting that dialyses, and a water is changed every 4h after 12h, every 8h after 24h
A water is changed, silk fibroin protein solution, concentration are obtained after 72h；
E) taking the solution after concentration adds the dithiothreitol, DTT solution 2.5ml alcohol that mass fraction is 1.7% to keep at 45 DEG C
17min, is cooled to the iodoacetamide solution 4.5ml that room temperature adds mass fraction to be 2.5%, and under dark surrounds 17min is kept
The chymotrypsin protein enzymatic solution of 65ul is added afterwards, is then diluted with formic acid solution；
F) mass spectral analyses are carried out to it using HPLC～MS/MS, result are contrasted with amino acid sequence database, compare A,
Fibroin aminoacid sequence in two groups of experimental results of B, B groups less, analyze A with amino acid sequence database contrast difference
Group, if aminoacid sequence has sequential structure of the obvious sequence difference explanation silkworm silk reinforcing to fibroin with B groups in A groups
Have an impact, if A groups aminoacid sequence reinforces unrelated with amino acid sequence structure with B groups no significant difference explanation silkworm silk.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107219370A (en) * | 2017-07-21 | 2017-09-29 | 浙江理工大学 | The radially-arranged method of amino acid in one kind analysis Bombyx silk albumen |
| CN113061157A (en) * | 2021-03-31 | 2021-07-02 | 浙江理工大学 | Protein extraction and enrichment method of silk proteomics |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070087024A1 (en) * | 2003-10-30 | 2007-04-19 | Knight David P | Article comprising fibers embedded in a glassy protein |
| CN102532253A (en) * | 2012-02-20 | 2012-07-04 | 浙江大学 | Method for extracting silk protein |
| CN103308616A (en) * | 2013-05-30 | 2013-09-18 | 中国丝绸博物馆 | Silk cultural relic fiber material identification method based on amino acid analysis |
| CN103983726A (en) * | 2014-05-29 | 2014-08-13 | 西南大学 | Application of glycine or metabolism regulator thereof in improving domestic silkworm silk output and method thereof |
| CN105842358A (en) * | 2016-03-28 | 2016-08-10 | 南通市纤维检验所 | Quantitative detection method of sericin content ratio in silk |
-
2016
- 2016-12-12 CN CN201611136630.7A patent/CN106596820B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070087024A1 (en) * | 2003-10-30 | 2007-04-19 | Knight David P | Article comprising fibers embedded in a glassy protein |
| CN102532253A (en) * | 2012-02-20 | 2012-07-04 | 浙江大学 | Method for extracting silk protein |
| CN103308616A (en) * | 2013-05-30 | 2013-09-18 | 中国丝绸博物馆 | Silk cultural relic fiber material identification method based on amino acid analysis |
| CN103983726A (en) * | 2014-05-29 | 2014-08-13 | 西南大学 | Application of glycine or metabolism regulator thereof in improving domestic silkworm silk output and method thereof |
| CN105842358A (en) * | 2016-03-28 | 2016-08-10 | 南通市纤维检验所 | Quantitative detection method of sericin content ratio in silk |
Non-Patent Citations (3)
| Title |
|---|
| QI WANG等: "Feeding Single-Walled Carbon Nanotubes or Graphene to Silkworms for Reinforced Silk Fibers", 《NANO LETTERS》 * |
| ZHAOMING DONG等: "Comparative Proteomics Reveal Diverse Functions and Dynamic Changes of Bombyx mori Silk Proteins Spun from Different Development Stages", 《JOURNAL OF PROTEOME RESEARCH》 * |
| 李玲玲等: "不同钙-醇溶解体系丝素蛋白的制备及表征研究", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107219370A (en) * | 2017-07-21 | 2017-09-29 | 浙江理工大学 | The radially-arranged method of amino acid in one kind analysis Bombyx silk albumen |
| CN113061157A (en) * | 2021-03-31 | 2021-07-02 | 浙江理工大学 | Protein extraction and enrichment method of silk proteomics |
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| CN106596820B (en) | 2019-01-22 |
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