CN106770751A - A kind of method for differentiating Ancient Silk Textile silk species - Google Patents

Abstract

The invention discloses it is a kind of differentiate Ancient Silk Textile silk species method, after obscuring three kinds of Ancient Silk Textiles packet, use mass fraction for 0.5% Na₂CO₃Solution degumming, decolourized and use cupri-ethylene diamine solution to dissolve fibroin albumen after de-oiling wax step, dialysis share purification with ultrafiltration, complete the extraction of fibroin albumen, digested using enzymatic cleavage methods in solution, then mass spectral analysis is carried out to it through HPLC MS/MS, result and database are carried out into contrast differentiates three kinds of silk species of Ancient Silk Textile；Instant invention overcomes the detection limit of common determination method so that the Ancient Silk Textile for even preserving out of order can also be distinguished identification；Ancient Silk Textile silk species is identified using proteomics method, uses the technology of digestion in solution to cause that experimental implementation is simple and convenient in detection；Ancient Silk Textile silk species is identified using proteomics method, sensitivity is high, and the degree of accuracy is high, and sample usage amount is few.

Description

A kind of method for differentiating Ancient Silk Textile silk species

Technical field

It is more particularly to a kind of to differentiate Ancient Silk Textile silkworm the present invention relates to a kind of method for differentiating silk species The method of silk species.

Background technology

Silk is one of ancient times Han nationality's civilization product, is secretion silk liquid concretionary continuous fiber when matured silkworm cocoons, Also referred to as " natural silk ", according to the difference of food, silkworm, tussah, cassava silkworm, wild silkworm, willow silkworm and giant silkworm etc. are divided again, various silks exist Sphere of life is widely used, and is also received much concern in sphere of learning, but the various silk goods being made up of different silks, often because Cause the various silk goods of label confused for careless or the time the extension of work, the research work to after is brought Very big inconvenience, can be by infrared if preserving preferable silk goods, and the various ways such as XRD are distinguish between its silk kind Class, the Ancient Silk Textile for preserving out of order is then difficult to discriminate between using common analysis and testing technology.

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, easy discriminating The method of Ancient Silk Textile silk species.

To achieve the above object, the invention provides following technical scheme：A kind of discriminating Ancient Silk Textile silk species Method, the method uses following steps：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 2~8g of Ancient Silk Textile that chestnut silk makes, It is divided into A, tri- groups of B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.4~0.6%₂CO₃Boiling boils 20 in the aqueous solution ~40min carries out degumming, 4~6 times repeatedly, then separates three groups of sample solutions with fibroin respectively, treats fibroin deionized water After flushing is squeezed out, then the hydrochloric acid solution that 50~80ml mass fractions are 1% is taken, 50~60min is soaked at room temperature, washed with warm water To solution in neutrality, dried in 60~80 DEG C of baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 50~70 DEG C, is put into The mass fraction of 90~110ml is 100~120min of leaching in 60~80% methanol solutions, then at a temperature of 50~70 DEG C, is put into 60~80min is soaked in 90~110ml absolute ethyl alcohols, then at a temperature of 30~50 DEG C, is put into the acetone soln of 90~110ml 60~80min of leaching, finally squeezes out 50~70min in the ether of 90~110ml of immersion, is dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 50~60min at 96~98 DEG C, 4~6 times repeatedly, at 60~80 DEG C Dried in baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, at 50~60 DEG C be incubated 1~ 2h；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 8~10, using dialysis and ultrafiltration Share carries out desalting processing to sample, is then concentrated into the 1/3~2/3 of original volume using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the two sulphur threoses that mass fraction is 1.5~2% In 15~20min is kept at 40~50 DEG C, it is 2.2~2.8% to be cooled to room temperature and add mass fraction to 2~3ml of alcoholic solution 4~5ml of iodoacetamide solution, adds the chymotrypsin protein enzyme solutions of 50~80ul right after 15~20min is kept under dark surrounds Diluted with formic acid solution afterwards；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid of tri- groups of experiments of B, C Sequence library is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

A kind of method for differentiating Ancient Silk Textile silk species, it is characterised in that as follows using step：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 5g of Ancient Silk Textile that chestnut silk makes is divided into Tri- groups of A, B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.5%₂CO₃Boiling is boiled 30min and is entered in the aqueous solution Row degumming, 5 times repeatedly, then separates three groups of sample solutions with fibroin respectively, after fibroin deionized water rinsing is squeezed out, then The hydrochloric acid solution that 65ml mass fractions are 1% is taken, 55min is soaked at room temperature, solution is washed till in neutrality with warm water, at 70 DEG C Dried in baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 60 DEG C, is put into 100ml Mass fraction to soak 110min in 70% methanol solution, then at a temperature of 60 DEG C, be put into 100ml absolute ethyl alcohols and soak 70min, Then at a temperature of 40 DEG C, it is put into the acetone soln of 100ml and soaks 70min, finally squeezes out 60min in the ether of immersion 100ml, Dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 55min at 97 DEG C, 5 times repeatedly, is dried in 70 DEG C of baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, it is incubated 1.5h at 55 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 9, shared using dialysis and ultrafiltration Desalting processing is carried out to sample, then the 1/2 of original volume is concentrated into using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the dithiothreitol (DTT) that mass fraction is 1.7% Solution 2.5ml is cooled to the iodoacetamide solution that room temperature adds mass fraction to be 2.5% in 17min is kept at 45 DEG C 4.5ml, adds the chymotrypsin protein enzyme solutions of 65ul then to be diluted with formic acid solution after 17min is kept under dark surrounds；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid of tri- groups of experiments of B, C Sequence library is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

The beneficial effects of the invention are as follows：Ancient Silk Textile silk species is differentiated using proteomics method, is overcome general The detection limit of logical determination method so that the Ancient Silk Textile for even preserving out of order can also be distinguished identification； Ancient Silk Textile silk species is identified using proteomics method, uses the technology of digestion in solution to cause that experiment is grasped in detection Make simple and convenient；Ancient Silk Textile silk species is identified using proteomics method, sensitivity is high, and the degree of accuracy is high, and sample makes Consumption is few.

Specific embodiment

The present invention is described in detail below in conjunction with specific embodiment：

A kind of method for differentiating Ancient Silk Textile silk species, the method uses following steps：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 2~8g of Ancient Silk Textile that chestnut silk makes, It is divided into A, tri- groups of B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.4~0.6%₂CO₃Boiling boils 20 in the aqueous solution ~40min carries out degumming, 4~6 times repeatedly, then separates three groups of sample solutions with fibroin respectively, treats fibroin deionized water After flushing is squeezed out, then the hydrochloric acid solution that 50~80ml mass fractions are 1% is taken, 50~60min is soaked at room temperature, washed with warm water To solution in neutrality, dried in 60~80 DEG C of baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 50~70 DEG C, is put into The mass fraction of 90~110ml is 100~120min of leaching in 60~80% methanol solutions, then at a temperature of 50~70 DEG C, is put into 60~80min is soaked in 90~110ml absolute ethyl alcohols, then at a temperature of 30~50 DEG C, is put into the acetone soln of 90~110ml 60~80min of leaching, finally squeezes out 50~70min in the ether of 90~110ml of immersion, is dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 50~60min at 96~98 DEG C, 4~6 times repeatedly, at 60~80 DEG C Dried in baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, at 50~60 DEG C be incubated 1~ 2h；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 8~10, using dialysis and ultrafiltration Share carries out desalting processing to sample, is then concentrated into the 1/3~2/3 of original volume using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the two sulphur threoses that mass fraction is 1.5~2% In 15~20min is kept at 40~50 DEG C, it is 2.2~2.8% to be cooled to room temperature and add mass fraction to 2~3ml of alcoholic solution 4~5ml of iodoacetamide solution, adds the chymotrypsin protein enzyme solutions of 50~80ul right after 15~20min is kept under dark surrounds Diluted with formic acid solution afterwards；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid of tri- groups of experiments of B, C Sequence library (seeing appendix 1) is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

Embodiment 1：A kind of method for differentiating Ancient Silk Textile silk species, the method uses following steps：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 2g of Ancient Silk Textile that chestnut silk makes is divided into Tri- groups of A, B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.4%₂CO₃Boiling is boiled 20min and is entered in the aqueous solution Row degumming, 4 times repeatedly, then separates three groups of sample solutions with fibroin respectively, after fibroin deionized water rinsing is squeezed out, then The hydrochloric acid solution that 50ml mass fractions are 1% is taken, 50min is soaked at room temperature, solution is washed till in neutrality with warm water, at 60 DEG C Dried in baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 50 DEG C, is put into 90ml Mass fraction to soak 100min in 60% methanol solution, then at a temperature of 50 DEG C, be put into 90ml absolute ethyl alcohols and soak 60min, Then at a temperature of 30 DEG C, it is put into the acetone soln of 90ml and soaks 60min, finally squeeze out 50min in the ether of immersion 90ml, makes Dried up with hair-dryer；

D) fibroin obtained by step c) is boiled into 50min at 96 DEG C, 4 times repeatedly, is dried in 60 DEG C of baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, it is incubated 1h at 50 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 8, shared using dialysis and ultrafiltration Desalting processing is carried out to sample, then the 1/3 of original volume is concentrated into using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the dithiothreitol (DTT) that mass fraction is 1.5% Solution 2ml is cooled to the iodoacetamide solution 4ml that room temperature adds mass fraction to be 2.2% in 15min is kept at 40 DEG C, black The chymotrypsin protein enzyme solutions of 50ul are added then to be diluted with formic acid solution after 15min is kept under dark situation；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and database of tri- groups of experiments of B, C Contrasted, be that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

Embodiment 2:A kind of method for differentiating Ancient Silk Textile silk species, the method uses following steps：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 5g of Ancient Silk Textile that chestnut silk makes is divided into Tri- groups of A, B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.5%₂CO₃Boiling is boiled 30min and is entered in the aqueous solution Row degumming, 5 times repeatedly, then separates three groups of sample solutions with fibroin respectively, after fibroin deionized water rinsing is squeezed out, then The hydrochloric acid solution that 65ml mass fractions are 1% is taken, 55min is soaked at room temperature, solution is washed till in neutrality with warm water, at 70 DEG C Dried in baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 60 DEG C, is put into 100ml Mass fraction to soak 110min in 70% methanol solution, then at a temperature of 60 DEG C, be put into 100ml absolute ethyl alcohols and soak 70min, Then at a temperature of 40 DEG C, it is put into the acetone soln of 100ml and soaks 70min, finally squeezes out 60min in the ether of immersion 100ml, Dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 55min at 97 DEG C, 5 times repeatedly, is dried in 70 DEG C of baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, it is incubated 1.5h at 55 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 9, shared using dialysis and ultrafiltration Desalting processing is carried out to sample, then the 1/2 of original volume is concentrated into using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the dithiothreitol (DTT) that mass fraction is 1.7% Solution 2.5ml is cooled to the iodoacetamide solution that room temperature adds mass fraction to be 2.5% in 17min is kept at 45 DEG C 4.5ml, adds the chymotrypsin protein enzyme solutions of 65ul then to be diluted with formic acid solution after 17min is kept under dark surrounds；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid of tri- groups of experiments of B, C Sequence library is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

Embodiment 3:A kind of method for differentiating Ancient Silk Textile silk species, the method uses following steps：

A) take respectively it is a be confused by mulberry silk, tussah silk, each 8g of Ancient Silk Textile that chestnut silk makes is divided into Tri- groups of A, B, C；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.6%₂CO₃Boiling is boiled 40min and is entered in the aqueous solution Row degumming, 6 times repeatedly, then separates three groups of sample solutions with fibroin respectively, after fibroin deionized water rinsing is squeezed out, then The hydrochloric acid solution that 80ml mass fractions are 1% is taken, 60min is soaked at room temperature, solution is washed till in neutrality with warm water, at 80 DEG C Dried in baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 70 DEG C, is put into 110ml Mass fraction to soak 120min in 80% methanol solution, then at a temperature of 70 DEG C, be put into 110ml absolute ethyl alcohols and soak 80min, Then at a temperature of 50 DEG C, it is put into the acetone soln of 110ml and soaks 80min, finally squeezes out 70min in the ether of immersion 110ml, Dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 60min at 98 DEG C, 6 times repeatedly, is dried in 80 DEG C of baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, will be processed by step d) Three groups of fibroin samples afterwards are respectively put into the cupri-ethylene diamine solution for preparing, and bath raio is 1:50, it is incubated 2h at 60 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 10, shared using dialysis and ultrafiltration Desalting processing is carried out to sample, then the 2/3 of original volume is concentrated into using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, and the dithiothreitol (DTT) for adding mass fraction to be 2% is molten Liquid 3ml is cooled to the iodoacetamide solution 5ml that room temperature adds mass fraction to be 2.8% in 20min is kept at 50 DEG C, dark The chymotrypsin protein enzyme solutions of 80ul are added then to be diluted with formic acid solution after 20min is kept under environment；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid of tri- groups of experiments of B, C Sequence library is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

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.

Annex one

Silkworm：Bombyx mori (5263aa)

1 mrvktfvilc calqyvaytn anindfdedy fgsdvtvqss nttdeiirda sgavieeqit

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

Tussah： Antheraea pernyi (2639aa)

1 mrviafvilc calqyatakn lrhhdeyvdn hgqlverftt rkhfernaat rphlsgnerl

61 vetivleedp yghediyeed vvikrvpgas ssaaaassas agsgqtiive rqashgagga

121 agaaagaaag ssarrgggfy ethnsyssyg sgsssaaags gaggvgggyg sdsaaaaaaa

181 aaaasgaggs ggyggygsds aaaaaaaaaa aaagsgaggs ggyggyggyg sdsaaaaaaa

241 aaaaaagssa ggagggygwg dggygsdsaa aaaaaaaaaa agsgaggsgg yggygsdsaa

301 aaaaaaaaaa agssaggagg gygwgdggyg sdsaaaaaaa aaaaassgag grgdggygsg

361 gssaaaaaaa aaaaarragh draagsaaaa aaaaaaaaas gaggsgggyg wgdggygsds

421 aaaaaaaaaa aaagsgagga gggygwgdsg ygsdsaaaaa aaaaaaaasg aggsggyggy

481 gsdsaaaaaa aaaaaaagag aggaggsygw gdggygsdsa aaaaaaaaaa agsgaggrgd

541 ggygsgssaa aaaaaaaasa arraghdsaa gsaaaaaaaa aaaaasgagg sgggygwgdg

601 gygsdsaaaa aaaaaaaaag sgaggagggy gwgdggygsd saaaaaaaaa aaaasgargs

661 ggyggygsds aaaaaaaaaa aaagsgaggv gggygwgdgg ygsdsaaaaa aaaaaaagsg

721 aggrgdggyg sgssaaaaaa aaaasaarra ghdsaagsaa aaaaaaaaaa asgaggsggg

781 ygwgdggygs dsaaaaaaaa aaaaagsgag gagggygwgd ggygsdsaaa aaaaaaaaaa

841 sgargsggyg gygsdsaaaa aaaaaaaaag sgaggvgggy gwgdggygsd saaaaaaaaa

901 aaagsgaggr gdggygsgss aaaaaaaaaa saarraghds aagsaaaaaa aaaaaaasga

961 ggsgggygwg dggygsdsaa aaaaaaaaaa agsgaggagg gygwgdggyg sdsaaaaaaa

1021 aaaaaasgar gsggyggygs dsaaaaaaaa aaaaagsgag gvgggygwgd ggygsdsaaa

1081 aaaaaaaaag sgaggrgdgg ygsgssaaaa aaaaaaaarr aghdraagsa aaaaaaaaaa

1141 aasgaggsgg gygwgdggyg sdsaaaaaaa aaaaaaasga ggsggyggyg sdsaaaaaaa

1201 aaaaaagsga ggagggygwg dggygsdsaa aaaaaaaaaa asgaggsggy ggyggygsds

1261 aaaaaaaaaa aaagsgagga gggygwgdgg ygsdsaaaaa aaaaaaagsg aggrgdggyg

1321 sgssaaaaaa aaaaaaarra ghdraagsaa aaaaaaaaaa asgaggsggg ygwgdggygs

1381 dsaaaaaaaa aaaaaasgag gsggyggygs dsaaaaaaaa aaaaagsgag gvgggygwgd

1441 ggygsdsaaa aaaaaaaaaa sgaggsggyg gygsdsaaaa aaaaaaaaaa sgaggaggyg

1501 gygsdsaaaa aaaaaaaaag sgaggagggy gwgdggygsy saaaaaaaaa aaagsgaggr

1561 gdggygsgss aaaaaaaaaa aarraghdra agsaaaaaaa aaaaaasgag gagggygwgd

1621 ggyssdsaaa aaaaaaaaaa gsgaggaggg ygwgddgygs dsaaaaaaaa aaaagsgagg

1681 rgggygwgdg gygsdsaaaa aaaaaaaags gaggrgdggy gsgssaaaaa aaaaaaarra

1741 ghdraagsaa aaaaaaaaaa sgaggsggsy gwgdggygsd saaaaaaaaa aaaasgaggs

1801 ggyggyggyg gygsdsaaaa aaaaaaaaaa agsgaggvgg gygwgdggyg sdsaaaaaaa

1861 aaaaagsgag grgdggygsg ssaaaaaaaa aaaarraghd raagsaaaaa aaaaaaaasg

1921 aggagggygw gdggygsdsa aaaaaaaaaa aagsgaggag ggygwgddgy gsdsaaaaaa

1981 aaaaaagsga ggrgggygwg dggygsdsaa aaaaaaaaaa gsgaggrgdg gygsgssaaa

2041 aaaaaaaaaa rraghdraag saaaaaaaaa aaaasgaggs ggyggyggyg sdsaaaaaaa

2101 aaaaaagsga ggaggyggyg gygsdsaaaa aaaaaaaaag sgaggvgggy gwgdggygsd

2161 saaaaaaaaa aaaagsgagg rgdggygsgs saaaaaaaaa aaarragher aagsaaaaaa

2221 aaaaaasgag rsggsygwgd ggygsdsaaa aaaaaaaaaa sgaggsggyg gyggygsdsa

2281 aaaaaaaaaa asgaggaggy ggyggygsyg sdsaaaaaaa aaaagsgagg vgggygwgdg

2341 gygsdsaaaa aaaaaaaags gaggrrgyga ygsdssaaaa aaaaaasgag gsgggygwgd

2401 ggygsdsaaa aaaaaaaaaa agsgaggigg gfgrgdggyg sgssaaaaaa aaaaaarrag

2461 hgrsagsaaa aaaaaaaaaa sgaggsggsy gwdyesygsg saaaaagsga ggsgggygwg

2521 dggygsgssa aaaaaaaaaa gsrrsghdra ygagsaaaaa aaaaagagas rqvgiygtdd

2581 gfvldggyds egsaaaaaaa aaaaasssgr steghpllsi ccrpcshshs yeasrisvh

Chestnut silkworm：Saturnia japonica (partial, 991aa)

1 mrvafvivcc vlqyasaktl nhiglhpkgn trideyvdkt gqlyeryttr eefernaatl

61 phlsrnerll etiiieedps ghekiykedv vlkrvpgass aasassaaaa gsgsrrlgrl

121 hgwdggaygs dsaaaaaaaa aassgaeglg glyglhsgvy gsdsaaaaaa aaaaasgssr

181 vgferahrad taaaasasaa aaatsdsdsr sysseesvld ihrssssrrd sssasavaaa

241 aaaaaaaaaa aessehvyes esaaaaaaaa aaaagsgagg lgglyglhgg yygsdsaaaa

301 aaaaaaaags gagglgglyg lhggvygsds aaaaaaaaaa tagsgaeglg glyglhggyy

361 gsdsgaaaaa aaaaaagsga gglgghyglh ggvygsdsaa aaaaaaaaaa gsgagglggl

421 yglhggvygs sssrrdsssa ssaaaaaaaa agssaasser vyesesaaaa aaaaaaaags

481 ssrrvgghyg wdggvygsds aaaaaaaaaa agsgsrglgg lygwdggvyg sdsaaaaaaa

541 aaaaagsgvs glgglygwdg gvygsdsaaa aaaaaaaaag sgsrglggly gwnggvygsd

601 saaaaaaaaa aaagsgvsgl gglygwdrgv ygsdsaaaaa aaaaaaagsg srglgglygw

661 nggvygsdsa aaaaaaaaaa agsgvsglgg lygwdgglyg sdsaaaaaaa aaaaagsgvs

721 glgglygwdg glygsdsaaa aaaaaaaaag sgagglggly gyhggvygsd saaaaaaaaa

781 aaagsgisgl gglygwdggl ygsdsaaaaa aaaaaaagsg srglgglygw dggvygsdsa

841 aaaaaaaaaa agsgsrglgg lygwdggvyg sdsaaaaaaa aaaaagsgvs glgglygwdg

901 glygsdssaa aaaaaaaaag sgsrrlggly gwdggvygsd saaaaaaaaa aaagsgvggl

961 gglygwdggv ygsdsaaaaa aaaaaaagsg v

Claims (2)

1. it is a kind of differentiate Ancient Silk Textile silk species method, it is characterised in that using step it is as follows：

A) take respectively be confused by mulberry silk, tussah silk, each 2~8g of Ancient Silk Textile that chestnut silk makes is divided into A, B, C Three groups；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.4~0.6%₂CO₃In the aqueous solution boiling boil 20~ 40min carries out degumming, 4~6 times repeatedly, then separates three groups of sample solutions with fibroin respectively, treats that fibroin is rushed with deionized water Wash after squeezing out, then take the hydrochloric acid solution that 50~80ml mass fractions are 1%, 50~60min is soaked at room temperature, be washed till with warm water Solution is dried in neutrality in 60~80 DEG C of baking oven；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 50~70 DEG C, it is put into 90~ The mass fraction of 110ml is to soak 100~120min in 60~80% methanol solutions, then at a temperature of 50~70 DEG C, it is put into 90~ 60~80min is soaked in 110ml absolute ethyl alcohols, then at a temperature of 30~50 DEG C, is put into the acetone soln of 90~110ml and is soaked 60 ~80min, finally squeezes out 50~70min in the ether of 90~110ml of immersion, is dried up using hair-dryer；

D) fibroin obtained by step c) is boiled into 50~60min at 96~98 DEG C, 4~6 times repeatedly, in 60~80 DEG C of baking oven Middle drying；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, after being processed by step d) Three groups of fibroin samples be respectively put into the cupri-ethylene diamine solution for preparing, bath raio is 1:50,1~2h is incubated at 50~60 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 8~10, shared using dialysis and ultrafiltration Desalting processing is carried out to sample, then the 1/3~2/3 of original volume is concentrated into using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, and the dithiothreitol (DTT) for adding mass fraction to be 1.5~2% is molten 2~3ml of liquid is cooled to the iodine second that room temperature adds mass fraction to be 2.2~2.8% in 15~20min is kept at 40~50 DEG C 4~5ml of amide solution, adds the chymotrypsin protein enzyme solutions of 50~80ul then to use after 15~20min is kept under dark surrounds Formic acid solution dilutes；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid sequence of tri- groups of experiments of B, C Database is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.

2. it is a kind of differentiate Ancient Silk Textile silk species method, it is characterised in that using step it is as follows：

A) take respectively be confused by mulberry silk, tussah silk, each 5g of Ancient Silk Textile that chestnut silk makes is divided into A, B, C tri- Group；

B) three groups of samples are with 1:100 bath raio is in the Na that mass fraction is 0.5%₂CO₃Boiling is boiled 30min and is taken off in the aqueous solution Glue, 5 times repeatedly, then separates three groups of sample solutions with fibroin respectively, after fibroin deionized water rinsing is squeezed out, then takes 65ml mass fractions are 1% hydrochloric acid solution, and 55min is soaked at room temperature, solution are washed till in neutrality with warm water, in 70 DEG C of baking Dried in case；

C) fibroin that will be dried by step b) desalinations continues to decolourize and de-oiling wax, first at a temperature of 60 DEG C, is put into the matter of 100ml Amount fraction is to soak 110min in 70% methanol solution, then at a temperature of 60 DEG C, is put into 100ml absolute ethyl alcohols and soaks 70min, then At a temperature of 40 DEG C, it is put into the acetone soln of 100ml and soaks 70min, finally squeeze out 60min in the ether of immersion 100ml, uses Hair-dryer is dried up；

D) fibroin obtained by step c) is boiled into 55min at 97 DEG C, 5 times repeatedly, is dried in 70 DEG C of baking oven；

E) cupri-ethylene diamine solution of 300ml is prepared by 19.8g Kocide SDs and 25.5ml ethylenediamines, after being processed by step d) Three groups of fibroin samples be respectively put into the cupri-ethylene diamine solution for preparing, bath raio is 1:50, it is incubated 1.5h at 55 DEG C；

F) after cooling use quality fraction be 10% vinegar acid for adjusting pH value to 9, shared to sample using dialysis and ultrafiltration Product carry out desalting processing, are then concentrated into the 1/2 of original volume using Macrogol 6000；

G) solution after a three groups of sample concentrations is taken respectively, adds the dithiothreitol (DTT) solution that mass fraction is 1.7% 2.5ml is cooled to the iodoacetamide solution 4.5ml that room temperature adds mass fraction to be 2.5% in 17min is kept at 45 DEG C, black The chymotrypsin protein enzyme solutions of 65ul are added then to be diluted with formic acid solution after 17min is kept under dark situation；

H) mass spectral analysis is carried out to it using HPLC-MS/MS, by A, the amino acid sequence and amino acid sequence of tri- groups of experiments of B, C Database is contrasted, and is that can determine that A according to feature peptide fragment, tri- groups of species of silk goods silk of B, C.