CN107519223B - Ginseng extract containing endogenous polypeptide, preparation and determination thereof, medicine or pharmaceutical composition containing ginseng extract and application thereof - Google Patents

Ginseng extract containing endogenous polypeptide, preparation and determination thereof, medicine or pharmaceutical composition containing ginseng extract and application thereof Download PDF

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CN107519223B
CN107519223B CN201610442864.8A CN201610442864A CN107519223B CN 107519223 B CN107519223 B CN 107519223B CN 201610442864 A CN201610442864 A CN 201610442864A CN 107519223 B CN107519223 B CN 107519223B
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张晓哲
刘欣欣
程孟春
赵楠
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a preparation method of a ginseng extract containing endogenous polypeptide, a ginseng extract, a determination method of polypeptide in the ginseng extract, a medicine or a pharmaceutical composition containing the ginseng extract and application thereof. The preparation method comprises the steps of grinding the ginseng medicinal materials into powder, extracting the powder by using an organic solvent or formic acid, separating an extracting solution by using a column, and concentrating an eluent to obtain the ginseng extract taking endogenous ginseng polypeptide as a main component. The determination method of the ginseng extract is characterized in that a Nano LC-LTQ-Orbitrap technology is utilized for analysis, a Swiss-Prot database is searched through PEAKS, 308 endogenous polypeptides are totally identified from the ginseng extract, the amino acid sequences of the polypeptides are SEQ ID No.1-125 and SEQ ID No. 1-183, and the molecular weight range is 300-7000 Da. The content of Ginseng radix polypeptide in the extract is greater than 30% by UHPLC-Q-TOF detection and Progenetics QI software analysis. The invention also carries out cell experiments on the ginseng extract, and the experimental result shows that the extract has the function of promoting the proliferation of PC12 cells.

Description

Ginseng extract containing endogenous polypeptide, preparation and determination thereof, medicine or pharmaceutical composition containing ginseng extract and application thereof
Technical Field
The invention belongs to the field of natural medicines. In particular to a preparation method of a ginseng extract containing endogenous polypeptide, a determination method of the ginseng extract and polypeptide in the ginseng extract, a medicine or a pharmaceutical composition containing the ginseng extract and application thereof, wherein the polypeptide is derived from the ginseng extract and is endogenous polypeptide.
Background
With the rapid development of modern society and economy, the pressure born by people in learning and working is increasing day by day, and the health problem becomes a non-negligible one, which can cause anxiety, insomnia, depression and the like, and finally cause memory decline and work efficiency reduction. In addition, in a normal physiological state, a decline in learning and memory ability occurs with aging and aging of the body. Therefore, it is necessary to develop health products with neuroprotective effect or improving learning and memory function.
The Ginseng radix is dried root and rhizome of Panax ginseng C.A. Mey belonging to Araliaceae family. Tranquilization and intelligence development, recovering pulse and relieving depletion, tonifying spleen and lung, promoting the production of body fluid and quenching thirst, and has a long medicinal history in China. In China, ginseng is mainly produced in the three provinces of northeast China, the yield of the first generation of Changbai mountain and Tunghua of Jilin is the highest, and the main components of the ginseng comprise saponin, polypeptide, polysaccharide, volatile oil and the like. Studies show that ginseng has the functions of exciting and inhibiting central nervous system, improving learning and memory, enhancing stress capacity of organism, improving immunity of organism, etc. (Leodan et al, ginseng research, 2013(01): 46-52; Cao et al, ginseng research, 2012(02): 39-43.). In recent years, with the development of molecular biology, biochemistry and other disciplines, research on bioactive peptides is becoming more extensive, and various animal or plant polypeptides with different physiological activities are extracted or developed into related products. The research of ginseng protein polypeptide starts in the 60 th 20 th century, but is limited by means of separation technology, analysis, sequencing and the like, the quantity of polypeptide for separating and determining accurate amino acid sequence from ginseng is quite limited, and in preparation aspect, most researches obtain enzymolysis products of ginseng protein through means of enzymolysis and the like, so that the method has profound scientific value and good application prospect by adopting advanced mass spectrometry technology and proteomics mass spectrometry data analysis software to analyze endogenous polypeptide in ginseng and prepare ginseng polypeptide-rich parts from ginseng.
The PC12 cell is a catecholamine cell differentiated from rat adrenal medulla pheochromocytoma, can synthesize, store and release a proper amount of catecholamine (mainly dopamine and norepinephrine), has typical nerve cell characteristics, is a cell model commonly used for in vitro research of neurological diseases, and screens traditional Chinese medicine active ingredients with neuroprotective effect.
Disclosure of Invention
The invention provides a preparation method of a ginseng extract containing polypeptide, a ginseng extract, a determination method of polypeptide in the ginseng extract, a medicine or a pharmaceutical composition containing the ginseng extract and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preparing Ginseng radix extract containing polypeptide,
grinding Ginseng radix into powder, extracting with organic solvent or formic acid, separating the extractive solution with column, and concentrating the eluate under reduced pressure to obtain Ginseng radix extract containing endogenous Ginseng radix polypeptide as main ingredient;
the method comprises the following specific steps:
1) solvent extraction
Grinding Ginseng radix, soaking in organic solvent or formic acid for 0.5-1.5 hr, ultrasonic extracting for 30-60 min, centrifuging, and collecting supernatant;
2) column separation
Separating the supernatant by a column, eluting with an organic solvent with the volume fraction of 0-20% to remove impurities, eluting with an eluent with the volume fraction of 20-70%, collecting the eluent with the volume fraction of 20-70%, concentrating and drying to obtain the ginseng extract.
The organic solvent in the step 1) is one or more than two of methanol, ethanol, acetonitrile or acetone with the volume fraction of 40-60%, the balance is water, the volume fraction of the formic acid is 70-90%, the balance is water, and the mass volume ratio of the ginseng to the organic solvent or the formic acid is 1g:10-20 mL; the volume of the concentrated and dried mixture is 5 to 15 percent of the original volume.
Step 2), separating the column by adopting one of a reversed phase C18 column, an MCI column or a macroporous adsorption resin column HPD 100; the organic solvent for eluting and impurity removing in the step 2) is selected from one of acetonitrile, methanol or ethanol with the volume fraction of 0-20% (when the organic solvent is 0%, water is directly used for eluting and impurity removing); the eluent is selected from one of acetonitrile, methanol or ethanol with the volume fraction of 20-70%; the elution volume is 2-3 times of the column volume.
The operating conditions for the separation of the C18 column in the step 2) are as follows: concentrating the supernatant after impurity removal, passing through a C18 column, eluting with 2 times of 10%, 30%, 40%, 50%, 70% and 90% acetonitrile in terms of volume fraction, at a flow rate of 0.5mL/min, collecting 40% acetonitrile eluate, concentrating, and drying to obtain Ginseng radix extract;
the operation conditions for the separation of the HPD100 resin column in the step 2) are as follows: concentrating the supernatant after impurity removal, passing through an HPD100 resin column, eluting with 2 times of 10%, 30%, 50%, 70% and 90% ethanol in volume fraction at a flow rate of 0.5mL/min, collecting 50% ethanol eluate, concentrating, and drying to obtain Ginseng radix extract;
the operating conditions for the MCI column separation in step 2) are as follows: concentrating the supernatant after removing impurities, passing through MCI (CHP20/P120) column, eluting with 10 times of column volume of water to remove acid, eluting with 2 times of column volume of 10%, 30%, 40%, 50%, 70%, 90% and 100% methanol at flow rate of 0.5mL/min, collecting 40% methanol eluate, concentrating, and drying to obtain Ginseng radix extract.
The ginseng extract obtained by the preparation method contains ginseng endogenous polypeptide, and the amino acid sequence of the polypeptide comprises SEQ ID No.1-125 and SEQ ID No. 1-183.
The content of polypeptide components in the ginseng extract is more than 30% and the molecular weight range is 300-7000 Da through UHPLC-Q-TOF determination.
The method utilizes the Nano LC-LTQ-Orbitrap technology for analysis, utilizes Progenetics QI software to determine the polypeptide components and content in the ginseng extract, further performs secondary mass spectrometry, searches a Swiss-Prot database through PEAKS, identifies 308 endogenous polypeptides from the ginseng extract, and analyzes and sequences the ginseng polypeptides in two modes of PEAKS DB and PEAKS PTM.
The specific operation is as follows:
1) and (3) analysis: ginseng extract obtained in claim 1 was dissolved with 0.1% formic acid, analyzed by Nano LC-LTQ-Orbitrap instrument, 8 μ L of sample was injected into a trap column (150 μm ID, 3cm, ReproSil-Pur C18 AQ) by a syringe pump, eluted with an aqueous solution containing 0.2% formic acid and 2% acetonitrile for 3 minutes and then switched to an analysis column (75 μm ID, ReproSil-Pur C18 AQ) for analysis, mobile phase a: 0.2% formic acid, mobile phase B: aqueous solution containing 0.2% formic acid and 95% acetonitrile, gradient elution procedure: 0-3.5 min, 5% B; 10% -20% of B in 3.5-6 min; 20% -50% of B in 6-35 minutes; 35-40 minutes, 50% -90% B; 40-45 minutes, 90% B;
2) polypeptide sequencing analysis: introducing original data into a proteomics mass spectrometry software Peaks Studio 7(BSI), respectively carrying out sequence analysis by using two modes of PEAKS DB and PEAKS PTM, wherein the filtering threshold value of the data is that the data quality value is more than 0.3, automatically sequencing the processed data from head, respectively setting the mass errors of parent ions and daughter ions to be 10ppm and 0.05Da, cracking without appointing enzyme participation, appointing common post-translational modifications such as C-terminal amidation, N-terminal acetylation, glutamic acid and glutamine cyclization (pyromalylation), and the like, searching and identifying the false positive rate (FDR) of less than 1% by using all proteins in Swiss-Prot as a database, and identifying 308 high-credibility polypeptides from near two thousand sequences obtained by searching by combining with the credibility (-10logP), the peptide chain fracture condition, the distribution condition of daughter ions and the error range one by one, the amino acid sequence of the polypeptide is SEQ ID No.1-125 and SEQ ID NO. 1-183.
A medicine containing the Ginseng radix extract as active ingredient or a pharmaceutical composition containing Ginseng radix extract as active ingredient and other pharmaceutically acceptable carriers and/or excipients is provided.
The application of the medicine or the medicine composition in the neuroprotection and/or the improvement of the learning and memory function and/or the cell proliferation promotion of PC12 measured by UHPLC-Q-TOF.
The solvent of the solution used in the invention is water except the solute, and the contents are volume fractions.
Compared with the literature, the ginseng extract prepared by the invention has the following advantages and progresses:
relevant literature is investigated, and there are two main methods for preparing ginseng polypeptide: one is an enzymatic hydrolysis method, which comprises extracting radix Ginseng with water, and hydrolyzing with enzyme (Chinese patent 2014103022318; Chinese patent 2015106273310); the other method is membrane separation, column separation and other means, such as extraction with Tris-HCl buffer solution, precipitation with ammonium sulfate, dialysis, hollow fiber ultrafiltration column and column separation and other steps (Tengtze et al, proceedings of Changchun university, 2005(03): 181-. The method has the advantages that the operation of the former is simple, but the ginseng component is complex, the main component of the ginseng is ginsenoside, the content of polypeptide is limited, the purity or the activity of the product cannot be guaranteed by the method, the prepared ginseng protein is an enzymolysis product of ginseng protein rather than endogenous polypeptide, and the endogenous polypeptide with potential activity is decomposed and destroyed in the enzymolysis process; the latter has the disadvantages of complicated steps, high extraction and separation cost, high extraction difficulty, no contribution to industrial production, suitability for being used as a basic research means, and limited quantity of extracted polypeptide. The present invention overcomes the above disadvantages.
1) Compared with the first method, the method provided by the invention has the advantages that the effective components are clear and controllable, the main component is endogenous ginseng polypeptide, the content is more than 30%, and the molecular weight range of the polypeptide is 300-7000 Da; the physiological activity is obvious, cell experiments show that the extract has the effect of promoting proliferation of PC12 cells, and the structure and the activity of polypeptide can be ensured in the extraction process;
2) compared with the second method, the invention provides a method which is simple to operate and does not use equipment with higher cost, thereby meeting the control requirement of industrial batch production on cost, promoting the smooth conversion from an experimental process to a production process, and in addition, the prepared extract polypeptide has more quantity and high content;
3) the invention is a ginseng extract, the main component is endogenous ginseng polypeptide, compared with the ginseng crude extract health care products, the ginseng crude extract health care products have definite and controllable components, the dosage is reduced, and the ginseng crude extract health care products are more beneficial to patients to accept; compared with ginsenoside medicines or health products, the final product of polypeptide metabolism in vivo is amino acid, so that the safety of medication is ensured, and the health products can be developed for long-term administration.
4) The polypeptides contained in the ginseng extract prepared by the invention are identified and sequenced to obtain 308 polypeptide sequences.
Description of the attached tables
The attached table 1 shows the influence of the action of the ginseng extract for 72 hours on the survival rate of PC12 cells; attached table 2 shows the content of endogenous polypeptide in the ginseng extract of preparation example 1 of the ginseng extract measured by UHPLC-Q-TOF detection and prognesis QI software analysis; accompanying tables 3 and 4 are information on 308 endogenous polypeptide sequences identified from ginseng using the PEAKS software for de novo sequencing.
Detailed Description
The present invention will now be described in further detail with reference to examples, which are given for illustration of the present invention and are not intended to limit the present invention.
Preparation example of Ginseng extract
Example 1
1) Solvent extraction: soaking Ginseng radix powder in 40% methanol for 1 hr, ultrasonic extracting for 30 min, centrifuging, and collecting supernatant;
2) c18 column separation: concentrating the supernatant under reduced pressure to 5% of the original volume, separating with C18 column, eluting with 2 times of 10% (v/v), 30% (v/v), 40% (v/v), 50% (v/v), 70% (v/v), and 90% (v/v) acetonitrile at flow rate of 0.5mL/min, collecting 40% acetonitrile eluate, concentrating, and drying to obtain Ginseng radix extract;
3) content determination: detecting the content of endogenous polypeptide in the extract by UHPLC-Q-TOF, and performing chromatographic column: agilent Prorpshell 120 SB C182.7 μm 3.0 × 150mm, mobile phase a: 0.5% formic acid, mobile phase B: acetonitrile, gradient elution: 0-15min, 5% -90% B, ion source: ESI source, detection mode: a positive ion; carrying out peak identification, peak filtration and peak alignment on UHPLC-Q-TOF data by adopting Progenetics QI software, and filtering strength (Filter strength): 0.3, bar graph/Resolution (Centroided date/Resolution): 60000, Absolute ion intensity/minimum intensity (Absolute ion intensity/minimum intensity): 1000, multiple charges (the Charge number is more than or equal to 2) are typical physical and chemical characteristics of the polypeptide, interfering ions are eliminated through parameter optimization and Mass Hunter analysis, information such as Mass-to-Charge ratio (m/z), Charge number (Charge), retention time (retention time) and abundance of the extract ions is finally obtained, the contents of polypeptide components and non-polypeptide components can be obtained through calculating the abundance and percentage of the multiple charges and the single charges, and the result shows that the endogenous polypeptide content in the extract is 32%, and the result is shown in the attached table 2.
Example 2
1) Solvent extraction: soaking the ginseng powder in 60% ethanol with the volume 10 times that of the ginseng powder for 1 hour, performing ultrasonic extraction for 30 minutes, centrifuging and collecting supernate;
2) HPD100 resin column separation: concentrating the supernatant under reduced pressure to 5% of the original volume, separating with HPD100 resin column, eluting with 2 times of 10% (v/v), 30% (v/v), 50% (v/v), 70% (v/v) and 90% (v/v) ethanol at flow rate of 0.5mL/min, collecting 50% ethanol eluate, concentrating, and drying to obtain Ginseng radix extract;
the content of endogenous polypeptide is 30% by UHPLC-Q-TOF detection.
Example 3
1) Solvent extraction: soaking the ginseng powder in 10 times of 80% formic acid by volume for 1 hour, performing ultrasonic extraction for 30 minutes in an ice bath, centrifuging and collecting supernate;
2) and (3) MCI column separation: concentrating the supernatant under reduced pressure to 5% of the original volume, separating with MCI (CHP20/P120) column, eluting with 10 times of column volume of water to remove acid, eluting with 2 times of column volume of 10% (v/v), 30% (v/v), 40% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) and 100% (v/v) methanol at flow rate of 0.5mL/min, collecting 40% methanol eluate, concentrating, and drying to obtain Ginseng radix extract;
the content of endogenous polypeptide detected by UHPLC-Q-TOF is 33%.
Example for the identification of endogenous Polypeptides of Ginseng
Example 1
1) And (3) analysis: "Ginseng extract preparation example 1" the obtained ginseng extract was dissolved with 0.1% (v/v) formic acid, analyzed by a Nano LC-LTQ-Orbitrap apparatus, and 8. mu.L of the sample was injected by a syringe pump into a trap column (150. mu.m ID, 3cm, Repuril-Pur C18 AQ), eluted with 2% acetonitrile (containing 0.2% formic acid) for 3 minutes, and then switched to an analysis column (75. mu.m ID, Repuril-Pur C18 AQ) for analysis, all in terms of volume fraction, mobile phase A: 0.2% formic acid, mobile phase B in volume fraction: 95% acetonitrile with 0.2% formic acid, gradient elution procedure: 0-3.5 min, 5% B; 10% -20% of B in 3.5-6 min; 20% -50% of B in 6-35 minutes; 35-40 minutes, 50% -90% B; 40-45 minutes, 90% B;
2) polypeptide sequencing analysis: the original data are imported into a protein group mass spectrum software Peaks Studio 7(BSI), sequence analysis is carried out by using two modes of PEAKS DB and PEAKS PTM respectively, and the filtering threshold value of the data is more than 0.3. The processed data were automatically sequenced from the head, the mass errors of the parent and daughter ions were set to 10ppm and 0.05Da, respectively, and no enzyme was assigned for cleavage. Common post-translational modifications are designated C-terminal amidation, N-terminal acetylation, and cyclization of glutamate and glutamine (pyro-glutamylation). Data retrieval was performed using all proteins in Swiss-Prot as a database, and the false positive rate (FDR) of the search identification was < 1%. And manually analyzing and confirming the searched peptide sequences one by one according to the reliability (-10logP), the peptide chain breakage condition, the distribution condition of the daughter ions and the error range. Finally, 308 highly reliable polypeptides were identified from the nearly two thousand sequences searched (see attached Table 3 and attached Table 4).
Effect of the pharmacodynamic examples on the proliferative Effect of PC12 cells
Example 1
Experimental samples Ginseng radix extracts obtained by concentrating 10% (v/v), 30% (v/v), 50% (v/v) and 70% (v/v) ethanol eluents prepared in example 2 were used.
The cells used in the experiment were PC12, rat adrenal pheochromocytoma cells, cultured in DMEM/F12(Sigma) containing 10% fetal bovine serum (ilex hangzhou), and digested with 0.05% trypsin (Solarbio). Cells were incubated at 37 ℃ with 5% CO2And culturing in an incubator with saturated humidity.
Reagents used for the experiment: MTT, sigma product. DMSO, tianjin komi.
The apparatus used for the experiment: carbon dioxide incubator (HEAL FORCE), clean bench (HEAL FORCE), microplate reader (sunrise, TECAN), inverted microscope (Olymbus).
The experimental method comprises the following steps:
the MTT method is used for detecting the influence of the ginseng extract on the survival rate of PC12 cells: PC12 cells were seeded into 96-well plates at 3200 cells per well, 100 μ L per well, and cultured overnight. Ginseng radix extract was prepared to the desired concentration (4 concentrations, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, respectively) in culture medium, 100. mu.L of culture medium was added to each well, and 3 parallel wells were set for the negative control wells. After culturing for 72h, adding 20 mu L of MTT (5mg/mL) into each well, after reacting for 4h, carefully throwing away the culture solution, adding 200 mu L of DMSO into each well, shaking for 10min for full dissolution, detecting the absorbance value at the wavelength of 570nm by using an enzyme-linked immunosorbent assay, calculating the cell survival rate, and calculating the cell survival rate formula: survival (PR%) (experimental-a blank)/(negative a-a blank) x 100%.
The results show that: the cell survival rate of the negative control group (the concentration is 0 point) is 100 percent, and the cell survival rate of each concentration of the drug-adding group is calculated. Therefore, when the concentration of the sample is 500 mug/mL, 30% of parts have the function of promoting the proliferation of the PC12 cells and have statistical significance; when the concentration of the sample is 250 mu g/mL, 50 percent and 70 percent of the sites have the function of promoting the proliferation of the PC12 cells and have statistical significance. The results suggest that: the Ginseng radix extract can promote cell proliferation of PC 12. See attached table 1.
TABLE 1 pharmacodynamics Effect of Ginseng radix extract in example 1 on survival of PC12 cells for 72h
Figure BDA0001022813850000051
Figure BDA0001022813850000052
P <0.05, compared to negative control group.
TABLE 2 accompanying Table 2 content of endogenous polypeptide of Ginseng radix extract in example 2, expressed as ion abundance and percentage (%)
Figure BDA0001022813850000053
Appendix 3 Table 3 sequence information of 125 endogenous polypeptides identified from Ginseng radix using PEAKS DB
Figure BDA0001022813850000054
Figure BDA0001022813850000061
Figure BDA0001022813850000071
Figure BDA0001022813850000081
Figure BDA0001022813850000091
aThe long peptide chain could not be completely cleaved, but there was clear MS/MS information.bPost-translational modification: poor quality-1.01, C is derived from disulfide bonds; poor quality-17.3, Q is derived from glutamic acid and glutamine residues; poor quality-18.01, E is derived from glutamic acid and glutamine residues; poor quality +42.01, N-terminal acetylation; poor mass-0.98, amidation.
Appendix 4 sequence information of 183 endogenous polypeptides identified from Ginseng by PEAKS PTM
Figure BDA0001022813850000092
Figure BDA0001022813850000101
Figure BDA0001022813850000111
Figure BDA0001022813850000121
Figure BDA0001022813850000131
Figure BDA0001022813850000141
aPost-translational modification: poor quality-1.01, C is derived from disulfide bonds; poor quality-17.3, Q is derived from glutamic acid and glutamine residues; poor quality-18.01, E is derived from glutamic acid and glutamine residues; poor quality +42.01, N-terminal acetylation; poor mass-0.98, amidation.
Sequence listing
<110> institute of chemistry and physics, large connection of Chinese academy of sciences
<120> ginseng extract containing endogenous polypeptide, preparation and determination thereof, medicine or pharmaceutical composition containing ginseng extract and application thereof
<140> 2016104428648
<160> 308
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Ser Ile Ile Gly Gly Asp Ile Leu Leu Asp Ile Ile Glu Ser Ile
1 5 10 15
Val Asn His Phe
20
<210> 2
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ser Ile Ile Gly Gly Asp Ile Leu Leu Asp Ile Ile Glu Ser Ile Val
1 5 10 15
Asn His Phe
<210> 3
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
His Leu Ala Trp Pro Ser His Glu Thr Asn Thr Glu Asp
1 5 10
<210> 4
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
His Leu Ala Trp Pro Ser His Glu Thr Asn Thr Glu
1 5 10
<210> 5
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys
35 40
<210> 6
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ile Ile Gly Gly Asp Ile Leu Leu Asp Ile Ile Glu Ser Ile Val Asn
1 5 10 15
His Phe
<210> 7
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Ala Ile Lys Lys His Leu Asn Ala Val Pro Glu Ile Asp Phe Thr
1 5 10 15
Lys Asn Glu Asn
20
<210> 8
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Val Gln Lys Thr Glu Val Glu Ala Thr Ser Thr Val Pro Ala Gln
1 5 10 15
Lys Leu
<210> 9
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Ala Ile Lys Lys His Leu Asn Ala Val Pro Glu Ile Asp Phe Thr Lys
1 5 10 15
Asn Glu Asn
<210> 10
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
His Leu Asn Ala Val Pro Glu Ile Asp Phe Thr Lys Asn Glu Asn
1 5 10 15
<210> 11
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10 15
<210> 12
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10 15
<210> 13
<211> 43
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 14
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
1 5 10
<210> 15
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Val Gln Lys Thr Glu Thr Gln Ala Ile Ser Pro Val Pro Ala Glu
1 5 10 15
Lys Leu Phe Lys Gly Ser Phe
20
<210> 16
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 17
<211> 43
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 18
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp
1 5 10 15
Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys
20 25
<210> 19
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Leu Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10 15
<210> 20
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys
35 40
<210> 21
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser
35 40
<210> 22
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys
35 40
<210> 23
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys
35 40
<210> 24
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10 15
Met Lys
<210> 25
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
His Leu Ala Trp Pro Ser His Glu Thr Asn Thr
1 5 10
<210> 26
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 27
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10 15
<210> 28
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
1 5 10
<210> 29
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
His Leu Ala Trp Pro Ser His Glu Thr Asn
1 5 10
<210> 30
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
1 5 10
<210> 31
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys
1 5 10 15
Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys
20 25
<210> 32
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10 15
Lys Met Lys
<210> 33
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys Pro
1 5 10 15
Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu
20 25
<210> 34
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
1 5 10
<210> 35
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Pro Glu Ile Asp Phe Thr Lys Asn Glu Asn
1 5 10
<210> 36
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 37
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Leu Ala Trp Pro Ser His Glu Thr Asn Thr Glu
1 5 10
<210> 38
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
1 5 10
<210> 39
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp
1 5 10 15
Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu
20 25
<210> 40
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Leu Arg Leu Gln Trp Pro Ala Gly Phe Cys Glu Val Asn Asn Ala Cys
1 5 10 15
Asp Thr Lys Ser Leu Leu Asn Thr
20
<210> 41
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys
1 5 10
<210> 42
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Ala Leu Arg Leu Gln Trp Pro Ala Gly Phe Cys Glu Val Asn Asn Ala
1 5 10 15
Cys Asp Thr Lys Ser Leu Leu Asn Thr Phe
20 25
<210> 43
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10
<210> 44
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe
1 5 10 15
Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys
20 25 30
<210> 45
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Phe Ala Leu Arg Leu Gln Trp Pro Ala Gly Phe
1 5 10
<210> 46
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Leu Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10 15
<210> 47
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Ala Val Pro Glu Ile Asp Phe Thr Lys Asn Glu Asn
1 5 10
<210> 48
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Phe Trp Glu His Glu Trp Lys Lys His
1 5
<210> 49
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Phe Val Arg Leu Pro Ser Lys Met Lys
1 5
<210> 50
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 51
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro
1 5 10
<210> 52
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10
<210> 53
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Phe Trp Glu His Glu Trp Lys Lys
1 5
<210> 54
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Ala Leu Arg Leu Gln Trp Pro Ala Gly Phe
1 5 10
<210> 55
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp
1 5 10 15
Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg
20 25 30
Leu Pro Ser Lys Met Lys
35
<210> 56
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 57
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
His Leu Ala Trp Pro Ser His Glu Thr
1 5
<210> 58
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Leu Gln Trp Pro Ala Gly Phe Cys Glu Val Asn Asn Ala Cys Asp Thr
1 5 10 15
Lys Ser
<210> 59
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys Pro Thr Asp
1 5 10 15
Asp Ala Thr Asp Asp Tyr Arg Leu
20
<210> 60
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser
35 40
<210> 61
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10 15
Lys
<210> 62
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Asp Tyr Arg Leu Lys Phe Val Arg
1 5
<210> 64
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10
<210> 65
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
His Leu Ala Trp Pro Ser His Glu
1 5
<210> 66
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Glu Ile Asp Phe Thr Lys Asn Glu Asn
1 5
<210> 67
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10
<210> 68
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Leu Arg Leu Gln Trp Pro Ala Gly Phe
1 5
<210> 69
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Leu Ala Trp Pro Ser His Glu Thr Asn
1 5
<210> 70
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Asn Ala Val Pro Glu Ile Asp Phe Thr Lys Asn Glu Asn
1 5 10
<210> 71
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe
1 5 10 15
Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
20 25 30
Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 72
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Leu Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 73
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys Met
1 5 10 15
Ile Lys
<210> 74
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 75
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 76
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp
1 5 10 15
Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg
20 25 30
Leu Pro Ser Lys Met Lys
35
<210> 77
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys Met
1 5 10 15
Lys
<210> 78
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro
1 5 10
<210> 79
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro
1 5 10
<210> 80
<211> 43
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 81
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys Pro
1 5 10 15
Thr Asp Asp Ala Thr Asp
20
<210> 82
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg
1 5 10
<210> 83
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Val Gln Val Leu Glu Gly Asn Gly Gly Val Gly Thr Ile Lys Asn
1 5 10 15
<210> 84
<211> 37
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys
1 5 10 15
Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu
20 25 30
Pro Ser Lys Met Lys
35
<210> 85
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe
1 5 10 15
Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
20 25 30
Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 86
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Ala Leu Arg Leu Gln Trp Pro Ala Gly Phe
1 5 10
<210> 87
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Leu Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro
1 5 10 15
<210> 88
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Asn Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10 15
<210> 89
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Pro Asn Asn Asp Leu Tyr Arg
1 5
<210> 90
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys
1 5 10
<210> 91
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5
<210> 92
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Phe Trp Glu His Glu Trp Lys Lys His Gly
1 5 10
<210> 93
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys
1 5 10 15
Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25
<210> 94
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Ala Leu Arg Leu Gln Trp Pro Ala Gly Phe Cys Glu Val Asn Asn Ala
1 5 10 15
Cys Asp Thr
<210> 95
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Thr Ile His Gly Leu Tyr Pro Tyr Asn Ala Lys
1 5 10
<210> 96
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu
1 5 10
<210> 97
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10 15
Lys
<210> 98
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp
1 5 10 15
Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg
20 25 30
Leu Pro Ser Lys Met Lys
35
<210> 99
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Asp Asp Ala Thr Asp Asp Tyr Arg Leu
1 5
<210> 100
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Glu Ala Ile Lys Lys His Leu Asn
1 5
<210> 101
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 102
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 103
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Gln Glu Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 104
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe
1 5 10 15
Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
20 25 30
Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 105
<211> 42
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Ser Glu Tyr Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala
1 5 10 15
Thr Arg Phe Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg
20 25 30
Leu Lys Phe Val Arg Leu Pro Ser Lys Met
35 40
<210> 106
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Ala Val Pro Glu Ile Asp Phe Thr Lys
1 5
<210> 107
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
1 5 10 15
<210> 108
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Val Cys Val Asn Gln Gln Ala Thr Arg Phe Val Asp Cys Pro Thr Asp
1 5 10 15
Asp Ala Thr Asp Asp Tyr Arg
20
<210> 109
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Asp Asp Ala Thr Asp Asp Tyr Arg
1 5
<210> 110
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Gly Ile Tyr Pro Asn Asn Asp Leu Tyr Arg Pro
1 5 10
<210> 111
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Ala Phe Arg Lys Ala Phe
1 5
<210> 112
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 112
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg
1 5 10
<210> 113
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 113
Phe Val Arg Leu Pro Ser Lys
1 5
<210> 114
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 114
Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser
1 5 10 15
Lys Met Lys
<210> 115
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 115
Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Arg Leu Pro Ser Lys
1 5 10 15
<210> 116
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 116
Ala Leu Arg Leu Gln Trp Pro Ala
1 5
<210> 117
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 117
Pro Tyr Asn Ala Lys Gly Thr Pro Ala
1 5
<210> 118
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 118
Arg Leu Pro Ser Lys Met Lys
1 5
<210> 119
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 119
Val Leu Thr Asp Ile Asn Val Cys Val Asn Gln Gln Ala Thr Arg Phe
1 5 10 15
Val Asp Cys Pro Thr Asp Asp Ala Thr Asp Asp Tyr Arg Leu Lys Phe
20 25 30
Val Arg Leu Pro Ser Lys Met Lys
35 40
<210> 120
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Asp Tyr Arg Leu Lys Phe
1 5
<210> 121
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 121
Leu Pro Ser Lys Met Lys
1 5
<210> 122
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 122
Leu Lys Phe Val Arg Leu Pro Ser Lys Met Lys
1 5 10
<210> 123
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 123
Asp Leu Tyr Arg Pro Lys
1 5
<210> 124
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 124
Phe Val Arg Leu Pro Ser
1 5
<210> 125
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 125
Val Arg Leu Pro Ser Lys Met Lys
1 5
<210> 126
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 126
Ala Asp Glu Val Val His His Pro Arg
1 5
<210> 127
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 127
Ala Asp Glu Val Val His His
1 5
<210> 128
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 128
Ala Asp Glu Val Val His His Pro Leu Asp
1 5 10
<210> 129
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 129
Ala Asp Glu Val Val His His Pro Leu Asp Lys
1 5 10
<210> 130
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 130
Ser Asp Glu Glu His Gln Phe Glu Ser Arg
1 5 10
<210> 131
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 131
Asp Glu Val Val His His Pro Arg
1 5
<210> 132
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 132
Ala Ala Ser His Glu Glu Ser Lys Pro Ser Glu Leu Asp Glu Pro Glu
1 5 10 15
Glu Lys
<210> 133
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 133
Glu Asp Glu Glu His Gln Phe Glu Ser Arg
1 5 10
<210> 134
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 134
Ser Leu Lys Glu Lys Leu His Lys Glu Glu Asp Thr Ser Val Pro Val
1 5 10 15
Glu Lys
<210> 135
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 135
Ala Asp Glu Val Val His His Pro Leu Asp Lys Ser
1 5 10
<210> 136
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 136
Phe Trp Glu His Glu Trp Asn Lys His Gly
1 5 10
<210> 137
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 137
Ala Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
<210> 138
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 138
Ala Asn Leu Leu His Lys Leu Glu Glu Thr
1 5 10
<210> 139
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 139
Thr Glu Asp Lys Gln Phe Trp Glu His Glu Trp Lys Lys His
1 5 10
<210> 140
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 140
Lys Pro Glu Val Asp Phe Thr Lys Asn Glu Asp
1 5 10
<210> 141
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 141
Pro Leu Thr Ala Pro Ala Pro Glu Glu Pro Lys Glu
1 5 10
<210> 142
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 142
Glu Glu Gly Leu His Pro Arg
1 5
<210> 143
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 143
Gly Phe Leu Asp Lys Leu Lys Glu Lys
1 5
<210> 144
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 144
Ala Glu His Gly Glu Leu Pro Glu Pro Arg
1 5 10
<210> 145
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 145
Ala Asn Leu Leu His Lys Leu Cys Arg Thr Leu Gly Met Gly Gly Asp
1 5 10 15
Lys
<210> 146
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 146
Phe Ala Leu Arg Leu Gln
1 5
<210> 147
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 147
Ala Asp Glu Val Val His His Pro Leu Arg
1 5 10
<210> 148
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 148
Ala Asn Leu Leu His Lys Leu Glu Glu
1 5
<210> 149
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 149
Ala Asn Leu Leu His Lys Leu Glu
1 5
<210> 150
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 150
Ala Asn Leu Leu His Lys Leu
1 5
<210> 151
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 151
Asp Lys Gln Phe Trp Glu His Glu Trp Asn Lys His
1 5 10
<210> 152
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 152
His Val Ser Asp Pro Glu Pro Lys Phe Glu Cys Asn
1 5 10
<210> 153
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 153
Phe Trp Glu His Glu Trp Asn Lys His Gly Arg
1 5 10
<210> 154
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 154
Phe Val Arg Leu Pro Ser Lys Met Lys Arg
1 5 10
<210> 155
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 155
Val His Val Ser Asp Pro Glu Pro Lys Phe
1 5 10
<210> 156
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 156
Ala Asn His Gly Glu Arg Tyr Gly Gln Glu Glu
1 5 10
<210> 157
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 157
Val His Val Ser Glu Thr Glu Pro Lys Phe Glu Glu Lys Ser
1 5 10
<210> 158
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 158
Asp Lys Gln Phe Trp Glu His Glu Trp Asn Lys
1 5 10
<210> 159
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 159
Ala Glu His Gly Glu Leu Pro Glu Pro Lys
1 5 10
<210> 160
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 160
Leu Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Thr Cys Leu Leu Asn
20
<210> 161
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 161
Leu Thr Val Thr Pro Glu Glu Pro Val Val Val Glu Lys
1 5 10
<210> 162
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 162
His Val Ser Asp Pro Glu Pro Lys Phe Glu Glu
1 5 10
<210> 163
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 163
Ala Asn Leu Leu His Lys Leu Glu Glu Thr Leu Gly Met Asn Asp Lys
1 5 10 15
<210> 164
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 164
Ala Asn His Gly Glu Arg Tyr Gly Gln Glu
1 5 10
<210> 165
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 165
Val His Val Ser Glu Thr Glu Lys Pro Phe Glu Glu Ser Lys
1 5 10
<210> 166
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 166
Asn Gly Tyr Ala Ala Gly Asp Glu Gln His His Val Gln
1 5 10
<210> 167
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 167
Leu Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Ser Glu Arg
20
<210> 168
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 168
Glu His Thr Asn Thr Glu Asp Lys Gln Phe Trp Glu His Glu
1 5 10
<210> 169
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 169
Ala Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser
1 5 10
<210> 170
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 170
Ser Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu Thr Thr
1 5 10 15
Asp Arg
<210> 171
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 171
Phe Ala Arg Leu Leu Glu
1 5
<210> 172
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 172
Glu Glu Gly Leu His Pro Asn Arg
1 5
<210> 173
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 173
Thr Glu Asp Lys Gln Phe Trp Glu His Glu Trp Asn Lys His Gly Arg
1 5 10 15
<210> 174
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 174
Pro Lys Phe Glu Glu Ser Lys
1 5
<210> 175
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 175
Asp Glu Val Val His His Pro Leu Asp Lys
1 5 10
<210> 176
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 176
His Glu Thr Asn Thr Glu Asp Lys Gln Phe Trp
1 5 10
<210> 177
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 177
Glu Glu Gly Leu His Pro Asp Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 178
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 178
Ala Glu His Gly Glu Leu Pro Glu Pro Lys Gly Glu
1 5 10
<210> 179
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 179
Thr Gly Leu Lys Ser Asp Gly Asp Val Phe His Glu
1 5 10
<210> 180
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 180
Val Thr Leu Pro Ser Lys Met Lys
1 5
<210> 181
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 181
Leu Ser Lys Glu Lys Leu His Lys Glu Glu Asp Thr Ser Val Pro Val
1 5 10 15
Glu Lys Leu Glu Glu
20
<210> 182
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 182
Ala Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Cys Leu Arg
20
<210> 183
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 183
Val Ser Ser Glu Pro Lys Leu Lys Asp Leu Val Thr Glu Glu Gly
1 5 10 15
<210> 184
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 184
Ala Tyr Gly Asn Pro Val Gly Gln Thr Glu Ala Gly Phe Gly Asn Leu
1 5 10 15
Leu His
<210> 185
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 185
Asp Lys Lys Phe Trp Glu His Glu Trp Asn Lys His Val Asn
1 5 10
<210> 186
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 186
Arg Ser Asp Tyr Pro Arg
1 5
<210> 187
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 187
Leu Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Ser Gly Asp Leu
20
<210> 188
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 188
Thr Leu Pro Ser Lys Met Lys
1 5
<210> 189
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 189
Ala Asp Glu Val Val His His Pro Leu
1 5
<210> 191
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 191
Lys Phe Phe Thr Arg Phe
1 5
<210> 191
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 191
Ala Tyr Gly Asn Pro Val Gly Lys Thr Glu Gln Phe Gly Asn Leu Leu
1 5 10 15
His Glu
<210> 192
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 192
Thr Glu Lys Asp Lys Phe Trp Glu His Glu Asp Ala Lys Lys
1 5 10
<210> 193
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 193
Arg Asp Tyr Pro Arg
1 5
<210> 194
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 194
Ala Thr Asp Asp Tyr Arg Leu Lys Phe Val Thr Leu Pro Ser Lys Met
1 5 10 15
Lys
<210> 195
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 195
Thr Glu Asp Lys Lys Phe Trp Glu His Glu Gly Glu Asn Lys His Gly
1 5 10 15
<210> 196
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 196
Ala Glu His Gly Glu Leu Pro Glu Pro Lys Gly Glu Ser
1 5 10
<210> 197
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 197
Asp Arg Tyr Pro Asp Arg
1 5
<210> 198
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 198
Ala Asn His Gly Glu Arg Tyr Gly Gln Glu Gln
1 5 10
<210> 199
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 199
Leu Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Thr Asp Arg Gly Leu Phe Asp
20
<210> 200
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 200
Thr Glu Asp Lys Lys Phe Gly Glu Glu His Glu Trp Asn Lys His Gly
1 5 10 15
Arg
<210> 201
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 201
Phe Val Thr Leu Pro Ser Lys Met Lys
1 5
<210> 202
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 202
Val His Val Ser Asp Pro Glu Pro Lys Phe Glu Glu
1 5 10
<210> 203
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 203
Gln Glu Gly Leu His Pro Asn Asn Asp Leu Tyr Arg
1 5 10
<210> 204
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 204
Asp Tyr Arg Leu Lys Phe Val Thr Leu Pro Ser Lys
1 5 10
<210> 205
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 205
Ala Asp Glu Val Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Cys Leu Arg Gly Leu
20
<210> 206
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 206
Leu Asp Glu Val Val His His Leu Pro Asp Lys Ser Ser Glu Val Glu
1 5 10 15
Thr Thr Asp Arg Gly Leu Phe
20
<210> 207
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 207
Asp Asp Tyr Arg Leu Arg
1 5
<210> 208
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 208
Asn Asp Leu Tyr Arg Pro Arg
1 5
<210> 209
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 209
Ala Asn Leu Leu His Lys Leu Glu Glu Thr Leu Gly
1 5 10
<210> 210
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 210
Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Thr Val Leu Asp Lys Leu
1 5 10 15
<210> 211
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 211
Asp Asp Ala Thr Asp Asp Arg Tyr Leu Arg
1 5 10
<210> 212
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 212
Thr Glu Asp Lys Lys Phe Glu Gly Glu His Glu Trp Lys Lys His Gly
1 5 10 15
<210> 213
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 213
Thr Thr Val Pro Gly Lys Leu Leu Pro Glu Thr Leu Glu Val Ser Leu
1 5 10 15
Leu Tyr Thr
<210> 214
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 214
Leu Asp Glu Val Val His His Pro Leu Asp Lys Ser Asp Thr Val Glu
1 5 10 15
Thr Thr Asp
<210> 215
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 215
His Glu Cys Leu Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp Asn
1 5 10 15
Lys His Gly
<210> 216
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 216
Trp Thr Leu Thr Gly Leu Glu Ala His His Leu Pro Lys
1 5 10
<210> 217
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 217
His Glu Thr Asn Thr Glu Asp Lys Lys Phe Gly Glu Glu His Glu Trp
1 5 10 15
Asn Lys His Gly
20
<210> 218
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 218
His Arg Pro Glu Tyr Ala Val Ser Val Leu Leu Arg
1 5 10
<210> 219
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 219
Leu Ser Val Val Ala Ser Asp Leu Glu Ala His His Leu Pro Lys
1 5 10 15
<210> 220
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 220
Thr Thr Val Pro Gly Lys Leu Leu Pro Glu Thr Leu Glu Trp Leu Leu
1 5 10 15
His Gln
<210> 221
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 221
His Glu Thr Cys Pro Ser Gly Asp Lys Lys Phe Trp Glu His
1 5 10
<210> 222
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 222
Pro Tyr Asn Glu Lys Gly Thr Pro Gly
1 5
<210> 223
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 223
Val Pro Glu Gly Asp Ala Ser Leu Asp Asp Pro Phe Glu Phe Glu Glu
1 5 10 15
Leu Gln
<210> 224
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 224
His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Glu Gly
1 5 10 15
Lys Lys His
<210> 225
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 225
Met Ser Arg Arg Gln Leu
1 5
<210> 226
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 226
His Glu Cys Leu Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp Asn
1 5 10 15
Lys His
<210> 227
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 227
Val His Val Ser Asp Pro Glu Pro Lys Arg Cys Phe Ser Lys
1 5 10
<210> 228
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 228
Leu Leu Leu Leu Leu Leu Leu Pro Leu
1 5
<210> 229
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 229
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Val Lys
1 5 10
<210> 230
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 230
Gln Val Leu Leu His Gln Leu Glu Glu Thr Arg
1 5 10
<210> 231
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 231
Ser His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp
1 5 10 15
Asn Lys His Gly Arg
20
<210> 232
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 232
Glu Phe Gly Glu Glu His Glu Ser Val Asn Lys His Gly Arg
1 5 10
<210> 233
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 233
Leu Leu Lys Glu Lys Leu Pro Gly Gly Cys Lys Pro
1 5 10
<210> 234
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 234
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Val Lys
1 5 10
<210> 235
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 235
Leu Leu Leu Leu Leu Leu Leu Leu Leu Val Lys
1 5 10
<210> 236
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 236
Leu Asn His Gly Gly Pro Gln Gln Gly Gln Gly Gln Glu Gln Gly Tyr
1 5 10 15
Arg Cys Arg Gly Thr Asp
20
<210> 237
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 237
Leu Leu Leu Leu Leu Leu Leu Val Lys
1 5
<210> 238
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 238
Leu Asn His Gly Glu Val Gly Tyr Gly Gln Glu Gln Gly Gln Gly Gln
1 5 10 15
Gln Glu
<210> 239
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 239
His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp Gln
1 5 10 15
Lys His
<210> 240
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 240
Leu Leu Leu Leu Leu Leu Leu Lys Val
1 5
<210> 241
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 241
Ala Asp Glu Val Val His His Pro Leu Asp Lys Val Cys Thr Val Glu
1 5 10 15
Thr Thr Asp
<210> 242
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 242
Val Lys Leu Leu Leu Leu Leu Leu Leu
1 5
<210> 243
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 243
Leu Leu Leu Leu Leu Leu Leu Leu Val Lys
1 5 10
<210> 244
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 244
Gln Asn His Gly Glu Leu Pro Glu Pro Lys Gly Glu
1 5 10
<210> 245
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 245
Leu Leu Leu Leu Leu Leu Leu Leu Leu Lys Val
1 5 10
<210> 246
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 246
Gln Pro Gln Gly His Pro Asn Asn Asp Leu Tyr Arg Pro Lys
1 5 10
<210> 247
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 247
Ala Asp His Gly Glu Arg Cys Thr Ser Gly Glu Lys Gly Gln Gly Gln
1 5 10 15
Gln Gln Arg
<210> 248
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 248
Leu Leu Leu Leu Leu Pro Leu
1 5
<210> 249
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 249
Leu Leu Leu Leu Lys Leu Leu Pro
1 5
<210> 250
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 250
Leu Asp His Gly Glu Asn Tyr Gln Val Glu Gln Gly Gln Gly Gln Gln
1 5 10 15
Gly Ala Arg
<210> 251
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 251
Leu Asn Leu Leu His Lys Asn Glu Glu Thr Leu
1 5 10
<210> 252
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 252
Ala Val Pro Glu Leu Asp Phe Thr Thr Thr Gly His Ala
1 5 10
<210> 253
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 253
Leu Asp His Gly Glu Gly Val Tyr Gly Gln Glu Gln Gly Gln Gly Gln
1 5 10 15
Gln Gly Ala Arg
20
<210> 254
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 254
His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Ser Val
1 5 10 15
Gln Asn His Gly Arg
20
<210> 255
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 255
Glu Lys Leu Pro Gly Glu Ala Trp
1 5
<210> 256
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 256
Pro Gly Asn Lys Lys His Pro
1 5
<210> 257
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 257
Asp Tyr Arg Leu Lys Phe Val Thr Leu Pro Ser Lys Met Lys
1 5 10
<210> 258
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 258
Ala Thr Val Glu Gly Glu Gly Leu Leu Ala Ala Gln Lys
1 5 10
<210> 259
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 259
Ala Asp His Gly Glu Ala Cys Cys Lys Val Glu Lys Gly Gln Gly Gln
1 5 10 15
Gln Gly Ala Arg
20
<210> 260
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 260
His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp Asn
1 5 10 15
Arg Pro Pro Gln
20
<210> 261
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 261
Leu Leu Leu Leu Leu Leu Leu Leu Leu
1 5
<210> 262
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 262
Glu Lys Leu Lys Glu Lys Leu Pro Gly His Thr Phe
1 5 10
<210> 263
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 263
Ala Asn His Trp Arg Tyr Gly Gln Glu Gln Gly Gln Gly Gln Lys Lys
1 5 10 15
Pro Cys Asn Asp Ala
20
<210> 264
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 264
Val Val Glu Pro Glu Leu Phe Leu Leu Cys Glu Val
1 5 10
<210> 265
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 265
Ser His Glu Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Met
1 5 10 15
Pro Leu Ser His Gly Arg
20
<210> 266
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 266
Ala Asp His Gly Glu Phe Pro Leu Phe Glu Gln Gly Gln Gly Gln Gln
1 5 10 15
Gln Arg Gly Thr Asp
20
<210> 267
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 267
Leu Asn His Gly His Gly Lys Gly Gln Glu Gln Gly Tyr Glu Phe Gln
1 5 10 15
His Phe
<210> 268
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 268
Leu Gln Thr Pro Thr Glu Asp Gln Gln Phe Trp Glu His Glu Trp Pro
1 5 10 15
Gly Ala Gly Pro
20
<210> 269
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 269
Leu Ala Tyr Ser Pro Val Gly Gly Gly Tyr Ala Ala Ala Asp Glu Gln
1 5 10 15
His Gly Pro Lys Ser Tyr Leu Ala
20
<210> 270
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 270
Thr Pro Gly Leu His Pro Asn Pro Asn Met Tyr Arg Pro
1 5 10
<210> 271
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 271
Leu Asp Glu Val Val His His Pro Asn Glu Tyr Arg Pro Val Glu Thr
1 5 10 15
Thr Asp Arg Gly Leu Phe Asp
20
<210> 272
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 272
Leu Leu Leu Leu Leu Leu Pro Leu
1 5
<210> 273
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 273
Gln Lys Leu Lys Glu Lys Leu Pro Gly Gly Lys Cys Pro
1 5 10
<210> 274
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 274
Thr Lys Leu Pro Ala Val Ser Pro Ala Gly Phe Tyr Glu Val Asn Asp
1 5 10 15
<210> 275
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 275
Ser Lys Gln Tyr Pro Gly Glu
1 5
<210> 276
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 276
Asp Leu Asp Tyr Pro Ser Val Arg
1 5
<210> 277
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 277
Lys Tyr Asp His Thr Ser Leu Tyr Arg Pro Arg
1 5 10
<210> 278
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 278
Glu His Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu His Tyr
1 5 10 15
Lys Val Asn His
20
<210> 279
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 279
Lys Cys Gly Pro Tyr Pro Trp
1 5
<210> 280
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 280
Thr Glu Asp Lys Lys Phe Trp Glu His Glu Leu Val His Gly Glu Thr
1 5 10 15
<210> 281
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 281
Trp Leu Tyr Pro Met Asp Trp Leu Tyr Pro Asn Asn
1 5 10
<210> 282
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 282
Val Lys Leu Leu Leu Leu Leu Leu
1 5
<210> 283
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 283
His Glu Thr Ser Gln Glu Asp Lys Lys Phe Trp Glu His Glu Asp Asn
1 5 10 15
Ala Lys His Gly
20
<210> 284
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 284
Glu His Thr Asn Thr Glu Asp Lys Lys Phe Trp Glu His Glu Trp Asn
1 5 10 15
Gln
<210> 285
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 285
Leu Leu Leu Leu Leu Leu Leu
1 5
<210> 286
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 286
Ala Asn His Gly His Gly Lys Gly Gln Glu Gln Gly Tyr Phe Glu Gln
1 5 10 15
Gln Arg
<210> 287
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 287
Thr Glu Asp Lys Lys Phe Val Ser Glu His Glu Ser Val Asn Lys Cys
1 5 10 15
Phe Thr
<210> 288
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 288
Glu Gly Ala Lys Tyr Pro Gly Glu
1 5
<210> 289
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 289
Cys Leu Leu Ala Ala Arg His Leu Gly Leu
1 5 10
<210> 290
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 290
Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu
1 5 10
<210> 291
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 291
Asp Val Leu Ala Cys Leu Leu Gly Thr Leu His Glu
1 5 10
<210> 292
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 292
Val Leu Asp Pro Pro Pro Gly Gln Phe Ala
1 5 10
<210> 293
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 293
Glu Tyr Phe Gly Ala Pro Val Ser Cys Lys Ala Ala Lys Leu Gly Leu
1 5 10 15
Ala Gly Trp Pro Ala Gly Phe Tyr Glu Val Asn Asp
20 25
<210> 294
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 294
Lys Lys Gln Ala Pro Lys Met Lys
1 5
<210> 295
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 295
Gln Glu Gly Leu Tyr Leu Asp Pro Asp Leu Tyr Arg Pro Arg
1 5 10
<210> 296
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 296
Val Thr Leu Pro Leu Asp Pro Lys Pro Leu
1 5 10
<210> 297
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 297
Glu His Cys Leu Thr Glu Asp Lys Lys Phe Trp Glu His Glu Val Asn
1 5 10 15
Ser His Gln Gly Arg
20
<210> 298
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 298
Asn Gly Tyr Ala Gly Ala Glu Glu Lys His Arg Cys Val Ala Gly Cys
1 5 10 15
Lys
<210> 299
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 299
Glu Val Asp Pro Gly Phe Gly Pro His Lys Gly Ser Leu Asp Ala Arg
1 5 10 15
<210> 300
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 300
Ala Asn His Gly His Gly Lys Gly Gln Glu Gln Gly Tyr Glu Phe Gln
1 5 10 15
Gly Ala Arg
<210> 301
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 301
Glu Gly Leu Tyr Pro Asn Lys Thr Ala Pro Tyr Thr Pro Val Gly
1 5 10 15
<210> 302
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 302
Gln Asn Ala Pro His Asn Gly Pro Gly Asp Leu Tyr Arg
1 5 10
<210> 303
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 303
Asn Gly Gly Gly Ala Pro Ala Arg Gly
1 5
<210> 304
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 304
Asp Leu Val His His Pro Leu Asp Lys Ser Ser Glu Val Glu Ser Pro
1 5 10 15
Arg Phe Arg Phe Asp
20
<210> 305
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 305
Asp Ala Ala Leu Ser Trp Pro Leu Leu Val Val Asp
1 5 10
<210> 306
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 306
Leu Leu Leu Leu Leu Leu Leu Leu
1 5
<210> 307
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 307
Gln Gly Gln Pro Ala Gly Pro Trp
1 5
<210> 308
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 308
Lys Arg Ala Phe Asp Leu Leu Gly
1 5

Claims (6)

1. A preparation method of a ginseng extract containing endogenous polypeptide is characterized in that:
grinding Ginseng radix into powder, extracting with organic solvent, separating the extractive solution with column, and concentrating the eluate under reduced pressure to obtain Ginseng radix extract containing endogenous Ginseng radix polypeptide as main ingredient;
the ginseng extract contains ginseng endogenous polypeptide, and the amino acid sequence of the polypeptide comprises SEQ ID No.1-125 and SEQ ID NO. 1-183;
the content of polypeptide components in the ginseng extract is more than 30 percent and the molecular weight range is 300-7000 Da through UHPLC-Q-TOF determination;
the method comprises the following specific steps:
1) solvent extraction
Grinding ginseng, soaking the ground ginseng in 40-60% by volume of one of methanol, ethanol, acetonitrile or acetone or 70-90% by volume of formic acid for 0.5-1.5 hours, ultrasonically extracting for 30-60 minutes, centrifuging and collecting supernatant;
2) column separation
Separating the supernatant by a column, eluting and removing impurities by using an organic solvent with the volume fraction of 0-20%, eluting by using an eluent with the volume fraction of 20-70%, collecting the eluent with the volume fraction of 20-70%, concentrating and drying to obtain a ginseng extract;
in the step 1), the mass volume ratio of the human participated methanol, ethanol, acetonitrile, acetone or formic acid is 1g:10-20mL, and the volume of the concentrated and dried human participated methanol is 5% -15% of the original volume;
step 2), separating the column by adopting one of a reversed phase C18 column, an MCI column or a macroporous adsorption resin column HPD 100; the organic solvent for eluting and impurity removing in the step 2) is selected from one of acetonitrile, methanol or ethanol with the volume fraction of 0-20%, and when the organic solvent is 0%, water is directly used for eluting and impurity removing; the eluent is selected from one of acetonitrile, methanol or ethanol with the volume fraction of 20% -70%; the elution volume is 2-3 times of the column volume.
2. The method of claim 1, wherein:
the operating conditions for the separation of the C18 column in the step 2) are as follows: concentrating the supernatant after impurity removal, passing through a C18 column, eluting with 2 times of 10%, 30%, 40%, 50%, 70% and 90% acetonitrile in terms of volume fraction, at a flow rate of 0.5mL/min, collecting 40% acetonitrile eluate, concentrating, and drying to obtain Ginseng radix extract;
the operation conditions for the separation of the HPD100 resin column in the step 2) are as follows: concentrating the supernatant after impurity removal, passing through an HPD100 resin column, eluting with 2 times of 10%, 30%, 50%, 70% and 90% ethanol in volume fraction at a flow rate of 0.5mL/min, collecting 50% ethanol eluate, concentrating, and drying to obtain Ginseng radix extract;
the operating conditions for the MCI column separation in step 2) are as follows: concentrating the supernatant after removing impurities, passing through MCI CHP20/P120 column, eluting with 10 times of column volume of water to remove acid, eluting with 2 times of column volume of 10%, 30%, 40%, 50%, 70%, 90% and 100% methanol in volume fraction, flowing at 0.5mL/min, collecting 40% methanol eluate, concentrating, and drying to obtain Ginseng radix extract.
3. A ginseng extract obtained by the production method according to claim 1, which is characterized in that: the ginseng extract contains ginseng endogenous polypeptide, and the amino acid sequence of the polypeptide comprises SEQ ID No.1-125 and SEQ ID NO. 1-183;
the content of polypeptide components in the ginseng extract is more than 30% and the molecular weight range is 300-7000 Da through UHPLC-Q-TOF determination.
4. A method for assaying ginseng extract according to claim 3, which comprises: analyzing by using a Nano LC-LTQ-Orbitrap technology, detecting polypeptide components in the ginseng extract by using Progenetics QI software, further performing secondary mass spectrometry, searching a Swiss-Prot database by PEAKS, identifying 308 endogenous polypeptides from the ginseng extract, and analyzing and sequencing the ginseng polypeptides by using two modes, namely PEAKS DB and PEAKS PTM;
the specific operation is as follows:
1) and (3) analysis: ginseng extract obtained by the method of claim 1 was dissolved in 0.1% formic acid, analyzed by Nano LC-LTQ-Orbitrap instrument, 8 μ L of sample was injected into a trap column 150 μm ID, 3cm, ReproSil-Pur C18 AQ by a syringe pump, eluted with 2% acetonitrile containing 0.2% formic acid for 3 minutes and then switched to an analytical column 75 μm ID, ReproSil-Pur C18 AQ, mobile phase a: 0.2% formic acid, mobile phase B: 95% acetonitrile with 0.2% formic acid, gradient elution procedure: 0-3.5 min, 5% B; 10% -20% of B in 3.5-6 min; 20% -50% of B in 6-35 minutes; 35-40 minutes, 50% -90% B; 40-45 minutes, 90% B;
2) polypeptide sequencing analysis: importing original data into a protein group mass spectrum software Peaks Studio 7, respectively carrying out sequence analysis by using two modes of PEAKS DB and PEAKS PTM, setting the filtering threshold value of the data as a data quality value >0.3, automatically sequencing the processed data from head, setting the mass errors of parent ions and daughter ions as 10ppm and 0.05Da respectively, cracking without the participation of specified enzymes, specifying C-terminal amidation, N-terminal acetylation and the cyclization common post-translational modification of glutamic acid and glutamine, carrying out data retrieval by using all proteins in Swiss-Prot as a database, searching and identifying the false positive rate <1%, carrying out manual analysis and confirmation on the searched peptide sequences one by one according to the credibility, the peptide chain breakage condition, the distribution condition of daughter ions and the error range, and finally identifying 308 high-credibility polypeptides from the searched nearly two thousand sequences, the amino acid sequence of the polypeptide is SEQ ID No.1-125 and SEQ ID NO. 1-183.
5. A pharmaceutical composition comprising the ginseng extract of claim 3 as an active ingredient or the ginseng extract of claim 3 as an active ingredient together with other pharmaceutically acceptable carriers and/or excipients.
6. Use of a medicament or pharmaceutical composition according to claim 5 for the preparation of a medicament with neuroprotective effect and/or for improving learning and memory function.
CN201610442864.8A 2016-06-20 2016-06-20 Ginseng extract containing endogenous polypeptide, preparation and determination thereof, medicine or pharmaceutical composition containing ginseng extract and application thereof Active CN107519223B (en)

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