CN107941943A - A kind of feature polypeptide using from heat shock protein GP96 differentiates the method that U.S.'s meat is joined - Google Patents

A kind of feature polypeptide using from heat shock protein GP96 differentiates the method that U.S.'s meat is joined Download PDF

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CN107941943A
CN107941943A CN201711179500.6A CN201711179500A CN107941943A CN 107941943 A CN107941943 A CN 107941943A CN 201711179500 A CN201711179500 A CN 201711179500A CN 107941943 A CN107941943 A CN 107941943A
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polypeptide
seq
meat
ginseng
albumen
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CN107941943B (en
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张晓梅
张鸿伟
崔淑华
王颖
徐彪
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
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    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract

The present invention relates to a kind of method for differentiating U.S.'s meat ginseng (Isostichopus badionotus) using specificity peptide fragment group, and one group when being used alone or in combination, it can be used to differentiate the feature polypeptide of U.S.'s meat ginseng, the feature polypeptide joins the low homology polypeptide fragment of albumen for U.S.'s meat, it is exclusive for U.S.'s meat ginseng that the low homology polypeptide fragment refers to the peptide fragment, and length is in the polypeptide fragment of 5~30 amino acid, although the albumen finds to have the species such as part and plant or bacterium consistent with its, but contrast plum blossom ginseng, Haiti melon, stichopus japonicus, when Mexico is joined, the feature polypeptide is that U.S.'s meat ginseng is exclusive.

Description

A kind of feature polypeptide using from heat shock protein GP96 differentiates what U.S.'s meat was joined Method
The application is that " CN201610410893.6, one kind differentiate U.S.'s meat using specificity peptide fragment group for Chinese invention application The divisional application of the method for ginseng ".
Technical field
The invention belongs to biological technical field, differentiates that U.S.'s meat is joined using specificity peptide fragment group in particular to one kind The method of (Isostichopus badionotus).
Background technology
Sea cucumber is rich in acidic mucopolysaccharide, there is immunomodulator, anticoagulation, control blood glucose and reducing blood lipid and other effects to human body. So sea cucumber enjoys the good reputation of " nutrition treasure-house ", modern is described as sea cucumber " first of seafood delights eight delicacies ".In the market sea cucumber species is numerous It is more, different sea cucumbers because its nutritive value, mouthfeel etc. are different and price difference is huge.U.S.'s meat ginseng is one kind of sea cucumber, naturally raw Grow in cold water marine site, its growth phase is longer, generally 7-10, therefore nutritive value is high, the richness possessed except the general sea cucumber of tool Outside containing protein and the advantages of various trace elements, also have that ginseng body is stout and strong, hair rises rate height, and ginseng meat is thick and solid, the spy such as smooth in taste Point.It is culinary art and medicinal all good superfine product.
The sea cucumber of different cultivars compares, and some glycosaminoglycan contents are high, are beneficial to cardiovascular and cerebrovascular；Some saponin contents Height is beneficial to anticancer.Sea cucumber is the same with ginseng, if saponin content is high, medical value is high, but may smell bad；And have Sea cucumber protein content it is high, such as eggplant ginseng and stichopus japonicus, can largely with hotel and Domestic dining table, protein is lacked, battalion Nutriment needed for undesirable crowd's offer body is provided.
Method due to lacking particularly effective identification sea cucumber kind at present, differentiates the method for sea cucumber species also simply by passing The visual inspection of system, lacks the support of technical merit, causes many bad businessman to adulterate, passes a fake product off as a genuine one and cheat consumer, Therefore a kind of method of the identification sea cucumber of efficiently and accurately is needed.
The content of the invention
The present invention studies the peptide fragment of U.S.'s meat ginseng (Isostichopus badionotus), establishes from more The technology that peptide level differentiates U.S.'s meat ginseng, has filled up the blank that domestic and international sea cucumber differentiates.
When being used alone or in combination present invention firstly relates to one group, it can be used to differentiate the feature polypeptide of U.S.'s meat ginseng, it is described Feature polypeptide joins the low homology polypeptide fragment of albumen for U.S.'s meat, and it is the U.S. that the low homology polypeptide fragment, which refers to the peptide fragment, Meat ginseng is exclusive, and length, in the polypeptide fragment of 5~30 amino acid, the albumen is the following albumen in U.S.'s meat ginseng：
(1) actin isoform 2, histone H2B and glyceraldehyde-3-phosphate Dehydrogenase albumen；
(2) arginine kinase, translationally controlled tumor protein and major 2 albumen of yolk protein；
(3) heat-shock protein 70 and heat-shock protein gp96 albumen；
(4) tropomyosin albumen.
Preferably, it is described from actin isoform 2, histone H2B and glyceraldehyde-3- The feature polypeptide of phosphate dehydrogenase albumen, its sequence are：
SEQ ID NO.1：EIAALAPPTMK；
SEQ ID NO.2：LCYVFLDFEQEMATAASSSSLEK；
SEQ ID NO.3：CDETLFQPSFIGMESAGIHETTYNSIMK；
SEQ ID NO.4：ETYSIYIYK；
SEQ ID NO.5：AGIALSENFVK；
SEQ ID NO.6：VPVPDVSVVDLTLR.
The m/z of above-mentioned peptide fragment 1-6 is respectively：
571.3；876.1；1069.5；393.9；574.8；754.9.
Preferably, it is described to derive from arginine kinase, translationally controlled tumor The feature polypeptide of 2 albumen of protein and major yolk protein, its sequence are：
SEQ ID NO.7：PYDDVLDPAYVISSR；
SEQ ID NO.8：IMDDVLDPAYVISSR；
SEQ ID NO.9：DVITGEELFSDSYK；
SEQ ID NO.10：LVDDLYWR；
SEQ ID NO.11：IGSVFESLNR；
SEQ ID NO.12：PVVFLNPER.
The m/z of above-mentioned peptide fragment 7-12 is respectively：
855.4；565.3；801.9；540.3；561.3；535.8.
Preferably, it is described from heat-shock protein 70 and heat-shock protein gp96 albumen Feature polypeptide, its sequence are：
SEQ ID NO.13：DAGVIAGLQVLR；
SEQ ID NO.14：SEIHELVLVGGSTR；
SEQ ID NO.15：VEIESFFDGEDFSETLTR；
SEQ ID NO.16：DDLVNNLGTIAK；
SEQ ID NO.17：EEAYDYLEPDTIEQLVK；
SEQ ID NO.18：YSQFINFPIYLWGSK.
The m/z of above-mentioned peptide fragment 13-18 is respectively：
606.4；748.9；1061.0；636.8；1028.0；932.0.
Preferably, the feature polypeptide from tropomyosin albumen, its sequence are：
SEQ ID NO.19：MSELESQVTSSK；
SEQ ID NO.20：MSELESQVMGSK；
SEQ ID NO.21：QLESELDDTSEK；
SEQ ID NO.22：YDDIEQSSEESER；
SEQ ID NO.23：YNDIEQSSEESER.
The m/z of above-mentioned peptide fragment 19-23 is respectively：
442.5；442.5；465.2；529.6；529.2.
The invention further relates to a kind of method for detecting U.S.'s meat ginseng, described method includes following steps：
(1) mass spectrum pre-treatment is carried out to sample to be tested, obtains candidate polypeptide filtrate：
(2) polypeptide moiety of sample to be tested is examined by mass spectrography, analyzes U.S.'s meat ginseng component in sample.
The mass spectrum pre-treatment step is as follows：
(1) 1g sea cucumber sample homogeneous is weighed into pulverulence, adds 10mL protein extracts (8M urea, 50mM NH4HCO3) concussion extraction albumen, high-speed low temperature centrifugation (20000r/min), takes supernatant 200ul to be transferred in 1ml EP pipes；
(2) take 2 μ L 1mol/L DTT be added in the 100 above-mentioned protein solutions of μ L 37 DEG C of reactions 1 it is small when；
(3) take the IAA (configuration process needs lucifuge, matching while using) for the 1mol/L that 10 μ L now match somebody with somebody to be added to and have cooled down to room In the above-mentioned reaction solution of temperature, when room temperature lucifuge reaction 1 is small；
(4) using 10K filter membrane 20000g ultrafiltration 30 minutes, rinsed repeatedly on filter membrane using 50mmol/L ammonium bicarbonate solns Layer, is transferred in new EP pipes；
(5) add ammonium bicarbonate soln and repeat this step, merge solution and complete protein extraction under film；
(6) 5ul Trypsin enzyme solutions (the Trypsin enzyme solutions of 1 μ g/ μ L) are taken to be added in above-mentioned protein solution 37 DEG C When enzymolysis 16-18 is small；
(7) using 10K filter membrane 14000g ultrafiltration 20 minutes, the peptide fragment filtrate of lower floor is collected, treats machine testing.
The mass spectrography is detected as, using AB SCIEX5600 detections,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.25mL/min,
TOF scanning ranges：350-1500Da,
Cation reaction pattern, GS1：35, GS2：45, Curtain Gas：35, ISVF：5500, TEM：500, DP：100, CE：10.
Detected using the triple level Four bars of AB SCIEX,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.3mL/min,
Electric spray ion source, cation reaction pattern, detection mode：MRM, spray voltage：5500V, ion transfer tube temperature Degree：475℃；Sheath atmospheric pressure：40；Assist gas pressure power：6.
U.S.'s meat ginseng component in the analysis sample is that the mass spectral results of sample to be tested are contrasted U.S.'s meat joins , there is the low homology of U.S.'s meat ginseng albumen in the standard mass spectrogram of the low homology polypeptide fragment (feature polypeptide) of albumen During the Mass Spectrometer Method spectrogram of polypeptide fragment, you can judge that the tissue samples join sample for U.S.'s meat.
Preferably, the low homology polypeptide fragment (feature polypeptide) of described U.S.'s meat ginseng albumen for SEQ ID NO.1~ Each bar specific polypeptide shown in 23.
The invention further relates to using the above-mentioned U.S.'s meat ginseng albumen exclusive feature polypeptide differentiate U.S.'s meat join method and The detection products such as the reagent/kit for being used to differentiate U.S.'s meat ginseng prepared using the feature polypeptide.
The invention further relates to the feature polypeptide to prepare the detection productions such as reagent/kit for differentiating U.S.'s meat ginseng Application in product.
Finally, although the albumen or the part of feature polypeptide have, the species such as plant or bacterium are consistent with its, only right Than plum blossom ginseng, Haiti melon, stichopus japonicus, Mexico's ginseng when sea cucumber belongs to kind, the feature polypeptide is that U.S.'s meat ginseng is exclusive.
Brief description of the drawings
Fig. 1, EIAALAPPTMK chromatogram
Fig. 2, LCYVFLDFEQEMATAASSSSLEK chromatogram
Fig. 3, CDETLFQPSFIGMESAGIHETTYNSIMK chromatogram
Fig. 4, ETYSIYIYK chromatogram
Fig. 5, AGIALSENFVK chromatogram
Fig. 6, VPVPDVSVVDLTLR chromatogram
Fig. 7, PYDDVLDPAYVISSR chromatogram
Fig. 8, IMDDVLDPAYVISSR chromatogram
Fig. 9, DVITGEELFSDSYK chromatogram
Figure 10, LVDDLYWR chromatogram
Figure 11, IGSVFESLNR chromatogram
Figure 12, PVVFLNPER chromatogram
Figure 13, DAGVIAGLQVLR chromatogram
Figure 14, SEIHELVLVGGSTR chromatogram
Figure 15, VEIESFFDGEDFSETLTR chromatogram
Figure 16, DDLVNNLGTIAK chromatogram
Figure 17, EEAYDYLEPDTIEQLVK chromatogram
Figure 18, YSQFINFPIYLWGSK chromatogram
Figure 19, MSELESQVTSSK chromatogram
Figure 20, MSELESQVMGSK chromatogram
Figure 21, QLESELDDTSEK chromatogram
Figure 22, YDDIEQSSEESER chromatogram
Figure 23, YNDIEQSSEESER chromatogram
Embodiment
Embodiment 1, the sequence source of specific polypeptide and comparison information
1. aforementioned polypeptides SEQ ID NO.1：EIAALAPPTMK；SEQ ID NO.2： LCYVFLDFEQEMATAASSSSLEK；SEQ ID NO.3：CDETLFQPSFIGMESAGIHETTYNSIMK is both from albumen Actin isoform 2 [Holothria glaberrima], its Accesion number on NCBI is gi | 215882299.By mass spectral analysis, polypeptide SEQ ID NO.1：EIAALAPPTMK；SEQ ID NO.2： LCYVFLDFEQEMATAASSSSLEK；SEQ ID NO.3：CDETLFQPSFIGMESAGIHETTYNSIMK is in U.S.'s meat ginseng It is implicitly present in, and peak shape is good, intensity is high.
Fig. 1 is chromatograms of the polypeptide EIAALAPPTMK in U.S.'s meat ginseng, its m/z is 571.3.
Fig. 2 is chromatograms of the polypeptide LCYVFLDFEQEMATAASSSSLEK in U.S.'s meat ginseng, its m/z is 876.1.
Fig. 3 is chromatograms of the peptide C DETLFQPSFIGMESAGIHETTYNSIMK in U.S.'s meat ginseng, its m/z is 1069.5。
2. aforementioned polypeptides SEQ ID NO.4：ETYSIYIYK comes from albumen histone H2B [Holothuria Rubulosa], its Accesion number on NCBI is gi | 559457.By mass spectral analysis, polypeptide SEQ ID NO.4：ETYSIYIYK is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Fig. 4 is chromatograms of the polypeptide ETYSIYIYK in U.S.'s meat ginseng, its m/z is 393.9.
3. aforementioned polypeptides SEQ ID NO.5：AGIALSENFVK；SEQ ID NO.6：VPVPDVSVVDLTLR comes from egg White glyceraldehyde-3-phosphate dehydrogenase [Apostichopus japonicus], it is on NCBI Accesion number be gi | 334821165.By mass spectral analysis, polypeptide SEQ ID NO.5：AGIALSENFVK；SEQ ID NO.6：VPVPDVSVVDLTLR is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Fig. 5 is chromatograms of the polypeptide A GIALSENFVK in U.S.'s meat ginseng, its m/z is 574.8.
Fig. 6 is chromatograms of the polypeptide VPVPDVSVVDLTLR in U.S.'s meat ginseng, its m/z is 754.9.
4. passing through mass spectral analysis, polypeptide SEQ ID NO.1-6 are not deposited in plum blossom ginseng, Haiti melon, stichopus japonicus, Mexico's ginseng .
Embodiment 2, the sequence source of specific polypeptide and comparison information
1. aforementioned polypeptides SEQ ID NO.7：PYDDVLDPAYVISSR；SEQ ID NO.8：IMDDVLDPAYVISSR comes It is from albumen arginine kinase [Apostichopus japonicus], its Accesion number on NCBI gi|4586462.By mass spectral analysis, polypeptide SEQ ID NO.7：PYDDVLDPAYVISSR；SEQ ID NO.8： IMDDVLDPAYVISSR is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Fig. 7 is chromatograms of the polypeptide PYDDVLDPAYVISSR in U.S.'s meat ginseng, its m/z is 855.4.
Fig. 8 is chromatograms of the polypeptide IMDDVLDPAYVISSR in U.S.'s meat ginseng, its m/z is 565.3.
2. aforementioned polypeptides SEQ ID NO.9：DVITGEELFSDSYK；SEQ ID NO.10：LVDDLYWR comes from albumen Translationally controlled tumor protein [Stichopus monotuberculatus], it is in NCBI On Accesion number be gi | 659902918.By mass spectral analysis, polypeptide SEQ ID NO.9： DVITGEELFSDSYK；SEQ ID NO.10：LVDDLYWR is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Fig. 9 is chromatograms of the polypeptide DVITGEELFSDSYK in U.S.'s meat ginseng, its m/z is 801.9.
Figure 10 is chromatograms of the polypeptide LVDDLYWR in U.S.'s meat ginseng, its m/z is 540.3.
3. aforementioned polypeptides SEQ ID NO.11：IGSVFESLNR；SEQ ID NO.12：PVVFLNPER comes from albumen Major yolk protein 2 [Apostichopus japonicus], its Accesion number on NCBI is gi | 240846033.By mass spectral analysis, polypeptide SEQ ID NO.11：IGSVFESLNR；SEQ ID NO.12：PVVFLNPER is in U.S. It is implicitly present in state's meat ginseng, and peak shape is good, intensity is high.
Figure 11 is chromatograms of the polypeptide IGSVFESLNR in U.S.'s meat ginseng, its m/z is 561.3.
Figure 12 is chromatograms of the polypeptide PVVFLNPER in U.S.'s meat ginseng, its m/z is 535.8.
4. passing through mass spectral analysis, polypeptide SEQ ID NO.7-12 are not deposited in plum blossom ginseng, Haiti melon, stichopus japonicus, Mexico's ginseng .
Embodiment 3, the sequence source of specific polypeptide and comparison information
1. aforementioned polypeptides SEQ ID NO.13：DAGVIAGLQVLR；SEQ ID NO.14：SEIHELVLVGGSTR；SEQ ID NO.15：VEIESFFDGEDFSETLTR is both from 70 [Apostichopus of albumen heat shock protein Japonicus], its Accesion number on NCBI is gi | 194465888.By mass spectral analysis, polypeptide SEQ ID NO.113：DAGVIAGLQVLR；SEQ ID NO.14：SEIHELVLVGGSTR；SEQ ID NO.15： VEIESFFDGEDFSETLTR is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Figure 13 is chromatograms of the polypeptide DAGVIAGLQVLR in U.S.'s meat ginseng, its m/z is 606.4.
Figure 14 is chromatograms of the polypeptide SEIHELVLVGGSTR in U.S.'s meat ginseng, its m/z is 748.9.
Figure 15 is chromatograms of the polypeptide VEIESFFDGEDFSETLTR in U.S.'s meat ginseng, its m/z is 1061.0.
2. aforementioned polypeptides SEQ ID NO.16：DDLVNNLGTIAK；SEQ ID NO.17：EEAYDYLEPDTIEQLVK； SEQ ID NO.18：YSQFINFPIYLWGSK is both from albumen heat shock protein gp96 [Apostichopus Japonicus], its Accesion number on NCBI is gi | 355344590.By mass spectral analysis, polypeptide SEQ ID NO.16：DDLVNNLGTIAK；SEQ ID NO.17：EEAYDYLEPDTIEQLVK；SEQ ID NO.18： YSQFINFPIYLWGSK is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Figure 16 is chromatograms of the polypeptide DDLVNNLGTIAK in U.S.'s meat ginseng, its m/z is 636.8.
Figure 17 is chromatograms of the polypeptide EEAYDYLEPDTIEQLVK in U.S.'s meat ginseng, its m/z is 1028.0.
Figure 18 is chromatograms of the polypeptide YSQFINFPIYLWGSK in U.S.'s meat ginseng, its m/z is 932.0.
3. passing through mass spectral analysis, polypeptide SEQ ID NO.13-18 are not deposited in plum blossom ginseng, Haiti melon, stichopus japonicus, Mexico's ginseng .
Embodiment 4, the sequence source of specific polypeptide and comparison information
1. aforementioned polypeptides SEQ ID NO.19：MSELESQVTSSK；SEQ ID NO.20：MSELESQVMGSK；SEQ ID NO.21：QLESELDDTSEK；SEQ ID NO.22：YDDIEQSSEESER；SEQ ID NO.23：YNDIEQSSEESER comes From in albumen tropomyosin [Apostichopus japonicus], its Accesion number on NCBI is gi | 302340969.By mass spectral analysis, polypeptide SEQ ID NO.19：MSELESQVTSSK；SEQ ID NO.20： MSELESQVMGSK；SEQ ID NO.21：QLESELDDTSEK；SEQ ID NO.22：YDDIEQSSEESER；SEQ ID NO.23：YNDIEQSSEESER is implicitly present in U.S.'s meat ginseng, and peak shape is good, and intensity is high.
Figure 19 is chromatograms of the polypeptide MSELESQVTSSK in U.S.'s meat ginseng, its m/z is 442.5.
Figure 20 is chromatograms of the polypeptide MSELESQVMGSK in U.S.'s meat ginseng, its m/z is 442.5.
Figure 21 is chromatograms of the polypeptide QLESELDDTSEK in U.S.'s meat ginseng, its m/z is 465.2.
Figure 22 is chromatograms of the polypeptide YDDIEQSSEESER in U.S.'s meat ginseng, its m/z is 529.6.
Figure 23 is chromatograms of the polypeptide YNDIEQSSEESER in U.S.'s meat ginseng, its m/z is 529.2.
2. passing through mass spectral analysis, polypeptide SEQ ID NO.19-23 are not deposited in plum blossom ginseng, Haiti melon, stichopus japonicus, Mexico's ginseng .
Embodiment 5, sea cucumber sample process and detecting step
The step of analyzing sea cucumber sample to be measured includes
(1) Sample pretreatment step：
(1) 1g sea cucumber sample homogeneous is weighed into pulverulence, adds 10mL protein extracts (8M urea, 50mM NH4HCO3) concussion extraction albumen, high-speed low temperature centrifugation (20000r/min), takes supernatant 200ul to be transferred in 1ml EP pipes；
(2) take 2 μ L 1mol/L DTT be added in the 100 above-mentioned protein solutions of μ L 37 DEG C of reactions 1 it is small when；
(3) take the IAA (configuration process needs lucifuge) for the 1mol/L that 10 μ L now match somebody with somebody to be added to and have cooled down to the above-mentioned of room temperature In reaction solution, when room temperature lucifuge reaction 1 is small；
(4) using 10K filter membrane 20000g ultrafiltration 30 minutes, rinsed repeatedly using 200 μ L 50mmol/L ammonium bicarbonate solns Filter membrane upper strata, is transferred in new EP pipes；
(5) 200 μ L ammonium bicarbonate solns are added and repeat this step, merges solution and completes protein extraction under film；
(6) 5ul Trypsin enzyme solutions (the Trypsin enzyme solutions of 1 μ g/ μ L) are taken to be added in above-mentioned protein solution 37 DEG C When enzymolysis 16-18 is small；
(7) using 10K filter membrane 14000g ultrafiltration 20 minutes, the peptide fragment filtrate of lower floor is collected, treats machine testing.
(2) machine testing on,
Using AB SCIEX5600 detections,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.25mL/min,
TOF scanning ranges：350-1500Da,
Cation reaction pattern, GS1：35, GS2：45, Curtain Gas：35, ISVF：5500, TEM：500, DP：100, CE：10.
Detected using the triple level Four bars of AB SCIEX,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.3mL/min,
Electric spray ion source, cation reaction pattern, detection mode：MRM, spray voltage：5500V, ion transfer tube temperature Degree：475℃；Sheath atmospheric pressure：40；Assist gas pressure power：6.
The mass spectrogram of each bar specific polypeptide in testing result comparative example 1-4, goes out matter described in current embodiment 1-4 During spectrum detection spectrogram, you can judge that the tissue samples join sample for U.S.'s meat.
Through further identification, the peptide fragment joins proprietary peptide fragment for U.S.'s meat, in other Haiti melons, Mexico's ginseng, stichopus japonicus, plum Expression is had no in the different sea cucumber kinds such as flower ginseng.
Finally it should be noted that above example is used only as helping skilled in the art to understand the essence of the present invention, It is not used as limiting the scope of the present invention.
SEQUENCE LISTING
<110>Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120>A kind of feature polypeptide using from heat shock protein GP96 differentiates the method that U.S.'s meat is joined
<160> 23
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213> Isostichopus badionotus
<400> 1
Glu Ile Ala Ala Leu Ala Pro Pro Thr Met Lys
1 5 10
<210> 2
<211> 23
<212> PRT
<213> Isostichopus badionotus
<400> 2
Leu Cys Tyr Val Phe Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala
1 5 10 15
Ser Ser Ser Ser Leu Glu Lys
20
<210> 3
<211> 28
<212> PRT
<213> Isostichopus badionotus
<400> 3
Cys Asp Glu Thr Leu Phe Gln Pro Ser Phe Ile Gly Met Glu Ser Ala
1 5 10 15
Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys
20 25
<210> 4
<211> 9
<212> PRT
<213> Isostichopus badionotus
<400> 4
Glu Thr Tyr Ser Ile Tyr Ile Tyr Lys
1 5
<210> 5
<211> 11
<212> PRT
<213> Isostichopus badionotus
<400> 5
Ala Gly Ile Ala Leu Ser Glu Asn Phe Val Lys
1 5 10
<210> 6
<211> 14
<212> PRT
<213> Isostichopus badionotus
<400> 6
Val Pro Val Pro Asp Val Ser Val Val Asp Leu Thr Leu Arg
1 5 10
<210> 7
<211> 15
<212> PRT
<213> Isostichopus badionotus
<400> 7
Pro Tyr Asp Asp Val Leu Asp Pro Ala Tyr Val Ile Ser Ser Arg
1 5 10 15
<210> 8
<211> 15
<212> PRT
<213> Isostichopus badionotus
<400> 8
Ile Met Asp Asp Val Leu Asp Pro Ala Tyr Val Ile Ser Ser Arg
1 5 10 15
<210> 9
<211> 14
<212> PRT
<213> Isostichopus badionotus
<400> 9
Asp Val Ile Thr Gly Glu Glu Leu Phe Ser Asp Ser Tyr Lys
1 5 10
<210> 10
<211> 8
<212> PRT
<213> Isostichopus badionotus
<400> 10
Leu Val Asp Asp Leu Tyr Trp Arg
1 5
<210> 11
<211> 10
<212> PRT
<213> Isostichopus badionotus
<400> 11
Ile Gly Ser Val Phe Glu Ser Leu Asn Arg
1 5 10
<210> 12
<211> 9
<212> PRT
<213> Isostichopus badionotus
<400> 12
Pro Val Val Phe Leu Asn Pro Glu Arg
1 5
<210> 13
<211> 12
<212> PRT
<213> Isostichopus badionotus
<400> 13
Asp Ala Gly Val Ile Ala Gly Leu Gln Val Leu Arg
1 5 10
<210> 14
<211> 14
<212> PRT
<213> Isostichopus badionotus
<400> 14
Ser Glu Ile His Glu Leu Val Leu Val Gly Gly Ser Thr Arg
1 5 10
<210> 15
<211> 18
<212> PRT
<213> Isostichopus badionotus
<400> 15
Val Glu Ile Glu Ser Phe Phe Asp Gly Glu Asp Phe Ser Glu Thr Leu
1 5 10 15
Thr Arg
<210> 16
<211> 12
<212> PRT
<213> Isostichopus badionotus
<400> 16
Asp Asp Leu Val Asn Asn Leu Gly Thr Ile Ala Lys
1 5 10
<210> 17
<211> 17
<212> PRT
<213> Isostichopus badionotus
<400> 17
Glu Glu Ala Tyr Asp Tyr Leu Glu Pro Asp Thr Ile Glu Gln Leu Val
1 5 10 15
Lys
<210> 18
<211> 15
<212> PRT
<213> Isostichopus badionotus
<400> 18
Tyr Ser Gln Phe Ile Asn Phe Pro Ile Tyr Leu Trp Gly Ser Lys
1 5 10 15
<210> 19
<211> 12
<212> PRT
<213> Isostichopus badionotus
<400> 19
Met Ser Glu Leu Glu Ser Gln Val Thr Ser Ser Lys
1 5 10
<210> 20
<211> 12
<212> PRT
<213> Isostichopus badionotus
<400> 20
Met Ser Glu Leu Glu Ser Gln Val Met Gly Ser Lys
1 5 10
<210> 21
<211> 12
<212> PRT
<213> Isostichopus badionotus
<400> 21
Gln Leu Glu Ser Glu Leu Asp Asp Thr Ser Glu Lys
1 5 10
<210> 22
<211> 13
<212> PRT
<213> Isostichopus badionotus
<400> 22
Tyr Asp Asp Ile Glu Gln Ser Ser Glu Glu Ser Glu Arg
1 5 10
<210> 23
<211> 13
<212> PRT
<213> Isostichopus badionotus
<400> 23
Tyr Asn Asp Ile Glu Gln Ser Ser Glu Glu Ser Glu Arg
1 5 10

Claims (10)

1. one group is reflected U.S.'s meat ginseng (Isostichopus badionotus) by being used alone or being used in any combination Another characteristic polypeptide, the feature polypeptide join the low homology polypeptide fragment of albumen, the low homology polypeptide for U.S.'s meat It is that meat ginseng in the U.S.'s is exclusive that fragment, which refers to the peptide fragment, and length is in the polypeptide fragment of 5~30 amino acid, U.S.'s meat ginseng egg White is heat-shock protein gp96 albumen；From the feature polypeptide of heat-shock protein gp96 albumen Sequence is：
SEQ ID NO.16：DDLVNNLGTIAK；
SEQ ID NO.17：EEAYDYLEPDTIEQLVK；
SEQ ID NO.18：YSQFINFPIYLWGSK.
2. feature polypeptide according to claim 1, it is characterised in that the polypeptide fragment of SEQ ID NO.16~18 M/z respectively be：636.8；1028.0；932.0.
3. a kind of method for detecting U.S.'s meat ginseng, described method includes following steps：
(1) mass spectrum pre-treatment is carried out to sample to be tested, obtains candidate polypeptide filtrate；
(2) polypeptide moiety of sample to be tested is examined by mass spectrography, analyzes the stichopus japonicus component in sample.
4. according to the method described in claim 3, it is characterized in that, the mass spectrum pre-treatment step is as follows：
(1) sea cucumber sample homogeneous is weighed into pulverulence, adds protein extract (8M urea, 50mM NH4HCO3) concussion extraction Albumen, high-speed low temperature centrifugation, takes supernatant to be transferred in EP pipes；
(2) DTT is taken to be added in above-mentioned protein solution, when 37 DEG C of reactions 1 are small；
(3) IAA that enchashment is matched somebody with somebody is added in the above-mentioned reaction solution for having cooled down to room temperature, when room temperature lucifuge reaction 1 is small；
(4) 10K ultrafiltration through membranes are used 30 minutes, rinses filter membrane upper strata repeatedly using ammonium bicarbonate soln, be transferred to new EP pipes In；
(5) add ammonium bicarbonate soln and repeat this step, merge solution and complete protein extraction under film；
(6) take Trypsin enzyme solutions to be added to above-mentioned protein solution, in 37 DEG C enzymolysis 16-18 it is small when；
(7) 10K ultrafiltration through membranes are used 20 minutes, collects the peptide fragment filtrate of lower floor, treat machine testing.
5. the method according to claim 3 or 4, it is characterised in that the mass spectrography is detected as：
Using AB SCIEX Triple5600 detections,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.25mL/min,
TOF scanning ranges：350-1500Da,
Cation reaction pattern, GS1：35, GS2：45, Curtain Gas：35, ISVF：5500, TEM：500, DP：100, CE： 10。
6. the method according to claim 3 or 4, it is characterised in that the mass spectrography is detected as：
Detected using the triple level Four bars of AB SCIEX,
Mobile phase A：0.1% formic acid-acetonitrile, Mobile phase B：0.1% formic acid-water,
Flow velocity：0.3mL/min,
Electric spray ion source, cation reaction pattern, detection mode：MRM, spray voltage：5500V, ion transfer tube temperature： 475℃；Sheath atmospheric pressure：40；Assist gas pressure power：6.
7. the method according to claim 3 or 4, it is characterised in that U.S.'s meat in the analysis sample joins component and is, By the mass spectrogram of each bar specific polypeptide of mass spectral results contrast SEQ ID NO.16~18 of sample to be tested, appearance and SEQ During the identical spectrogram of the Mass Spectrometer Method spectrogram of each bar specific polypeptide of ID NO.16~18, you can judge the tissue samples for U.S. State's meat ginseng sample.
8. any feature polypeptide of claim 1 or 2 is preparing the products such as reagent/kit for differentiating U.S.'s meat ginseng In application.
9. apply according to claim 8, it is characterised in that the feature polypeptide of claim 1 or 2 is with being selected from following each group Feature polypeptide be jointly used in the discriminating of U.S. meat ginseng,
(1) the feature polypeptide of 2 albumen of actin isoform is derived from, its sequence is as shown in SEQ ID NO.1-3；
(2) the feature polypeptide of 2 albumen of major yolk protein is derived from, its sequence is as shown in SEQ ID NO.11-12； And/or
(3) the feature polypeptide of tropomyosin albumen is derived from, its sequence is as shown in SEQ ID NO.19-23.
10. reagent or kit for differentiating U.S.'s meat ginseng, it is characterised in that the reagent or kit will comprising right The feature polypeptide described in 1 or 2 is sought, or includes feature polypeptide SEQ ID NO.1-3,11-2 and/or 19-23 at the same time.
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