A characteristic polypeptide of Long Thick-nosed Sea Dragon and its application

Abstract

The present invention relates to a characteristic polypeptide of Longus scaly-nosed Sea Dragon, its application, and a method for identifying Longus scaly-nosed Sea Dragon, belonging to the field of biotechnology detection technology. The present invention provides a characteristic polypeptide of Longus scaly-nosed Sea Dragon, the amino acid sequence of which is shown in SEQ ID NO. 1. The characteristic polypeptide provided by the present invention has excellent specificity and stability for Longus scaly-nosed Sea Dragon, has high specificity, can be used for identifying Longus scaly-nosed Sea Dragon, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology detection, and in particular to a characteristic polypeptide of a long-snouted sea dragon, an application thereof, and a method for identifying the long-snouted sea dragon. Background Art

[0002] Sea dragon is an important animal medicinal material in my country, known for its kidney-warming, yang-strengthening, and swelling-reducing properties. It has a long history of medicinal use and numerous sources. The Chinese Pharmacopoeia defines sea dragon as the dried body of the Syngnathidae family members Solenognathus hardwickii (Gray), Syngnathoides biaculeatus (Bloch), or Syngnathusa cusLinnaeus.

[0003] As a tonic medicinal material, the market demand for sea dragons is increasing year by year. However, due to the current inability to effectively cultivate sea dragons artificially, the wild resources of sea dragons are decreasing, and they are often confused with other varieties or sold as inferior products. At present, the sales proportion of sea dragons from different sources that are not included in the Chinese Pharmacopoeia is increasing year by year, resulting in chaotic circulation in the sea dragon medicinal material market. Long thick-snouted sea dragon is a common mixed variety in the market due to its large catch and low price. As an individual complete sea dragon medicinal material, long thick-snouted sea dragon is easy to distinguish from Diao sea dragon, pseudo sea dragon, and Shu's sea dragon due to the large differences in their characteristics. However, once they are powdered or processed into extracts, the characteristics disappear and they cannot be distinguished. In addition, long thick-snouted sea dragon is similar to thick-snouted sea dragon and Ge's sea bracelet fish in characteristics, so it is not easy to distinguish them simply by identifying based on their characteristics.

[0004] Currently, the main methods for identifying and controlling the species and quality of sea dragons include trait identification, HPLC fingerprinting, and molecular biology. Trait identification generally requires intact sea dragon individuals and extensive experience as the identifier, which is subject to some subjectivity. HPLC fingerprinting has low specificity. Molecular biology, while highly specific, is limited by the preservation of genetic material in the sample. DNA extraction is difficult for sea dragon medicinal liquors, processed products, extracts, samples damaged by insects and mold, and samples containing multiple individuals. In recent years, an increasing number of methods and standards have used signature peptides for the identification or quantification of natural medicinal products. Proteins from different species differ in amino acid residues at specific sites. These sites can be extracted using appropriate proteases to create peptides with identifying properties, known as signature peptides. Signature peptides are typically detected using mass spectrometry, which eliminates the need to consider protein activity, is simple to prepare, and offers relatively stable peptide properties. Existing sea dragon identification methods have numerous limitations, hindering their application to sea dragon derivatives such as powders, processed products, and extracts. Signature peptides from sea dragon proteins have the potential to serve as indicators for sea dragon species identification. At present, no protein sequence information related to the long-snouted sea dragon is included in various databases, and differential peptides cannot be obtained through protein sequence comparison. Therefore, there is no relevant record of the characteristic polypeptides of the long-snouted sea dragon. Summary of the Invention

[0005] The present invention aims to provide a characteristic polypeptide of Longus stoloniferum, its application, and a method for identifying Longus stoloniferum. The characteristic polypeptide provided by the present invention has excellent specificity and stability for Longus stoloniferum, and has strong specificity. It can be used to identify the species of sea dragon medicinal materials, specifically Longus stoloniferum, and has good application prospects.

[0006] The present invention provides a characteristic polypeptide of the long rough snout sea dragon, and the amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying the long rough snout sea dragon.

[0008] Preferably, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 779.6→1095.6.

[0009] The present invention also provides a method for identifying the long-snout sea dragon, using the characteristic polypeptide shown in SEQ ID NO.1 as a reference substance.

[0010] The present invention also provides a method for identifying the long-snout sea dragon based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0011] (1) Preparation of the test solution: The pretreated sea dragon extract or the pretreated sea dragon powder extract was mixed with trypsin to obtain a mixed solution, the mixed solution was mixed with an aqueous ammonium bicarbonate solution, and the mixture was enzymatically hydrolyzed and filtered to obtain the test solution;

[0012] (2) Preparation of reference solution: using the characteristic peptide of the long-thick-rhinated sea dragon described in the above technical solution as a reference substance, dissolving it in water to obtain a reference solution;

[0013] (3) Detection and analysis: Detection and analysis were performed using triple quadrupole mass spectrometry;

[0014] There is no time sequence limitation between the steps (1) and (2).

[0015] Preferably, in step (1), the method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water and decocting it three times, with the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting them with an aqueous ammonium bicarbonate solution to obtain the pretreated sea dragon extract;

[0016] The preparation method of the pretreated sea dragon powder extract comprises the following steps: mixing sea dragon powder with water and decocting three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting with an aqueous ammonium bicarbonate solution to obtain the pretreated sea dragon powder extract;

[0017] Preferably, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml.

[0018] Preferably, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0019] Preferably, the enzymatic hydrolysis condition is 37° C. for 2 h.

[0020] Preferably, the triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute.

[0021] Preferably, the triple quadrupole mass spectrometry uses a mass spectrometer detector in electrospray positive ion mode for multiple reaction monitoring, and selects a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 and a qualitative ion with a mass-to-charge ratio of m / z 779.6→1095.6 for SEQ ID NO.1 as the detection ion pair.

[0022] The present invention provides a common characteristic polypeptide of the long-snouted sea dragon. This characteristic polypeptide, as an indicator component for identifying long-snouted sea dragons, can distinguish them from other sea dragon species, laying the foundation for the development of identification methods for long-snouted sea dragons and related products. Specifically, the characteristic polypeptide of the present invention has the following beneficial effects:

[0023] (1) The present invention extracts differential polypeptide segments from the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and the morphology and processing of the sea dragon sample are not affected. This fills the gap in the identification of the long-snouted sea dragon species and the detection method of the long-snouted sea dragon in sea dragon products.

[0024] (2) The characteristic peptides and detection methods provided by the present invention provide a reference for finding characteristic peptides in closely related species that are lacking in the database;

[0025] (3) The characteristic polypeptide provided by the present invention has excellent specificity and stability for the long-snouted sea dragon, strong specificity, can be used for the identification of the long-snouted sea dragon medicinal material, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawing is a specific map of the characteristic peptide SEQ ID NO.1 of the long rough rhinoplastus provided by the present invention;

[0027] in, Figure 1 : SEQ ID NO.1 characteristic peptide control; Figure 2 : Sample of Long Thick-snouted Sea Dragon; Figure 3 : Sample of Ge's sea bracelet fish; Figure 4 : Sample of Roughnose Sea Dragon; Figure 5 : Diao Hailong sample; Figure 6 : Sample of Pseudohalodon; Figure 7 : Sample of Multispined Sea Dragon; Figure 8 : Sample of Schuler's Sea Dragon; Figure 9 : Baojia spear-kissed sea dragon sample. DETAILED DESCRIPTION

[0028] The present invention provides a characteristic polypeptide of a long thick-snouted sea dragon, the amino acid sequence of which is shown in SEQ ID NO.1: GDSGFTGPSGPAGPAGAR. The present invention provides water extracts of different species of sea dragons, such as a long thick-snouted sea dragon, a thick-snouted sea dragon, a Ge's sea dragon, a Diao sea dragon, and a Shu's sea dragon, which are enzymatically hydrolyzed with trypsin and then analyzed using a nano-liquid chromatography-high-resolution mass spectrometer to obtain a mass spectrum of the full peptide segment of the sea dragon protein; the mass spectrum data are subjected to chemometric analysis, and the specificity of the experimental results is verified using a triple quadrupole mass spectrometer; and finally, a characteristic ion pair is provided, corresponding to a characteristic peptide segment, and the specificity verification result is good.

[0029] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying the long rough snout sea dragon.

[0030] In the present invention, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 779.6→1095.6.

[0031] The present invention also provides a method for identifying the long-snout sea dragon based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0032] (1) mixing the pretreated sea dragon extract or the pretreated sea dragon powder extract with trypsin to obtain a mixed solution, mixing the mixed solution with an aqueous ammonium bicarbonate solution, performing enzymatic hydrolysis and filtering, and taking the filtrate to obtain a test solution;

[0033] (2) using the common characteristic peptide of the long-thick-rhined sea dragon described in the above technical solution as a reference substance, adding an ammonium bicarbonate aqueous solution to dissolve it to obtain a reference solution;

[0034] (3) Detection and analysis using triple quadrupole mass spectrometry;

[0035] There is no time sequence limitation between the steps (1) and (2).

[0036] The method of the present invention extracts differential polypeptide segments from the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and it is not affected by the morphology and processing of the sea dragon sample. It fills the gaps in the identification of the long-snouted sea dragon species and the detection method of the long-snouted sea dragon in sea dragon products, and provides a reference for the search for characteristic peptide segments in closely related species that are lacking in the database.

[0037] The present invention mixes the pretreated sea dragon extract or the pretreated sea dragon powder extract with trypsin to obtain a mixed solution, mixes the mixed solution with an aqueous solution of ammonium bicarbonate, filters after enzymatic hydrolysis, and takes the filtrate to obtain a test solution. In the present invention, the preparation method of the pretreated sea dragon extract preferably includes the following steps: crushing the long thick snout sea dragon, mixing it with water and decocting it three times, the decoction time is 4h, 3h and 2h respectively, combining the decoctions, and diluting it with an aqueous solution of ammonium bicarbonate to obtain a pretreated sea dragon extract. The present invention preferably takes 100g of crushed sea dragon, and the decoction is preferably 500mL in total. In the present invention, the dilution is preferably diluted 10 times. Specifically, the present invention preferably takes 5mL of the decoction, places it in a 50mL measuring flask, dilutes it to the scale with an aqueous solution of ammonium bicarbonate, shakes well, and obtains a pretreated sea dragon extract. In the present invention, the method for preparing the pretreated sea dragon powder extract preferably comprises the following steps: mixing sea dragon powder with water and decocting three times for 4 hours, 3 hours, and 2 hours, respectively; the decocting times are then combined to obtain the pretreated sea dragon powder extract. In the present invention, preferably, 1 gram of sea dragon powder is used, and the decoction is preferably 50 mL in total. Specifically, 5 mL of the decoction is preferably placed in a 50 mL volumetric flask, diluted to the mark with aqueous ammonium bicarbonate, and shaken to obtain the pretreated sea dragon powder extract. After obtaining the pretreated sea dragon extract and pretreated sea dragon powder extract, the present invention preferably measures 1-5 mL of the sea dragon extract or sea dragon powder extract, preferably adds 100 μL of trypsin solution, shakes well, then adds aqueous ammonium bicarbonate to 50 mL, shakes well, seals, and then performs enzymatic hydrolysis. In the present invention, the enzymatic hydrolysis is preferably performed at 37°C for 2 hours. After enzymatic hydrolysis, the flask is preferably cooled, more preferably cooled to room temperature, which is preferably in the range of 10-30°C. After cooling, the present invention performs filtration, and takes the filtrate as the test solution.

[0038] In the present invention, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml. In the present invention, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0039] The present invention uses the common characteristic peptide of the long-snapping sea dragon described in the above technical solution as a reference substance, dissolves it in water, and obtains a reference solution. The present invention does not specifically limit the source of the long-snapping sea dragon characteristic peptide, and artificial synthesis methods can be used. For example, the long-snapping sea dragon characteristic peptide in the present invention was synthesized according to a specified amino acid sequence by Nanjing Source Peptide Biotechnology Co., Ltd.

[0040] The present invention utilizes triple quadrupole mass spectrometry for detection and analysis. In the present invention, the triple quadrupole mass spectrometry preferably uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute. In the present invention, the triple quadrupole mass spectrometry preferably uses a mass spectrometer detector, electrospray positive ion mode, and multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 and a qualifier ion with a mass-to-charge ratio of m / z 779.6→1095.6.

[0041] The following is a further detailed introduction to the characteristic polypeptide of the long-snouted sea dragon, its application, and the method for identifying the long-snouted sea dragon in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0042] Triple quadrupole mass spectrometry conditions

[0043] Gradient elution was performed (0-3 min, 5% A → 8% A) using 0.1% formic acid in acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; the flow rate was 0.5 ml / min. Multiple reaction monitoring (MRM) was performed using a mass spectrometer in electrospray positive ionization (ESI+) mode. For SEQ ID NO. 1, a quantification ion with a mass-to-charge ratio of m / z 779.6 → 994.5 and a qualifier ion with a mass-to-charge ratio of m / z 779.6 → 1095.6 were selected.

[0044] Example 1: Searching for characteristic peptides of Long Thick-nosed Sea Dragon

[0045] (1) Preparation of test solution

[0046] Sea dragon water extract: Take 100g of the test sample sea dragon, grind it, place it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively. Combine the decoctions to make 500mL in total, boil gently and concentrate until the liquid becomes viscous, transfer it to a silica gel bowl, and dry it in a 60℃ electric constant temperature forced air drying oven until it becomes solid to obtain the sea dragon water extract;

[0047] Test solution: Weigh 0.1 g of the water extract of the sea dragon, add 50 ml of 1% ammonium bicarbonate aqueous solution, and sonicate for 30 min to dissolve it. Filter with a microporous membrane, take 100 μL of the filtrate, add 10 μL of 1 mg / ml trypsin aqueous solution, and enzymatically hydrolyze at 37°C for 2 h. Take out and cool to room temperature.

[0048] (2) Selection of characteristic ions and sequence inference

[0049] After the sample solution was analyzed by nano-liquid chromatography-high-resolution mass spectrometry, the mass spectrometry data was imported into PEAKS 8.5 software for de novo sequencing and sequence prediction of all peptides in the sample, and the results were analyzed. From the analysis results, peptides that were only detected in E. longissima and almost not detected in other species of sea dragons were selected as parent ions. Their secondary spectra were analyzed one by one, and the daughter ions with better responses were selected. This analysis yielded the mass spectrometric ion pair information representing the differences of E. longissima: m / z 779.6 (doubly charged) → 994.5, 1095.6. Through further analysis, the corresponding peptide sequence of E. longissima was deduced to be the characteristic peptide sequence of E. longissima, SEQ ID NO. 1: GDSGFTGPSGPAGPAGAR.

[0050] According to the deduced amino acid sequence, a characteristic peptide reference substance of the long-snout sea dragon was commissioned to be synthesized. The reference substance and the long-snout sea dragon test solution were tested simultaneously. The retention time and secondary mass spectrometry information of the two were consistent, thus confirming the correctness of the sequence.

[0051] Example 2: Study on the specificity of the characteristic peptide of Long Thick Snout Sea Dragon

[0052] The water extracts of all species of sea dragons were tested using triple quadrupole mass spectrometry. The results showed that the three batches of long-spined sea dragons all had the characteristic peptide chromatographic peak of long-spined sea dragons, while other sea dragons such as multi-spined sea dragons, pseudo-sea dragons, schumeri sea dragons, and sea dragons did not have the corresponding chromatographic peak. Figure 1-9 The results showed that the characteristic peptide was unique to the long-snout sea dragon and could be used as an indicator component to identify the long-snout sea dragon.

[0053] Example 3: Application of the characteristic peptide of Long Thick Snout Sea Dragon in Sea Dragon Extract and Sea Dragon Powder

[0054] (1) Sample preparation

[0055] Preparation of the test solution of sea dragon extract: Take 100 g of each long-snout sea dragon test sample, crush it, place it in a conical flask, add water and boil it three times, for 4 hours, 3 hours, and 2 hours respectively, and combine the decoctions to make 500 mL in total; take 5 ml of sea dragon extract, place it in a 50 ml volumetric flask, add 1% ammonium bicarbonate aqueous solution to dilute to the scale, shake well, measure 1 ml to a 5 ml volumetric flask, add 100 μl of 1 mg / ml trypsin aqueous solution, shake well, and then add 1% ammonium bicarbonate aqueous solution to make up to the scale, shake well, seal it, and hydrolyze it at 37°C for 2 hours. Let it cool, filter it, and take the filtrate as the test solution.

[0056] Preparation of the test solution of sea dragon powder extract: grind 1 g of each long-snout sea dragon test sample, place it in a conical flask, add water and boil three times, for 4 h, 3 h, and 2 h respectively. Combine the decoctions to make a total of 50 mL, measure 1 ml into a 5 ml volumetric flask, and prepare according to the above method starting from "add 100 μl of 1 mg / ml trypsin aqueous solution".

[0057] Reference solution of the characteristic peptide of the long-scratch-rhinoplastus: weigh 10 mg of the characteristic peptide of the long-scratch-rhinoplastus SEQ ID NO. 1, place it in a 50 ml volumetric flask, add water to dissolve it and dilute to the scale to obtain the solution.

[0058] (2) The determination was performed using triple quadrupole mass spectrometry conditions.

[0059] (3) Sample measurement

[0060] Ten batches of collected sea dragon extract samples and five batches of sea dragon powder samples were tested, and the results showed that the characteristic peptide of sea dragon was detected in all of them, indicating that this characteristic peptide can be used to identify the long snout sea dragon components in sea dragon products.

[0061] Example 4: Three ion information with identification potential was screened out from 370 candidate ions. After verification by triple quadrupole mass spectrometry, it was found that not all ions can be used to identify the unique components of the long-snouted sea dragon. Some ions are not specific enough for the long-snouted sea dragon. Only the ion with a mass-to-nuclear ratio of 779.6 has good specificity. The specific results are shown in Table 1.

[0062] Table 1 Specificity verification results

[0063]

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A polypeptide characteristic of the long rough snout sea dragon, characterized in that: The amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.

1.

2. Use of the characteristic polypeptide according to claim 1 in identifying the long rough snout sea dragon.

3. The use according to claim 2, characterized in that The identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 779.6→1095.

6.

4. A method for identifying a long-snout sea dragon, characterized in that: The following steps are involved: (1) Preparation of the test solution: the pretreated sea dragon extract or the pretreated sea dragon powder extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an aqueous ammonium bicarbonate solution, and the mixture is enzymatically hydrolyzed and filtered, and the filtrate is collected to obtain the test solution; (2) Preparation of reference solution: using the characteristic peptide of the long-thick-rhined sea dragon as the reference substance according to claim 1, dissolving it in water to obtain a reference solution; (3) Detection and analysis: Detection and analysis are performed using triple quadrupole mass spectrometry; There is no time sequence limitation between the steps (1) and (2); In step (1), the method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water, and decocting it three times, with the decocting time being 4 hours, 3 hours, and 2 hours respectively, combining the decoctions, and diluting them with an aqueous ammonium bicarbonate solution to obtain the pretreated sea dragon extract; The preparation process of the pretreated sea dragon powder filtrate comprises the following steps: mixing sea dragon powder with water, decocting for 1 hour, filtering, and taking the filtrate to obtain the pretreated sea dragon powder filtrate; The trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml; The mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%; the enzymatic hydrolysis condition is 37° C. for 2 hours; The triple quadrupole mass spectrometry method used 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution: 0-3 min, 5% A→8% A; the flow rate was 0.5 ml per minute; The triple quadrupole mass spectrometry method uses a mass spectrometer detector in electrospray positive ion mode to perform multiple reaction monitoring. For SEQ ID NO.1, a quantitative ion with a mass-to-charge ratio of m / z 779.6→994.5 and a qualitative ion with a mass-to-charge ratio of m / z 779.6→1095.6 are selected as detection ion pairs.

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