Pearl powder protein polypeptide CCP sequence and drug application thereof

Abstract

The invention discloses a pearl powder protein polypeptide CCP sequence and a medicine application of the pearl powder protein polypeptide CCP sequence. The amino acid sequence of the pearl powder protein polypeptide CCP sequence is CGPCPTVTNAYILDGPYLIGTDREYQCATGYGLVGSAYITCQPDGTWTKPNLQC; the compound has the effects of promoting the proliferation activity of A375 melanoma cells and improving the concentrations of C3 and C4 of the A375 cells, can be used for preparing medicines or foods for regulating complement system activation and enhancing the immunity of a human body, and can also be used for preparing targeted medicines for regulating a complement system.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a pearl powder protein polypeptide CCP sequence and its pharmaceutical applications. Background Technology

[0002] Proteins containing complement control protein modules (CCP modules) are generally classified into two categories: regulators of complement activation (RCA) and non-RCA proteins. The CCP domains are relatively conserved across different proteins; each protein may contain multiple CCP domains, although the number can vary. Studies have shown that these proteins may contain other domains besides CCP domains, such as transmembrane domains and receptor-binding domains. CCP domains have important functions, including binding antigen molecules and participating in cell signal recognition. In addition to their role in immune regulation and cell signal transduction, CCP domains may also play a role in the regulation of cellular carcinogenesis.

[0003] Pearl powder contains a large amount of protein and is often believed to enhance human immunity. If it can be confirmed that it contains CCP sequences and peptides can be synthesized from them, its effects on the complement system and immune function can be explored at the cellular level. This would provide a molecular biological basis for the traditional Chinese medicine theory that pearl powder enhances human immunity, and would also provide a basis for the development of drugs that regulate complement system activation and enhance human immunity, which is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pearl powder protein polypeptide CCP sequence and its pharmaceutical applications. The polypeptide has the effect of promoting the proliferation of A375 melanoma cells and increasing the concentration of C3 and C4 in A375 cells. It can be used to prepare drugs or foods that regulate complement system activation and enhance human immunity, and can also be used to prepare targeted drugs that regulate the complement system.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A pearl powder protein polypeptide CCP sequence, the amino acid sequence of which is as follows:

[0007] CGPCPTVTNAYILDGPYLIGTDREYQCATGYGLVGSAYITCQPDGTWTKPNLQC.

[0008] The CCP sequence of the pearl powder protein polypeptide is selected from a fragment of the pearl powder protein polypeptide sequence.

[0009] A pharmaceutical application of a pearl powder protein polypeptide CCP sequence, which is used to prepare drugs that regulate complement system activation, enhance human immunity, and target drugs that regulate the complement system.

[0010] The beneficial effects of this invention are as follows: This invention selects the CCP sequence from proteins identified in pearl powder and synthesizes polypeptides based on it. When applied to human A375 melanoma cells, it not only does not inhibit cell proliferation (i.e., it is non-cytotoxic), but also protects cells from UV damage and promotes cell proliferation after the cells are exposed to ultraviolet radiation. This further confirms that the CCP structure is related to the activation of the complement system, and also provides molecular-level evidence that pearl powder, as a traditional Chinese medicine, can enhance human immunity. Therefore, it can be used to prepare drugs that regulate complement system activation and enhance human immunity. In addition, it can also increase the concentration of intracellular C3 and C4, and can become a targeted drug for regulating the complement system. Attached Figure Description

[0011] Figure 1 The graph illustrates the effect of CCP on the proliferative activity of A375 melanoma cells without ultraviolet light irradiation.

[0012] Figure 2 The graph illustrates the effect of CCP on the proliferative activity of A375 melanoma cells under ultraviolet light irradiation.

[0013] (In the figure, Group A: different concentrations of CCP peptide were added after UV irradiation; Group B: different concentrations of CCP peptide were added before UV irradiation; * indicates P<0.05, ** indicates P<0.01)

[0014] Figure 3 A graph illustrating the effect of CCP on the concentration of human complement component C3 in A375 melanoma cells;

[0015] Figure 4 The graph illustrates the effect of CCP on the concentration of human complement component C4 in A375 melanoma cells. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0017] Example 1

[0018] The amino acid sequence of the pearl powder protein polypeptide CCP sequence is as follows:

[0019] CGPCPTVTNAYILDGPYLIGTDREYQCATGYGLVGSAYITCQPDGTWTKPNLQC.

[0020] Example 2

[0021] The specific steps for determining the effect of the pearl powder protein polypeptide CCP sequence on the proliferation activity of human A375 melanoma cell line are as follows:

[0022] (a) Preparation of polypeptide solution:

[0023] Take 1 mg of the polypeptide sample with the CCP sequence of pearl powder protein polypeptide shown above from the refrigerator and let it stand for 20 min to return to room temperature. Centrifuge to allow the polypeptide to settle to the bottom of the sample tube. Slowly open the tube cap, add 2 ml of DMEM culture medium, close the tube cap, and shake thoroughly to dissolve. Centrifuge to allow the solution to settle to the bottom of the sample tube. Use a 5 ml syringe to draw up the solution and filter it through a 0.22 μm microporous membrane into a 2 ml centrifuge tube to obtain 2 ml of 500 μg / ml solution. Then perform serial dilutions to prepare polypeptide solutions of the following concentrations: 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 μg / ml.

[0024] (b) Assay for cell proliferation activity:

[0025] Experiment 1

[0026] A375 cells were placed in DMEM medium, and FBS (containing 10 v / v % FBS) was added to adjust the cell concentration to 2 × 10⁻⁶ cells / year. 5 Inoculate 100 μl of each peptide solution per well into a 96-well plate and incubate for 24 h, then discard the culture medium. Add 100 μl of peptide solutions of 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 μg / ml into different wells, with each concentration set up in 5 replicates. Incubate for another 20 h, then add 10 μl of 5 mg / ml MTT to each well and incubate for another 4 h. After that, aspirate the culture medium, add 90 μl of DMSO to each well, place the 96-well plate in a microplate shaker and shake for 10 min, then measure the absorbance at 490 nm.

[0027] Experiment 2

[0028] Experimental Group A: A375 cells treated with ultraviolet light (UV irradiation conditions: UV light emitted by the lamp has a wavelength of 290–390 nm, a peak wavelength of 365 nm, and an UV index of 150 μW·cm at 30 cm vertically below the lamp). -2 Cells were placed in DMEM medium (with UV irradiation for 90 seconds) and FBS (containing 10 v / v% FBS) was added to adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 5Inoculate 100 μl of each peptide solution per well into a 96-well plate and incubate for 24 h, then discard the culture medium. Add 100 μl of peptide solutions of 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 μg / ml into different wells, with each concentration set up in 5 replicates. Incubate for another 20 h, then add 10 μl of 5 mg / ml MTT to each well and incubate for another 4 h. After that, aspirate the culture medium, add 90 μl of DMSO to each well, place the 96-well plate in a microplate shaker and shake for 10 min, then measure the absorbance at 490 nm.

[0029] Experimental Group B: A375 cells were placed in DMEM medium, and FBS (containing 10 v / v% FBS) was added to adjust the cell concentration to 2 × 10⁻⁶ cells / year. 5 Inoculate 100 μl of each peptide solution per well into a 96-well plate and incubate for 24 h, then discard the culture medium. Add 100 μl of peptide solutions at concentrations of 500, 250, 125, 62.5, 31.25, 15.63, and 7.81 μg / ml to different wells, with five replicates per concentration. Then subject the plates to UV irradiation (UV irradiation conditions: UV light emitted by the lamp has a wavelength of 290–390 nm, a peak wavelength of 365 nm, and a UV index of 150 μW·cm⁻¹ at 30 cm vertically below the lamp). -2 After irradiating with UV lamp for 90 s, and continuing to incubate for 20 h, add 10 μl of 5 mg / ml MTT to each well and continue incubating for 4 h. Then, aspirate the culture medium, add 90 μl of DMSO to each well, place the 96-well plate in a microplate shaker and shake for 10 min, and then measure the absorbance value at a wavelength of 490 nm.

[0030] Example 3

[0031] The effect of the pearl powder protein polypeptide CCP sequence on the concentration of human complement component C3 in human A375 melanoma cell line was determined by the following steps:

[0032] (a) Preparation of polypeptide solution:

[0033] Peptide solutions of 250, 125, 62.5, 31.25, and 0 μg / ml were prepared by serial dilution according to step (a) of Example 2.

[0034] (b) Determination of the concentration of human complement component C3:

[0035] A375 cells were placed in DMEM medium, and FBS (containing 10 v / v % FBS) was added to adjust the cell concentration to 2 × 10⁻⁶ cells / year. 5Cells were seeded at 1 ml / well in 24-well plates and cultured for 24 hours. The culture medium was then aspirated. 1 ml of prepared peptide solutions (250, 125, 62.5, 31.25, and 0 μg / ml) were added to each well, with five replicates per concentration. Cells were cultured for another 24 hours. After culture, cells were digested with 0.25% trypsin solution, collected, and centrifuged for 10 minutes (1000 rpm). The supernatant was discarded, and the cells were washed three times with sterile phosphate-buffered saline (PBS). Cells were then seeded at 1 × 10⁻⁶ cells / ml. 6 Add 200 μl of PBS to each centrifuge tube, resuspend the cells, and lyse them using a cell disruptor. Centrifuge each extract for 10 min (1000 r / min) to complete the preliminary treatment. Use the supernatant as the test sample and perform detection using a human complement component 3 (C3) enzyme-linked immunosorbent assay kit purchased from Sangon Biotech (Shanghai) Co., Ltd. Statistical analysis was performed using SPSS 21.0 software. One-way ANOVA was used for comparisons between groups, with P < 0.05 considered statistically significant. Graphs were generated using GraphPad Prism 8.0.2.

[0036] The specific operating steps for detecting human complement component 3 (C3) using an enzyme-linked immunosorbent assay kit are as follows:

[0037] (1) Preparation of washing solution: Dilute the 25-fold concentrated washing solution with ultrapure water to make a 1-fold application solution.

[0038] (2) Gradual dilution of standard: Add 1 ml of standard diluent to the lyophilized standard, dissolve it completely, and prepare a standard working solution of 100 ng / ml. Then perform serial dilution to prepare standard working solutions of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0 ng / ml.

[0039] (3) Remove the pre-coated ELISA plate and add 100 μl of the working solution of different concentrations of standard solution and the test samples after different concentrations of peptide solution have been applied to A375 cells to the reaction wells. Perform two replicates for each standard and sample. Seal the plate and incubate at 37°C for 90 min. (When adding samples, place them at the bottom of the ELISA plate, avoiding contact with the well walls as much as possible. Gently shake to mix, avoiding air bubbles; the sample addition time should be controlled within 10 min.)

[0040] (4) Discard the liquid, spin dry, add 100 μl of biotin-labeled complement component 3 antibody working solution to each reaction well, seal the plate and incubate at 37°C for 60 min.

[0041] (5) Washing: Discard the liquid, spin dry, add 350 μl of the washing solution prepared in step (1) to each reaction well, soak for 1-2 min, spin dry the washing solution; repeat 4 times.

[0042] (6) Add 100 μl of HRP-labeled streptavidin working solution to each reaction well, seal the plate and incubate at 37°C for 30 min.

[0043] (7) Washing: Add 300 μl of the washing solution prepared in step (1) to each reaction well, and shake off the washing solution after 30 s intervals. Repeat 4 times.

[0044] (8) Add 90 μl of color developer (protected from light) to each reaction well, seal the plate, and develop the color at 37°C in the dark for about 15 min.

[0045] (9) Add 50 μl of stop solution to each reaction well and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 min).

[0046] (10) OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0047] (11) Calculate the average OD value of the standard and sample: the OD value of each standard and sample should be subtracted from the OD value of the zero well.

[0048] Example 4

[0049] The effect of the pearl powder protein polypeptide CCP sequence on the concentration of human complement component 4 (C4) in human A375 melanoma cell line was determined using an enzyme-linked immunosorbent assay kit for human complement component 4 (C4) purchased from Sangon Biotech (Shanghai) Co., Ltd., following the steps described in Example 3. However, the step (2) of standard serial dilution in the supernatant detection was changed to: adding 2 ml of standard diluent to the lyophilized standard, dissolving it completely, and preparing a 200 ng / ml standard working solution, and then performing serial dilutions to prepare 200, 100, 50, 25, 12.5, 6.25, 3.13, and 0 ng / ml standard working solutions. The measured data were statistically analyzed using SPSS 21.0 software, and the intergroup comparisons were performed using one-way ANOVA. P < 0.05 was considered statistically significant. The statistically analyzed data were plotted using GraphPad Prism 8.0.2.

[0050] Experimental results:

[0051] 1. Effects of CCP sequence peptides on the proliferation activity of A375 cells

[0052] Under conditions without ultraviolet light irradiation, A375 cells were treated with solutions of pearl powder protein CCP sequence fragments at concentrations of 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 μg / ml for 24 h. The changes in cell proliferation activity were as follows: Figure 1 As shown. By Figure 1 It was found that, compared with the blank control group (with 0 μg / ml CCP sequence peptide solution), the cell proliferation activity changed very little after the addition of CCP sequence peptide solution, and there was a significant difference from the blank control group only at a concentration of 125 μg / ml (P<0.05). The cell proliferation activity was not significantly correlated with the concentration of CCP sequence peptide, indicating that CCP does not inhibit the growth of A375 cell line and has no drug toxicity to the cell line.

[0053] Pearl powder protein CCP sequence fragment polypeptide solutions at concentrations of 250, 125, 62.5, 31.25, 15.63, and 7.81 μg / ml were added to A375 cells after UV irradiation (Group A) and before UV irradiation (Group B) for 24 h. Changes in cell proliferation activity were observed as follows: Figure 2 As shown in the figure. Compared with the control group (containing 0 μg / ml CCP sequence peptide solution), experimental group A (A375 cells irradiated with ultraviolet light followed by CCP) showed significantly increased cell proliferation activity, which increased with increasing concentration, reaching its highest level at a CCP concentration of 125 μg / ml (P<0.05). Meanwhile, compared with the control group, experimental group B (A375 cells irradiated with ultraviolet light after CCP addition) also showed significantly increased cell proliferation activity, but this did not change significantly with increasing concentration (P<0.05). Compared with experimental group A, when CCP was added at the same concentration, group B showed significantly higher cell proliferation activity than group A at CCP concentrations of 7.81 and 15.63 μg / ml (P<0.05). However, with increasing concentrations of 31.25, 62.5, 125, and 250 μg / ml, group A showed significantly higher cell proliferation activity than group B, with significant differences at CCP concentrations of 62.5 and 125 μg / ml (P<0.05). The experiment demonstrates that whether CCP was added before or after UV irradiation, the CCP sequence fragment peptide of pearl powder protein effectively protected the proliferation activity of A375 cell line.

[0054] 2. Effects of CCP sequence peptides on the concentrations of human complement components C3 and C4 in A375 cells

[0055] A375 cells were treated with CCP sequence peptide solutions at concentrations of 250, 125, 62.5, and 31.25 μg / ml for 24 h. The intracellular concentrations of human complement components C3 and C4 were as follows: Figure 3 , 4 As shown. Figure 3The concentration of complement component C3 in humans increased with increasing concentration of CCP sequence peptide solution. The difference between the peptide concentration of 31.25 μg / ml and the control group was small, but the concentrations of 62.5, 125, and 250 μg / ml were significantly different from the control group (P<0.05). Figure 4 The concentration of complement component C4 in humans was not significantly different from that in the control group when the peptide solution concentration was 31.25 and 62.5 μg / ml, but there were significant differences compared with the control group when the concentration was 125 and 250 μg / ml (P<0.05), and the higher the peptide concentration, the higher the C4 concentration.

[0056] The above results show that the CCP sequence polypeptide of pearl powder, when applied to human A375 melanoma cells, not only did not inhibit cell proliferation (i.e., it was not cytotoxic), but also protected cells from UV damage and promoted cell proliferation after exposure to ultraviolet light. This further confirms that the CCP structure is related to the activation of the complement system, and provides molecular-level evidence that pearl powder, as a traditional Chinese medicine, can enhance human immunity. Therefore, it can be used to prepare drugs that regulate complement system activation and enhance human immunity, and can also increase the concentration of intracellular C3 and C4, making it a potential target drug for regulating the complement system, such as immunoglobulins, lectins, etc.

[0057] In this embodiment of the invention, fetal bovine serum (FBS), DMEM culture medium, and MTT cell proliferation assay kit were purchased from BBI Corporation, USA.

[0058] In this invention, the CCP sequence of the pearl powder protein polypeptide is a CCP sequence fragment selected from proteins identified in pearl powder, and the polypeptide sample with the CCP sequence of the pearl powder protein polypeptide used in the examples was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific method for identifying proteins and screening CCP sequences from pearl powder is as follows: (A) Sample pretreatment

[0059] Pearl powder with a particle size of less than 48 μm after sieving was washed with ultrapure water, centrifuged, and dried to obtain a pearl powder sample.

[0060] (B) Extraction of organic matrix

[0061] B1. Take 100g of the treated pearl powder sample, stir it in 200ml of ultrapure water at 8℃ for 48h, and centrifuge it at 8000g for 20min at 4℃, and then collect the supernatant.

[0062] B2. The supernatant obtained by separation and collection is filtered at room temperature using a microporous membrane with a pore size of 0.22 μm.

[0063] B3. The obtained supernatant was dried at -84°C using a vacuum freeze dryer to obtain a water-soluble matrix (WSM) of pearl powder.

[0064] B4. Wash the lower precipitate three times with ultrapure water, then slowly add 10% acetic acid while stirring continuously until no more CO2 gas is produced.

[0065] B5. The obtained suspension (final pH value is about 4.0) was centrifuged at 8000g for 30min at 4℃, the supernatant was collected and dialyzed with 1L of ultrapure water at 8℃ for 4 days.

[0066] B6. The dialysis solution was filtered through a microporous membrane with a pore size of 0.22 μm at room temperature and freeze-dried at -84 °C to obtain the acid-soluble matrix (ASM).

[0067] B7. The final precipitate was washed three times with 37% acetic acid and ultrapure water, centrifuged at 8000g at 4°C and freeze-dried at -84°C to obtain the acid-insoluble matrix (AIM).

[0068] (C)SDS-PAGE electrophoresis

[0069] C1. Prepare the separating gel solution (12% 15ml PAGE) as shown in Table 1; prepare the stacking gel solution (5% 6ml PAGE) as shown in Table 2; place the reagents shown in Table 3 in a 1L beaker, add 800ml H2O to dissolve, and then bring the volume to 1L with a volumetric flask to obtain 5× electrophoresis buffer. Store at room temperature and dilute to 1× electrophoresis buffer before use.

[0070] Table 1

[0071] Element Added amount Tetramethylethylenediamine (TEMED) 6μl 10% Ammonium Persulfate (APS) 150μl 10% Sodium Dodecyl Sulfonate (SDS) 150μl 1.5M 4×Tris-HCl separating gel preparation buffer (pH 8.8) 3.8ml Acrylamide / N-methylenebisacrylamide 30% solution (30% Acr / Bis) 6ml <![CDATA[H2O]]> 4.9ml

[0072] Table 2

[0073] Element Added amount Tetramethylethylenediamine (TEMED) 6μl 10% Ammonium Persulfate (APS) 60μl 10% Sodium Dodecyl Sulfonate (SDS) 60μl 0.5M 4×Tris-HCl separating gel preparation buffer (pH 6.8) 0.75ml Acrylamide / N-methylenebisacrylamide 30% solution (30% Acr-Bis) 1ml <![CDATA[H2O]]> 4.1ml

[0074] Table 3

[0075] Element Added amount Tris(hydroxymethyl)aminomethane (Tris) 15.1g Glycine 94g Sodium dodecyl sulfonate (SDS) 5g

[0076] C2. Sample preparation, electrophoresis, staining and destaining

[0077] C2-1 Sample preparation: During the stacking gel polymerization, mix the 4× gel loading buffer (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with the sample to be electrophoresed and boil for 5 minutes to denature the proteins.

[0078] C2-2, Electrophoresis: After the stacking gel has polymerized (30 min), remove the comb, rinse the sample wells thoroughly with distilled water, add an appropriate amount of sample (20 μl) and three-color pre-stained protein marker (5 μl), connect the electrophoresis apparatus to the power supply (note that the red positive electrode should be added to the lower tank), first use a voltage of 80 V / cm to make the sample migrate in the stacking gel, after the bromophenol blue enters the separating gel, increase the voltage to 100 V / cm until the bromophenol blue enters the bottom of the separating gel, then stop electrophoresis.

[0079] C2-3. Staining: After electrophoresis, pry open the glass plate, mark the gel plate, place it in a large petri dish, and stain using a low-background silver staining kit.

[0080] Protein identification on gel strips involves separating sample proteins by gel electrophoresis and then obtaining protein gel strips at different locations on a membrane.

[0081] (D) Protein digestion and high-performance liquid chromatography (HPLC) purification

[0082] Samples were digested with 5 μg of trypsin in 10-fold volume of 50 mM NH4HCO3 at 37 °C for 4 h. The digested samples were desalted using a C18 filter and dried in a vacuum concentrator for LC-MS / MS analysis. The dried peptide samples were remixed with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20000 g for 10 min, and the supernatant was injected. Separation was performed using a Thermo UltiMate 3000 UHPLC. The sample was first enriched and desalted in a trapping column, then fed into a C18 column (75 μm inner diameter, 3 μm column diameter, 25 cm length) and separated at a flow rate of 300 nL / min using the following effective gradients: 0–5 min, 5% mobile phase B (98% CAN, 0.1% FA); 5–45 min, mobile phase B linearly increasing from 5% to 25%; 45–50 min, mobile phase B increasing from 25% to 35%; 50–52 min, mobile phase B increasing from 35% to 80%; 52–54 min, 80% mobile phase B; 54–60 min, 5% mobile phase B. The nanoliter liquid chromatography end was directly connected to the mass spectrometer.

[0083] (E) Mass spectrometry detection

[0084] Peptides separated by liquid chromatography were ionized by a nanoESI source and then detected by DDA (data correlation acquisition) mode using a Q-Exactive HF X tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA).

[0085] Key parameter settings: Ion source voltage set to 1.9 kV; MS1 scan range 350–1500 m / z; resolution set to 60000; MS2 starting m / z fixed at 100; resolution set to 15000. MS2 fragmentation ion screening criteria: the top 30 precursor ions with charges 2+ to 6+ and peak intensities exceeding 10000. Ion fragmentation mode set to HCD; fragment ions detected in Orbitrap. Dynamic exclusion time set to 30 s. AGC settings: MS1 3E6, MS2 1E5.

[0086] (F) Database search and protein identification

[0087] Mass spectrometry data were searched using the MASCOT search engine in a local database [see Shen W, Hu Y, He Z, et al. Histological and Comparative Transcriptome Analyses Provide Insights Into the Immune Response in Pearl Sac Formation of Hyriopsis cumingii[J]. Frontiers in Marine Science, 2020, 7:256.]. The peptide mass tolerance was 20 ppm, and the fragment mass tolerance was 0.05 Da. Other search parameters were carbamate methylation as a fixed modification, methionine oxidation, N-terminal Gln→pyro-Glu, and deamidation as variable modifications. In this pipeline, results from the search engine were preprocessed and re-scored using Percolator to improve matching accuracy. The output results were then filtered by 1% spectral-level FDR (PSM-level FDR <= 0.01) to obtain a significant identification spectrum and peptide list. Protein inference was then performed on the peptides according to the resolution principles, generating a series of proteomes.

[0088] The obtained protein peptides were manually compared with laboratory databases to determine their DNA sequences, and compared with other sequences of mollusc shell proteins.

[0089] Specifically, the Mascot 2.1 search engine was used to search the translated pearl sac transcriptome database of *Saccharomyces cerevisiae*, with aminomethanized cysteine ​​as a fixed modification and oxidized methionine and tryptophan as variable modifications. The mass tolerances for precursors and fragments were set to 10 ppm and 0.5 Da, respectively. Finally, sequences with a Mascot score of at least 10.0 and at least two matching peptide fragments were considered valid. Using the established pearl sac transcriptome as a database, the local blastn program was used to query for genes corresponding to the proteins identified in the LC-MS / MS experiments described above; complement control proteins (CCPs) in the proteins were identified based on the biological function of the protein annotations and the domains predicted by SMART.

[0090] Proteomics was used to identify proteins in the GenBank nonredundant protein sequence (NR) database using the BLASTp tool from the National Center for Biotechnology Information (NCBI, http: / / blast.ncbi.nlm.nih.gov / blast.cgi) and the UniProKB and SwissProt databases using the BLAST tool from UniProt (https: / / www.uniprot.org / blast / ). Functional domains were predicted using the Translate tool from the Expasy server (https: / / web.expasy.org / translate / ) and the SMART analysis service (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?NORMAL=1), and confirmed by another domain prediction online service, InterProScan (https: / / www.ebi.ac.uk / interpro / ). The secondary structure of the protein sequence was predicted by PSIPRED 4.0 (http: / / bioinf.cs.ucl.ac.uk / psipred), and its three-dimensional structure was predicted by the CI-TASSER online service (http: / / bioinf.cs.ucl.ac.uk / psipred).

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pearl powder protein polypeptide CCP sequence, characterized in that, Its amino acid sequence is CGPCPTVTNAYILDGPYLIGTDREYQCATGYGLVGSAYITCQPDGTWTKPNLQC.

2. The pearl powder protein polypeptide CCP sequence as described in claim 1, characterized in that, The CCP sequence of the pearl powder protein polypeptide is selected from a fragment of the pearl powder protein polypeptide sequence.

3. The pharmaceutical application of the pearl powder protein polypeptide CCP sequence as described in any one of claims 1-2, characterized in that, The CCP sequence of this pearl powder protein polypeptide can be used to prepare drugs that regulate complement system activation, enhance human immunity, and target drugs that regulate the complement system.

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