An anti-tumor active protein SVAP derived from Phellinus igniarius mycelium and its application

Abstract

The present invention discloses an anti-tumor active protein SVAP derived from the mycelium of Phellinus igniarius and its application, belonging to the fields of genetic engineering and microbial technology. The amino acid sequence of the anti-tumor active protein SVAP is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene SVAP is shown in SEQ ID NO.1. The encoding gene SVAP of the protein SVAP in poplar Phellinus igniarius is expressed in the P. pastoris system, and the obtained recombinant protein has a certain inhibitory effect on human cervical cancer cells HeLa, human liver cancer cells Hep G2, mouse liver cancer cells Hepa 1-6, etc., showing the potential for medicinal development. The present invention utilizes a gene heterologous expression approach to realize the recombinant production of the anti-tumor active protein of Phellinus igniarius, providing a technical basis and data support for subsequent industrial development.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and microbial technology, and in particular to an anti-tumor active protein SVAP derived from phellinus igniarius mycelium and its application. Background Art

[0002] Edible and medicinal fungi are gaining increasing attention due to their safety and effectiveness, particularly their outstanding anti-tumor performance, demonstrating the enormous potential for discovery as natural anti-tumor drugs. Phellinus igniarius is one of the known edible and medicinal fungi with significant anti-tumor efficacy. Its polysaccharides, triterpenes, and other bioactive components have been shown to exhibit significant anti-tumor activity, but no reports have yet identified its anti-tumor active proteins.

[0003] Due to the scarcity of wild resources of Phellinus igniarius, the long cultivation cycle, the low natural protein content, and the difficulty in separation and purification, large-scale development technology is not yet mature enough to meet market demand. With the continuous maturation of molecular biology technology, the recombinant production of active proteins through heterologous gene expression pathways is expected to achieve industrial large-scale production of this medicinal resource.

[0004] Therefore, discovering anti-tumor active proteins in Phellinus igniarius and using gene heterologous expression pathways to recombinantly produce anti-tumor active proteins in Phellinus igniarius are of great significance for the industrial large-scale production of Phellinus igniarius, a scarce medicinal resource. Summary of the Invention

[0005] The present invention aims to provide an anti-tumor protein derived from the mycelium of Phellinus igniarius and its application to address the problems of the prior art. The present invention obtains a novel anti-tumor protein derived from the poplar plant Phellinus igniarius, SVAP, and utilizes a heterologous gene expression pathway to achieve recombinant production of the anti-tumor protein, providing a technical foundation and data support for subsequent industrial development.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an anti-tumor active protein SVAP derived from Phellinus igniarius mycelium. The amino acid sequence of the anti-tumor active protein SVAP is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding an anti-tumor active protein SVAP derived from the mycelium of Phellinus igniarius. The nucleotide sequence of the gene encoding SVAP is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for constructing a recombinant bacterium that heterologously expresses the anti-tumor protein SVAP, comprising the steps of transforming a recombinant plasmid containing the SVAP gene into a eukaryotic microorganism, so that the obtained eukaryotic microorganism can heterologously express the anti-tumor protein SVAP;

[0010] The nucleotide sequence of the gene SVAP is shown in SEQ ID NO.1;

[0011] The amino acid sequence of the anti-tumor active protein SVAP is shown in SEQ ID NO.2.

[0012] Furthermore, the eukaryotic microorganism includes yeast.

[0013] Furthermore, the yeast includes Pichia pastoris.

[0014] The present invention also provides a recombinant bacterium obtained by the construction method.

[0015] The present invention also provides the use of the anti-tumor active protein SVAP, the coding gene SVAP or the recombinant bacteria in the preparation of anti-tumor drugs.

[0016] Furthermore, the tumors include cervical cancer and liver cancer.

[0017] The present invention also provides an anti-tumor drug, the active ingredient of which includes the anti-tumor active protein SVAP.

[0018] The present invention discloses the following technical effects:

[0019] The present invention has obtained a novel anti-tumor protein, SVAP, derived from the poplar plant phellinus. The gene encoding the SVAP protein from the poplar plant phellinus was expressed in P. pastoris to investigate the anti-tumor effect of the recombinant protein. The results showed that the SVAP gene could be successfully heterologously expressed in Pichia pastoris GS115. The expressed recombinant protein had a certain inhibitory effect on human cervical cancer cells HeLa, human liver cancer cells Hep G2, and mouse liver cancer cells Hepa 1-6, demonstrating potential for pharmaceutical development.

[0020] The present invention utilizes a gene heterologous expression pathway to achieve the recombinant production of mulberry igniarius anti-tumor active protein, providing a technical basis and data support for subsequent industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 is the protein concentration standard curve;

[0023] Figure 2 The mycelial protein yield (A) and antitumor activity (B) of different cell disruption methods, where Cont.: blank control group; 5-FU: positive control group; E: enzymatic hydrolysis method; B: bead beating method; L: liquid nitrogen grinding method; ac: different letters indicate significant differences;

[0024] Figure 3 Comparison of the extraction and anti-tumor activity of crude protein from poplar mulberry linterin. A is the inhibition rate of mulberry linterin against HeLa cells (within the concentration range of 0 μg / mL-100 μg / mL); B is the inhibition rate of mulberry linterin against different tumor cells at a concentration of 50 μg / mL; different letters indicate significant differences.

[0025] Figure 4 The effect of SVCP at different concentrations on the proliferation of L929 cells; Cont.: blank control group; 5, 10, 25, 50: different concentrations of poplar linalool (μg / mL); 5-FU: positive control group; ad: different letters indicate significant differences;

[0026] Figure 5 The inhibitory activity of the purified fraction of (NH4)2SO4 on HeLa cells;

[0027] Figure 6 The inhibitory activity of each component on HeLa cells; Cont.: control group; SP: cationic column purified flow-through fraction; LSP3: cationic column purified eluate fraction (tube 3); LSP4: cationic column purified eluate fraction (tube 4); 5-FU: positive control group; ab: different letters indicate significant differences;

[0028] Figure 7 The figure shows the analysis of purified components by anion exchange chromatography and the in vitro activity verification. A is the SDS-PAGE image of the purified components by anion exchange chromatography; B is the inhibitory activity of each component against HeLa cells; C is the morphological changes of HeLa cells; Cont.: blank control group; Q15, Q16, Q17: tubes 15, 16, and 17 of the purified components by anion column; 5-FU: positive control group; ab: different letters indicate significant differences.

[0029] Figure 8 Effects of SVAP on tumor cell apoptosis (A) and cell cycle arrest (B);

[0030] Figure 9 Western Blot was used to detect the effect of SVAP on proteins related to the apoptosis pathway in Hela cells;

[0031] Figure 10 is the protein phylogenetic tree;

[0032] Figure 11 The recombinant plasmid pPIC9K-SVAP was verified by gel electrophoresis, where M: Marker; 1-4: recombinant plasmid pPIC9K-SVAP; plasmid pPIC9K; SVAP; double enzyme digestion of pPIC9K-SVAP;

[0033] Figure 12 For the screening of high-copy transformants, A is the screening of transformants on an MD plate without G418; B is the screening of transformants on an MD plate containing 2 mg / mL G418; C is the screening of transformants on an MD plate containing 4 mg / mL G418; D is the screening of transformants on an MD plate containing 10 mg / mL G418;

[0034] Figure 13 PCR verification of positive transformants;

[0035] Figure 14 Analysis of protein expression levels of recombinant strains of P. pastoris;

[0036] Figure 15 The inhibitory effect of rSVAP on different tumor cell lines. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] General Notes:

[0043] The Phellinus linteus strain Sanghuangporus vaninii (SH2, from Shandong) used in the examples of the present invention was preserved in the Fermentation Engineering Laboratory of Huazhong Agricultural University.

[0044] Example 1 Extraction of six phellinus proteins and verification of their in vitro antitumor activity

[0045] 1. Pretreatment of Phellinus igniarius mycelium

[0046] Six strains of Phellinus igniarius were activated and cultured on PDA plates. After the mycelium had grown all over the plate, 1 cm 2 Four to five clumps of the fungus were placed in a pre-prepared liquid culture medium (3% soybean meal, 3% soluble starch, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, natural pH) and incubated at 28°C and 150 rpm for 14 days for liquid fermentation. The mycelia of the six species of Phellinus linteus from the liquid fermentation were sieved through a 60-mesh sieve, collected, and freeze-dried. The resulting freeze-dried mycelial powder was stored at -80°C until ready for use.

[0047] 2. Extraction of Phellinus lintein

[0048] Three cell wall disruption methods (enzymatic method, bead milling method and liquid nitrogen grinding method) were used to extract Phellinus igniarius protein. The protein extraction yield was used as an indicator to optimize the Phellinus igniarius mycelial cell disruption method.

[0049] 2.1 Cell disruption

[0050] Enzymatic method: Dissolve 10 mg of snail enzyme in 1 mL of ultrapure water to prepare a stock solution. Hydrolyze 0.2 g of mycelium with 5% enzyme. Incubate at 40°C for 1.5 h. Centrifuge at 8000 rpm for 10 min. Remove the supernatant and prepare three replicates.

[0051] Bead milling (tissue grinder): Place 0.2 g of mycelium into a 10 mL centrifuge tube. Place two small glass beads soaked in 20% ethanol into each tube. After quick freezing in liquid nitrogen, place the tubes into an adapter and grind for 10 minutes at 60 Hz, running for 50 seconds with a 10-second pause. The ground powder is placed in Tris-HCl buffer and extracted at 4°C for 8 hours. After extraction, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and prepare three replicates.

[0052] Liquid nitrogen grinding: 0.2 g of mycelium was placed in a mortar and ground with liquid nitrogen. 5 mL of Tris-HCl buffer was added and the mixture was extracted at 4°C for 8 h. The mixture was then centrifuged at 8000 rpm for 10 min. The supernatant was collected and triplicate samples were prepared for repeated experiments.

[0053] 2.2 Protein concentration determination

[0054] A protein concentration standard curve was developed with protein concentration as the horizontal axis and absorbance as the vertical axis, and the fitting formula was obtained: Y = 0.1839X + 0.3461, R 2 =0.9963( Figure 1 When measuring the sample, substitute the absorbance value into the formula to calculate the protein concentration. Protein concentration was determined using the Bradford protein concentration assay kit.

[0055] The results of the test are as follows Figure 2 As shown in Figure A, the protein yield after bead milling was 3.02%, significantly higher than that of the enzymatic method (1.55%) and liquid nitrogen grinding method (2.36%) (p < 0.05). This may be due to the following reasons: bead milling keeps the mycelium powder within a sealed centrifuge tube, preventing additional cell loss, and the mechanical force allows for more complete cell disruption; whereas, liquid nitrogen grinding causes mycelium to splash outward due to the influence of liquid nitrogen, resulting in partial cell loss; and, since the enzymatic method requires high-temperature inactivation of the enzyme, which can affect protein activity, and the high cost of the enzyme, it is not suitable for large-scale cell disruption.

[0056] The crude proteins obtained by the three cell disruption methods were compared for their in vitro antitumor activity (refer to “3. In vitro antitumor activity of Phellinus igniarius crude protein”). Figure 2As shown in B. Overall, the three disruption methods did not have a significant impact on protein activity. The inhibition rate of the extracted proteins on the proliferation of HeLa cells in vitro reached more than 88.00%. Among them, the protein extracted by the bead milling method had the highest activity (inhibition rate 98.52%), slightly higher than the effect of the positive drug 5-FU (inhibition rate 92.57%). The protein obtained by the enzymatic hydrolysis method had an inhibition rate of 88.14% on HeLa cells, which was significantly lower than that obtained by the bead milling and liquid nitrogen grinding methods (p < 0.05). Considering the protein extraction yield and tumor cell inhibitory activity, the bead milling method was determined to be the appropriate cell disruption method for Phellinus igniarius mycelium.

[0057] 3. In vitro anti-tumor activity detection of Phellinus igniarius crude protein

[0058] The tumor cells used were HeLa (human cervical cancer cells), Hep G2 (human liver cancer cells), and Hepa 1-6 (mouse liver cancer cells). Hep G2 and Hepa 1-6 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin; HeLa cells were cultured in R / MINI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All tumor cells were cultured in T-25 vented cell culture flasks in a 37°C, 5% CO2, saturated humidity incubator.

[0059] This example uses a CCK-8 kit (Cell Counting Kit-8) to measure cell proliferation. CCK-8 is a kit based on WST-8 and widely used for rapid, highly sensitive detection of cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT that can be reduced to an orange-yellow formazan by some dehydrogenases in the mitochondria in the presence of an electron coupling agent. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is linearly related to the number of cells.

[0060] The experiment set up blank control group, positive control group and experimental group, and 100 μL of 6×10 4 Cells were plated at 400 μg / mL, with six replicate wells per group. The cells were placed in a 37°C incubator. After 12 hours of cell culture, the positive drug and protein sample were added, mixed, and continued to be cultured in a 37°C incubator. After 24 hours, 10 μL of CCK-8 solution was added to each well and incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the inhibitory rate of the drug on the cells was calculated.

[0061] The calculation formulas for cell proliferation survival rate and cell proliferation inhibition rate are as follows:

[0062] Cell viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100;

[0063] Cell inhibition rate (%) = 1-[(OD of experimental group-OD of blank group) / (OD of control group-OD of blank group)]×100.

[0064] 3.1 In vitro antitumor activity of Phellinus igniarius crude protein

[0065] The crude protein of mycelium of Phellinus igniarius (denoted as SVCP) was obtained by bead milling. To compare the anti-tumor activity of different Phellinus igniarius proteins, the inhibitory effect of SVCP protein on the proliferation of HeLa cells in vitro was investigated in a concentration gradient range of 0μg / mL-100μg / mL. The results showed that the activity of crude protein SVCP of the poplar Phellinus igniarius strain was equivalent to that of the positive drug 5-FU, and the effect was dose-dependent. At a drug concentration of 50μg / mL, the inhibition rate of SVCP on HeLa cells was as high as 71.91% ( Figure 3 A).

[0066] In order to verify whether SVCP has broad-spectrum anti-tumor ability, Hep G2 and HT29 cells were added as test subjects on the basis of HeLa cells to investigate the inhibitory effect of each protein (50 μg / mL) on different tumor cells. Figure 3 As shown in Figure B, the inhibition rates of mulberry linterin SVCP on the three tumor cells were 83.05% (HeLa), 51.78% (Hep G2), and 79.87% (HT29), respectively, and were comparable to the effect of the positive drug 5-FU (p>0.05).

[0067] 3.2 Safety evaluation of six types of Phellinus igniarius crude proteins

[0068] In order to evaluate the safety of mulberry linterin, mouse fibroblast L929 cells were selected as the research object to assess the feasibility of the application of anti-tumor active protein. Figure 4 As shown, mulberry linterin SVCP showed the ability to promote normal cell proliferation at concentrations of 5μg / mL and 10μg / mL, and showed slight inhibition on cells at concentrations of 25μg / mL and 50μg / mL, suggesting slight toxicity. On the contrary, 5-FU showed high toxicity to L929 cells. Even though mulberry linterin SVCP has a slight inhibitory effect on the survival of normal cells L929, its toxicity is very weak compared with the clinical chemotherapy drug 5-FU, and mulberry linterin SVCP has a strong inhibitory effect on tumor cells. Therefore, mulberry linterin SVCP is a good choice as an anti-tumor active protein.

[0069] Example 2 Isolation and purification of poplar phellin, preliminary exploration of anti-tumor properties and protein identification

[0070] 1. Preliminary purification by (NH4)2SO4 precipitation method

[0071] The (NH4)2SO4 precipitation method is a simple and effective neutral salt precipitation method for initial protein purification. It separates different proteins based on their solubility in salt solutions. The amount of (NH4)2SO4 to be weighed is calculated based on the volume of the supernatant. (NH4)2SO4 powder is slowly added to achieve concentrations of 40%, 60%, and 80% saturation. The precipitate of each component is collected, reconstituted with PBS, and dialyzed before being tested for anti-tumor activity.

[0072] The results are as follows Figure 5 The inhibition rate of histones precipitated with (NH4)2SO4 at 40%-60% saturation reached 79.31% against HeLa cells, and that of histones precipitated with 60%-80% saturation reached 84.26%. However, the anti-tumor activity of histones precipitated with 0%-40% saturation was significantly reduced, reaching only 35.38% (p<0.05). The fraction with the highest anti-tumor activity, 60%-80% saturation, was selected for further purification via ion exchange chromatography and designated SVP3.

[0073] 2. Further purification by cation exchange chromatography

[0074] Ion exchange chromatography is the most commonly used method for protein separation and purification. Its principle is to separate different proteins by utilizing their different surface charges. In this example, HiTrap SP cation chromatography was first used to purify SVP3. When the pH value of the buffer solution is less than the isoelectric point of the protein, the protein is positively charged and can be bound by the cation exchange resin. The results showed that the elution peak of the cation column binding part had a single peak shape and a wide peak distance, which suggested that the protein components were not well separated and purified. The purified components of the flow-through and eluate were collected, and the anti-tumor active components therein were tracked. The results are as follows. Figure 6 Compared with the control, the eluate fraction (SP) had essentially no antitumor activity, while the LSP3 and LSP4 fractions in the flow-through exhibited significant HeLa cell inhibitory activity (97.66% and 97.54%), indicating that the active fractions were not bound to the cationic column, indicating that the target protein was negatively charged under the current conditions.

[0075] 3. Further purification by anion exchange chromatography

[0076] Based on the aforementioned analysis, the target protein possesses a negative charge, which can exchange with the active ions of the anion exchange resin. Therefore, SVP3 was dissolved in loading buffers ranging from pH 5 to pH 9. This allowed the different protein components to acquire different charges across a range of buffers, exhibiting varying degrees of binding to the anion exchange resin and, consequently, different elution orders. The optimal loading buffer was selected through comprehensive analysis of SDS-PAGE gel electrophoresis and UV absorption peaks of the eluted fractions. When the loading buffer pH was 5-6, the UV absorption peak was broad and absent a single peak. While distinct absorption peaks appeared at pH 7 and 9, none were narrow. At pH 8, the UV absorption peak was single and narrow, resulting in optimal protein separation.

[0077] 4. In vitro activity verification of purified fractions

[0078] The precipitated sample was subjected to anion exchange chromatography using Tris-HCl buffer (pH 8) as the loading buffer and 1 M NaCl as the eluent for linear elution. Protein was collected based on UV absorption, with 1.5 mL collected per tube, and analyzed for band changes and antitumor activity.

[0079] The results are as follows Figure 7 As shown, the Q17 component has a single band around 50 kDa ( Figure 7 A). By Figure 7 As shown in Figure B, the anti-tumor activity of the collected Q15-Q17 protein purified fractions gradually increased, with the Q17 fraction showing the strongest activity, achieving an inhibition rate of 91.30% against HeLa cells, slightly higher than the effect of the positive drug 5-FU (inhibition rate of 81.58%). Given that the purified active fraction of Q17 is derived from S. vaninii, it will be referred to as S. vaninii active protein, or SVAP for short. Figure 7 As shown in Figure C, SVAP can cause morphological changes in HeLa cells. Compared with the blank control, the tumor cells became smaller, the cells shrank, the tentacles disappeared, and they showed apoptosis, which was consistent with the results of the positive control 5-FU.

[0080] 5. Effects of SVAP on tumor cell apoptosis and cell cycle arrest

[0081] Apoptosis is one of the main anti-tumor mechanisms. Flow cytometry is used to detect apoptosis and cell cycle arrest to better understand the anti-tumor mechanism. After Annexin V-FITC / PI double staining, apoptosis is detected by flow cytometry, and cell cycle arrest is detected by PI.

[0082] The results are as follows Figure 8 As shown, Figure 8In Figure A, the upper left quadrant (Q1) represents mechanically damaged cells, the lower left quadrant (Q4) represents normal cells, the upper right quadrant (Q2) represents late apoptotic cells, and the lower right quadrant (Q3) represents early apoptotic cells. Compared with the control group, SVAP and the positive drug 5-FU induced apoptosis in HeLa tumor cells, primarily in late apoptotic cells, with a lower proportion of early apoptotic cells. At a concentration of 50 μg / mL, SVAP had an apoptosis rate of 8.99%, slightly lower than that of 5-FU (11.9%), and the effect was concentration-dependent.

[0083] Detection of SVAP blocking effect on HeLa cells, such as Figure 8 As shown in Figure B, SVAP treatment significantly altered the cell cycle compared to the control group, with a significant increase in cells in the G0 / G1 phase and a decrease in the proportion of cells remaining in the G2 / M and S phases. This concentration-dependent effect suggests that DNA replication is blocked. These results demonstrate that SVAP can induce apoptosis and arrest cells in the G0 / G1 phase.

[0084] 6. Effects of SVAP on tumor cell apoptosis pathway proteins

[0085] P53 is a key transcription factor in apoptosis, regulating multiple pro-apoptotic factors. Increased expression of P53 indicates active apoptosis. Cytochrome C is a pro-apoptotic protein. When stimulated to initiate apoptosis, cytochrome C translocates from the mitochondria to the cytoplasm through the mitochondrial transition pore. Increased cytochrome C expression is also a signal for anti-tumor efficacy.

[0086] The experiment set up a blank control group and an experimental group. Take tumor cells in the logarithmic growth phase and adjust the cell density to 2×10 5 / mL, inoculated into 6-well plates, added 1.5mL to each well, and placed in a cell culture incubator for 24 hours. Prepare different concentrations of protein solutions, add them to 24-well plates, 1.5mL per well, and continue incubating in the incubator for 24 hours. Western Blot was used to detect the expression levels of the above tumor cell apoptosis pathway proteins. The results are as follows Figure 9 As shown, at a SVAP concentration of 10 μg / mL, P53 expression increased compared with the normal group, but the difference was not significant (p>0.05). Cytochrome C expression was significantly different from the normal group (p<0.05). At SVAP concentrations of 25 μg / mL and 50 μg / mL, P53 and cytochrome C expression gradually increased, with significant differences (p<0.05). There was no significant difference with the positive drug 5-FU (p>0.05).

[0087] 7. Bioinformatics analysis of SVAP gene

[0088] The SVAP protein, purified by (NH₄)₂SO₄ precipitation and ion exchange chromatography, was subjected to SDS-PAGE electrophoresis. After gel excision, the target band was subjected to mass spectrometry. Based on the results of mass spectrometry identification, the amino acid sequence was aligned with the S. vaninii transcriptome data. The protein with the highest coverage was 51.483 kDa, and 30 peptides were detected, accounting for 49.60% of all peptide coverage (Table 1). It was hypothesized that the protein with the highest coverage was the target protein, and the corresponding gene was the target gene. Further bioinformatics analysis of the gene revealed that the SVAP gene sequence is 1419 bp long, encoding 472 amino acids, with a protein size of approximately 51.48 kDa and a theoretical isoelectric point of 7.16. It contains 56 negatively charged residues and 56 positively charged residues. The instability coefficient and aliphatic coefficient are 34.19 and 79.53, respectively. Theoretical analysis indicates that the SVAP protein is stable. The nucleotide sequence of the SVAP gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0089] Table 1 Mass spectrometry identification results

[0090]

[0091] SEQ ID NO.1:

[0092]

[0093] SEQ ID NO.2:

[0094] MATPTWPTPQDLNDLQADVVLGIPKRAEDFIFFNIQDAAAFKKDLKELIPAITTTAQVKKIRTDITNHKQQGKTDLIKVAAINIAFSVAGLKKLGITESLGDDAFNQGQLAHAQNLG DTGKTENGSFVPEWIDAFKNEIDGVVLVAGDSKISVDEGVNKVEHTFGKSIKEVFKVNGHVRPNKEKGHEHFGFKDGISFPAIDALTGHLPGQIVVDPGVLVCGTNGDTVQNRPAWAK NGSFLVYRQLQQLVPEFNKFLTDNPVNVPGLPRDKGSELLGARLFGRWKSGAPIELAPTADDPKLAEDKQRNNNFDFKDSFSDQTKCPFAAHIRKTNPRNDLIQPFGDGALKPHMIV RQSITYGPEVTPEEAHSNKTKVDRGLAFVCYQSNLANGFEFVQESWANATGFPPQKPVTPGFDPIIGQKNGQARETAGLQIDNQSANTTLPIEFVVSKGGAYFFSPSITALKTKLSA.

[0095] The obtained SVAP protein sequence was compared with the currently reported medicinal fungal anti-tumor proteins to analyze their sequence similarity. Figure 10 As shown, the phylogenetic tree results showed that the SVAP protein was distantly homologous to other medicinal fungal proteins, proving that a new anti-tumor active protein was isolated from S. vaninii.

[0096] Example 3 Heterologous expression and activity verification of the anti-tumor protein gene of Poplar Phellinus igniarius

[0097] 1. Expression of SVAP gene in Pichia pastoris

[0098] 1. Construction of recombinant plasmid PIC9K-SVAP

[0099] PIC9K was used as the eukaryotic expression vector and Pichia pastoris GS115 was used as the expression strain to express the SVAP gene. Using the cDNA of the poplar strain Phellinus linteus as the template, primers were designed and the target gene SVAP was successfully amplified.

[0100] The specific sequences of the primers are as follows:

[0101] SVAP-F: CGCGGATCCATGACTACTCCCGCCTG (SEQ ID NO.3);

[0102] SVAP-R: AAGGAAAAAAGCGGCCGCTTAGTGATGATGATGATGATGAGCAGTAAGCTTCGTCTTAAG (SEQ ID NO. 4).

[0103] After the amplified DNA fragments were recovered, the target fragment and PIC9K vector were double-digested with BamH I and Not I, respectively. The target fragment after digestion was ligated with the linearized vector. The ligation system is shown in Table 2.

[0104] Table 2 Connection system

[0105]

[0106] Remove competent E. coli DH5α cells from a -80°C freezer and thaw on ice for 5 minutes. Add 10 μL of the ligation system to the competent cells, gently tap to mix, and place on ice for 30 minutes. Heat shock at 42°C for 45 seconds, place on ice for 2 minutes, add 500 μL of antibiotic-free LB solution, and incubate at 37°C at 150 rpm for 1 hour. Spread 100 μL of the recovered bacterial suspension onto an LB plate (100 μg / mL ampicillin) and incubate at 37°C inverted to observe for positive clones.

[0107] Positive clones were selected for expansion and PCR. After PCR, the bacterial solution was subjected to 1% agarose gel electrophoresis to exclude false positive clones. The PCR stock solution was then sent to Qingke Biotechnology Co., Ltd. (Wuhan) for sequencing. Positive clones that were sequenced correctly were stored at -80°C, and the extracted plasmids were stored at -20°C for later use.

[0108] Select the positive clones for PCR identification. The gel electrophoresis results show a band at 1500bp, which is consistent with the target band in size. After extracting the recombinant plasmid, double enzyme digestion verification is performed. Figure 11 Lane 1 is the recombinant plasmid pPIC9K-SVAP. After double enzyme digestion, the recombinant plasmid has two bands (lane 4), one is consistent with the size of pPIC9K (lane 2), and the other is consistent with the size of SVAP (lane 3). The recombinant plasmid pPIC9K-SVAP was successfully constructed.

[0109] 2. Screening of high-copy transformants

[0110] The recombinant plasmid pPIC9K-SVAP linearized by SacⅠ was electroporated into P. pastoris GS115, and positive transformants were screened on MD plates ( Figure 12These transformants were gradually inoculated onto MD plates containing 2 mg / mL, 4 mg / mL, and 10 mg / mL G418 ( Figure 12 Theoretically, G418 concentration is proportional to copy number. After obtaining high-copy positive transformants, streak them onto plates with higher G418 concentrations to further screen for high-copy transformants. Select well-growing transformants from strains #15, #18, and #22 for PCR verification.

[0111] 3. PCR verification of positive transformants

[0112] The bacterial solution of the selected high-copy transformants was lysed with alkaline and then PCR was performed to verify the results. Figure 13 Transformants #15, #18, and #22 all amplified bands at 1500 bp, the sizes of which were consistent with expectations, demonstrating that the recombinant plasmid had been successfully integrated into the P. pastoris genome. Positive strains #15 and #22, which had the brightest bands, were selected for shake flask fermentation.

[0113] 4. Fermentation of recombinant strains of P. pastoris

[0114] Fermentation testing of recombinant proteins

[0115] 1) Select the positive transformant cultures #15 and #22 and inoculate 1% of the culture medium into 100 mL of BMGY (500 mL Erlenmeyer flask) and culture overnight at 30°C and 250 rpm for 12 h.

[0116] 2) Centrifuge at 8000 rpm for 10 min to collect the cells;

[0117] 3) Transfer the cells to 100 mL of BMMY medium (500 mL Erlenmeyer flask) and culture at 30°C and 250 rpm;

[0118] 4) Methanol was added every 24 h to a final concentration of 1%;

[0119] 5) Take 1 mL of sample every 24 hours, centrifuge at 8000 rpm for 5 minutes, collect the supernatant, store at -20°C, and measure the activity.

[0120] After the bacteria were harvested, SDS-PAGE gel electrophoresis was performed. Using the empty Pichia pastoris / pPIC9K as a negative control, the proteins of strains #15 and #22 both had the target protein band (denoted as rSVAP) at 55 kDa, and the size was in line with expectations. The protein expression level of strain #22 was higher than that of strain #15 ( Figure 14 ).

[0121] The recombinant protein obtained after fermentation of the #22 recombinant strain was separated and purified by anion column chromatography. After multiple purification and enrichment, a protein rSVAP with a size of 55 kDa was obtained for subsequent anti-tumor activity experiments.

[0122] 5. Antitumor activity of rSVAP

[0123] To investigate the anti-tumor activity of the recombinant protein rSVAP, three tumor cell lines, HeLa, Hep G2, and Hepa 1-6, were selected for in vitro anti-tumor tests, with 5-FU as a positive control. Figure 15 As shown, at a concentration of 50 μg / mL, rSVAP inhibited the proliferation of HeLa, Hep G2, and Hepa 1-6 cell lines by 63.98%, 70.28%, and 81.71%, respectively, compared to 72.61%, 75.40%, and 77.97% in the positive control group (p < 0.05). These results demonstrate that the recombinant protein rSVAP possesses potent in vitro antitumor activity.

[0124] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of an anti-tumor active protein SVAP derived from the mycelium of Phellinus linteus in the preparation of an anti-tumor drug, characterized in that: The tumor is cervical cancer or liver cancer; The amino acid sequence of the anti-tumor active protein SVAP is shown in SEQ ID NO.2.

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