Earthworm protein with anti-tumor activity and application thereof

Abstract

The invention relates to the field of protein, in particular to earthworm protein with anti-tumor activity and application of the earthworm protein. The earthworm protein with antineoplastic activity has at least one of amino acid sequences as shown in 1)-3): 1) at least one of amino acid sequences as shown in SEQ ID NO: 1 or SEQ ID NO: 2; 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and 3) an amino acid sequence having at least 90% consistency with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The earthworm protein has anti-tumor activity, can inhibit growth and proliferation of cancer cells, and can induce apoptosis of the cancer cells. The compound can be widely applied to preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to the field of proteins, and in particular to earthworm protein with anti-tumor activity and application thereof. Background Art

[0002] Earthworms, also known as earth dragons, are rich in functional ingredients such as protein, amino acids, trace elements, enzymes, hormones, etc. The protein content of dried earthworms reaches 70%. They are the raw material for preparing medicines, cosmetics, health foods, etc. and have great economic value.

[0003] Earthworm extract is a bioactive substance with multiple physiological effects and functions. It is widely used in the fields of medicine, health products and agriculture, and shows significant therapeutic effects and promotion. In medicine, earthworm extract can enhance human immunity, promote the production and activation of white blood cells, and increase the resistance of immune cells. In addition, earthworm peptides also have antibacterial, antiviral and anti-tumor properties. It can inhibit the growth of bacteria and viruses, reduce the occurrence of infection and disease, and is currently often used in the preparation of cardiovascular and anti-tumor drugs.

[0004] Earthworm extract plays an important role in killing tumor cells, radiation enhancement, chemical enhancement and improving the body's immune function. However, the mechanism of action is not yet fully understood, and most of the extracts currently developed are compound or mixed extracts, which have not yet reached the level of complete purification. If a single active ingredient can be further isolated, its pharmacological effect will be further clarified. In addition, there is a lack of research on the specificity and targetedness of earthworm extracts in anti-tumor treatment. These are all worthy of our in-depth discussion. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an isolated and purified anti-tumor active protein, which is a single-component protein that can inhibit the growth and proliferation of cancer cells and induce apoptosis of cancer cells. It has high specific activity and can be used in the preparation of anti-tumor drugs.

[0006] The specific scheme of the present invention is as follows:

[0007] The first aspect of the present invention provides an earthworm protein having anti-tumor activity, wherein the earthworm protein is selected from at least one of the following amino acid sequences:

[0008] 1) at least one of the amino acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0009] 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0010] 3) An amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] The molecular weight of the earthworm protein with a sequence as shown in SEQ ID NO: 1 is 24.95 KDa, and the molecular weight of the earthworm protein with a sequence as shown in SEQ ID NO: 2 is 26.67 KDa.

[0012] The second aspect of the present invention provides a nucleic acid encoding any of the earthworm proteins. In a specific embodiment of the present invention, a nucleotide sequence is designed according to the codon preference of Escherichia coli. Specifically, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4.

[0013] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following I-III:

[0014] I. A vector comprising a nucleic acid encoding at least one of said earthworm proteins;

[0015] II. A transformant comprising the vector described in I;

[0016] III. A recombinant cell capable of producing at least one earthworm protein, and / or containing at least one nucleic acid encoding the earthworm protein.

[0017] The fourth aspect of the present invention provides the use of the earthworm protein or the nucleic acid or the biological material in the preparation of anti-tumor drugs.

[0018] The fifth aspect of the present invention provides a composition, which comprises the earthworm protein. Preferably, the composition further comprises an adjuvant acceptable in the field of medicine, pharmacy or food. Preferably, the dosage form of the composition comprises at least one of powder, granules, pills, tablets, capsules, pastes or decoctions.

[0019] The sixth aspect of the present invention provides a method for preparing the earthworm protein by genetic engineering, the method comprising the step of introducing at least one nucleotide sequence encoding the earthworm protein into a target cell or a target microorganism. The target cell or target microorganism is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells.

[0020] The beneficial effects of the present invention include but are not limited to:

[0021] The present invention separates a new single-component protein from earthworms. The protein has anti-tumor activity, can inhibit the growth of cancer cells and promote apoptosis of cancer cells, and can be used in the preparation of food, medicine or health products for treating tumor diseases. It also lays a foundation for large-scale production by recombinant protein expression, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 1 is an electrophoresis diagram of two earthworm protein sequences in the embodiment of the present invention, wherein 1 is an electrophoresis diagram of an earthworm protein with a sequence as shown in SEQ ID NO: 1, and 2 is an electrophoresis diagram of an earthworm protein with a sequence as shown in SEQ ID NO: 2;

[0024] Figure 2 Schematic diagram of the morphology of liver cancer cell 7402 in which lumbrokinase and earthworm proteins QY-1 and QY-2 were added and incubated for 24 hours in the embodiment of the present invention, wherein 1 is the result of blank control, 2 is the result of adding lumbrokinase, 3 is the result of adding QY-1, and 4 is the result of adding QY-2;

[0025] Figure 3 Schematic diagram of the morphology of lung cancer A549 cells in which lumbrokinase and earthworm proteins QY-1 and QY-2 were added and incubated for 24 hours in an example of the present invention, wherein 1 is the result of the blank control, 2 is the result of adding lumbrokinase, 3 is the result of adding QY-1, and 4 is the result of adding QY-2. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0027] Example 1 Separation and purification of earthworm protein

[0028] (1) Wash fresh earthworms, extract the earthworms and pure water at a ratio of 1:20 at 35° C. for 16 hours to obtain a solution containing earthworm extracts, wherein the earthworms are Eisenia fetida;

[0029] (2) adjusting the pH value of the solution containing the earthworm extract obtained in step (1) to 7.5, stirring for 15 minutes, and collecting the supernatant by centrifugation; adding anhydrous ethanol to the supernatant until the ethanol concentration is 45%, and refrigerating in a refrigerator at 4°C for 12 hours; collecting the supernatant by centrifugation, adding anhydrous ethanol to the supernatant until the ethanol concentration is 75%, and refrigerating in a refrigerator at 4°C for 12 hours; collecting the precipitate by centrifugation, adding an appropriate amount of deionized water to dissolve the precipitate, and freeze-drying the solution to obtain an earthworm protein primary extract;

[0030] (3) Isolation and purification of earthworm protein primary extract: The obtained earthworm protein primary extract was dissolved in pH 7.8 phosphate buffer, passed through DEAE-Sepharose FF (16 mm × 200 mm), first rinsed with 0.1 mol / L saline, and then gradient eluted with 0.1-0.6 mol / L saline. The eluate of the active component was then passed through Sephadex G-75 (16 mm × 960 mm), eluted with clean water, and the eluate was collected and freeze-dried to obtain earthworm protein.

[0031] (4) The active part of the fibrinolytic protein was separated and purified by molecular sieve chromatography: the NGC protein chromatography system was used as the separation and purification device, the chromatographic column was a molecular sieve gel chromatography column (Superdex 75 Increase 10 / 300 GL) of GE Company, the detector wavelength was set to 280 nm, 20 mM Tris-HCl and 0.3 M NaCl (pH 7.5) solution was used as the elution solvent; the flow rate was 0.5 mL / min; the main peak corresponding fraction was collected, and deionized water was used as the replacement solvent and ultrafiltration was performed by ultrafiltration tube (cut-off molecular weight 3 KDa) for desalting until no white floccules were produced when 1% AgNO3 was added to the filtrate; finally, the protein solution was concentrated by ultrafiltration to obtain a certain concentration of anti-tumor active protein, and the anti-tumor active protein powder was obtained by freeze drying. The obtained anti-tumor active protein powder had the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO:2 as verified by sequencing. The earthworm protein with the amino acid sequence shown in SEQ ID NO.1 is named QY-1, and the earthworm protein with the amino acid sequence shown in SEQ ID NO.2 is named QY-2. After comparison, the amino acid sequences of QY-1 and QY-2 have 80% homology with lumbrokinase.

[0032] Example 2 Preparation of earthworm protein by genetic engineering

[0033] The plasmid, endonuclease, PCR enzyme, column DNA extraction kit and DNA gel recovery kit used in this example are commercial products, and the specific operations are carried out according to the instructions of the kit.

[0034] Conventional operations such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0035] (1) Construction of vector and recombinant engineering bacteria

[0036] The obtained earthworm protein was optimized according to the codon preference of Escherichia coli to obtain the corresponding nucleotide sequence, and the whole gene was synthesized and constructed into the plasmid vector pET-32a(+) (purchased from Hunan Fenghui Biotechnology Co., Ltd.), and then transformed into competent cells BL21(DE3) (purchased from Shanghai Maokang Biotechnology Co., Ltd.), and then the genetically engineered bacteria were obtained by restriction digestion and sequencing. Specifically, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4.

[0037] (2) Fermentation and cultivation of engineered bacteria

[0038] The culture medium formula used in this example is:

[0039] LB medium formula: peptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L, ampicillin 100mg / L.

[0040] Trace element formula: ferrous sulfate heptahydrate 2g / L, manganese sulfate 0.52g / L, zinc sulfate 0.75g / L, cobalt chloride 0.13g / L, copper sulfate 0.1g / L, sodium molybdate 0.13g / L, calcium chloride dihydrate 8.37g / L.

[0041] Basic culture medium formula: glucose 2g / L, yeast powder 12g / L, potassium dihydrogen phosphate 15g / L, magnesium sulfate 0.25g / L, trace elements 10mL / L.

[0042] Feed medium formula: trace elements 100mL / L, glucose 400g / L, yeast powder 100g / L, magnesium sulfate 15.5g / L.

[0043] The engineered bacteria obtained above were inoculated into LB medium at a 1% inoculum and cultured at 37°C and 220 rpm in a shaking incubator until OD 600≥1.2, obtain seed solution; transfer the seed solution to a 50L fermenter containing 25L basal medium at a 5% inoculation rate, set the temperature to 37°C, ventilation to 25L / min, pH to 7.0, pressure to 0.050Mpa, speed to 300rpm, and control mode to automatic. During fermentation, adjust the speed and ventilation to control the dissolved oxygen to more than 30%. When OD 600 When it stops growing, start feeding; when OD 600 When OD ≥30, stop feeding, lower the tank temperature to 20℃ within 1 hour, add IPTG to a final concentration of 0.5mmol / L for induction culture, observe the change of dissolved oxygen, continue feeding, and control the feeding rate to maintain the dissolved oxygen content at about 30%. Ferment until OD 600 When the wet weight stops increasing, the material is collected and can be put into the tank.

[0044] (3) Protein separation and purification

[0045] Step (4) in Example 1 was used to separate and purify earthworm protein for subsequent experiments.

[0046] Example 3 Physical and chemical properties test of earthworm protein

[0047] Test 1

[0048] Test the molecular weight of earthworm protein: Use 15% SDS-PAGE denaturing gel electrophoresis Coomassie brilliant blue staining method to analyze the protein of earthworm protein. Figure 1 As shown, it is shown that the molecular weight of the protein shown in SEQ ID NO: 1 is about 24.95 KDa, and the molecular weight of the protein shown in SEQ ID NO: 2 is about 26.67 KDa.

[0049] Test 2

[0050] Detection of the inhibitory effect of earthworm protein on cell proliferation: liver cancer 7402 cells and lung cancer A549 were provided and identified by the Biological Center of the Institute of Oncology, Chinese Academy of Medical Sciences; 5 mg / ml MTT solution: accurately weigh an appropriate amount of MTT (purchased from Gibco) and dissolve it in an appropriate amount of PBS solution to make the concentration of MTT solution 5 mg / ml, blow and dissolve it fully, filter through a 0.22 μm filter membrane, dispense 1 ml into each tube, wrap it in tin foil and store it at -20°C away from light.

[0051] Cell culture: Hepatoma cell 7402 and lung cancer cell A549 were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0052] (1) MTT assay to detect cell proliferation inhibition: Hepatocellular carcinoma cells 7402 and lung cancer cells A549 in the logarithmic growth phase were routinely digested, and the cell concentration was adjusted to 2.5×104 / mL, respectively. 200 μL was inoculated into each well of a 96-well culture plate. After 24 hours of adherence, earthworm proteins QY-1 and QY-2 were added as experimental groups 1 and 2, respectively. Lumbrokinase was prepared in culture medium, and three concentrations were set. Three parallel wells were set up in each group, and 200 μL was added to each well. A control group 1 (without any protein) and a control group 2 (with the same concentration of lumbrokinase purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were set up. After adding protein, the cells were cultured for 24 hours, and then 20 μL of 5 mg / mL MTT solution was added. The cells were cultured in an incubator for another 4 hours, the supernatant was discarded, 200 μL of DMSO was added to each well to dissolve, and the cells were shaken thoroughly. The absorbance at 570 nm (A value) was measured by an enzyme-labeled instrument, and the inhibition rate and half-maximal inhibitory concentration IC50 value of earthworm protein on cell growth were calculated.

[0053] The inhibition rate calculation formula is: inhibition rate = number of dead cells / total number of cells*100%.

[0054] The results are shown in Table 1-2.

[0055] Table 1 Effect of earthworm protein on proliferation of hepatoma cell line 7402

[0056]

[0057] Table 2 Effect of earthworm protein on proliferation of lung cancer cell A549

[0058]

[0059]

[0060] It can be seen from Table 1 and Table 2 that the earthworm proteins QY-1 and QY-2 of the present invention can both inhibit the proliferation of cancer cells, and have a better inhibitory effect than lumbrokinase.

[0061] (2) Observation of cell morphological changes under an inverted microscope: Liver cancer cells 7402 and lung cancer cells A549 in the logarithmic growth phase were routinely digested, the cell concentration was adjusted to 1×106 cells / mL, and inoculated into a 6-well plate, 2 mL per well. After culturing for 24 hours, the original solution was discarded, and 2 mL of 50 μg / mL earthworm protein QY-1 and QY-2 of the present invention were added to the experimental group; 2 mL of the same concentration of lumbrokinase was added to the control group 1; and 2 mL of serum-free culture medium was added to the control group 2. There were 4 groups in total. After incubation for 24 hours, the overall cell morphological changes were observed under a 10x microscope and photographed.

[0062] The results are as follows Figure 2-3As shown, it was found that the cells in the control group 1 with lumbrokinase and the experimental groups with earthworm proteins QY-1 and QY-2 increased in shrinkage and floating, and decreased in adherent cells, and the inhibitory effect of earthworm proteins QY-1 and QY-2 was better than that of lumbrokinase.

[0063] Test 3

[0064] Flow cytometric detection of cell cycle: liver cancer cells 7402 and lung cancer cells A549 were grouped and then lumbrokinase and earthworm protein were added for experiments. The grouping and dosage were the same as test 2, and each group had 3 replicates. After 24 hours of incubation with drugs, the cells of each group were collected, marked, and rinsed twice with 2mL of PBS solution precooled at 4℃, and the supernatant was discarded. 1mL of PBS solution was added to resuspend the cells, and they were slowly added to 2mL of 70% (φ) ethanol (precooled at -20℃), mixed and fixed at 4℃ overnight. Centrifuge at 1000r / min for 2min, discard the 70% ethanol solution, rinse twice with 1mL of PBS solution precooled at 4℃, discard the supernatant, and add 800μL of 20μg / mL PI staining solution (200μg / mL RNase) to each group according to 1×106 cells, mix, incubate at 37℃ in the dark for 30min, and then detect on the machine. The results are shown in Tables 3 and 4.

[0065] Table 3 Distribution ratio of 7402 cell cycle after earthworm protein treatment

[0066]

[0067]

[0068] Table 4 Distribution ratio of A549 cell cycle after earthworm protein treatment

[0069]

[0070] It can be seen from Table 3-4 that earthworm proteins QY-1 and QY-2 can induce apoptosis of cancer cells.

[0071] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An earthworm protein with anti-tumor activity, characterized in that: The earthworm protein is selected from at least one of the following amino acid sequences: 1) at least one of the amino acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2; 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; 3) An amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid, characterized in that The nucleic acid encodes any one of the earthworm proteins described in claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO:

4.

4. A biomaterial, characterized in that: The biological material is selected from any one of the following I-III:

1. A vector comprising at least one nucleic acid according to claim 2 or 3; II. A transformant comprising the vector described in I; III. A recombinant cell capable of producing at least one earthworm protein according to claim 1 and / or containing at least one nucleic acid according to claim 2 or 3.

5. Use of the earthworm protein according to claim 1, the nucleic acid according to claim 2 or 3, or the biomaterial according to claim 4 in the preparation of anti-tumor drugs.

6. A composition, characterized in that The composition comprises the earthworm protein of claim 1.

7. The composition according to claim 6, characterized in that The composition further comprises excipients acceptable in the fields of medicine, pharmacy or food.

8. The composition according to claim 6, characterized in that The dosage form of the composition includes at least one of powder, granules, pills, tablets, capsules, pastes or decoctions.

9. A method for preparing the earthworm protein according to claim 1 by genetic engineering, characterized in that: The method comprises the step of introducing the nucleic acid according to claim 2 or 3 into a target cell.

10. The method according to claim 9, characterized in that The target cell or target microorganism is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells.

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