Proteins for reducing tumor invasiveness and relieving immunosuppression and uses thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a protein for reducing tumor invasion ability and relieving immunosuppression and application thereof. The protein for reducing tumor invasion ability and relieving immunosuppression provided in the present application has an amino acid sequence as shown in SEQ ID NO:1. The protein can simultaneously bind to cytokeratin 81 (K81) and phosphatidylserine (PS) in tumor cells, inhibit tumor cell metastasis, relieve immunosuppression, and thus improve the development process of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a protein used to reduce tumor invasiveness and relieve immunosuppression, and its applications. Background Technology

[0002] Malignant tumors remain one of the most deadly killers threatening human life due to their high treatment costs, high rate of metastasis and recurrence, drug resistance, and immunosuppression. Tumor antigens can be broadly classified into extracellular antigens and intracellular antigens. The vast majority of antibody-based research focuses on cell surface antigens, but cell surface targets are limited because intracellular tumor antigens constitute the majority of tumor targets, and the vast treasure trove of intracellular tumor targets remains untapped. In 2009, the National Cancer Institute (NCI) conducted a pilot project to list and score cancer antigens to determine the priority of subsequent cancer vaccine projects. Of the 76 tumor antigens identified, 42 were membrane proteins or soluble secreted proteins, and 34 were intracellular proteins. Surprisingly, 11 of the 20 top targets were intracellular protein antigens, indicating the enormous potential of intracellular proteins as tumor targets. Therefore, multi-target therapy based on intracellular protein antigens is a promising approach for the efficient treatment of tumors.

[0003] Intermediate keratin filaments are a crucial component of the cytoskeleton of epithelial cells, forming a stable yet dynamic filamentous network that provides resistance to mechanical stress. While keratin has long been used as a diagnostic and prognostic marker, numerous studies have demonstrated its active regulatory role in tumorigenesis and development, closely related to cancer invasion and metastasis. For example, research has shown that cytokeratin 81 (K81) within tumor cells can promote tumor cell metastasis. Phosphatidylserine (PS) is a ubiquitous phospholipid, typically located in the inner lamina of cell membranes. PS exposed on the surface of tumor cells and tumor vascular endothelial cells can recruit suppressive cells and M2 macrophages and produce anti-inflammatory cytokines, thereby promoting the creation of an immunosuppressive microenvironment. Based on this, proteins designed to bind K81 and PS in tumor cells may be beneficial in reducing tumor invasiveness and altering immunosuppressive states. Summary of the Invention

[0004] To improve the efficacy of tumor treatment, this invention provides a protein for reducing tumor invasiveness and relieving immunosuppression, named sKA511, whose amino acid sequence is shown in SEQ ID NO: 1.

[0005] The present invention also provides a gene encoding the above-mentioned protein (sKA511).

[0006] In some embodiments of the present invention, the nucleotide sequence of the above-mentioned gene is shown in SEQ ID NO: 2.

[0007] Expression cassettes, vectors, bacteria, or fungi containing the aforementioned genes are also within the scope of protection of this invention. The vector may be a cloning vector or an expression vector. The bacteria may be *Escherichia coli*. The fungus may be yeast.

[0008] The use of the above-mentioned proteins in the preparation of drugs for intracellular tumor therapy is also within the scope of protection of this invention.

[0009] The present invention also provides a medicament for intracellular tumor therapy comprising the aforementioned protein (sKA511).

[0010] In some embodiments of the present invention, the dosage form of the above-mentioned drug is an injection.

[0011] The present invention also provides a method for preparing the above-mentioned protein (sKA511), comprising: introducing a gene encoding the above-mentioned protein into an expression vector to obtain a recombinant vector; introducing the recombinant vector into an expression host bacterium to obtain a recombinant bacterium; culturing the recombinant bacterium and inducing protein expression; and extracting and purifying the protein.

[0012] In vitro self-assembly experiments showed that even low concentrations of sKA511 could assemble well with cytokeratin 81 (K81). Figure 2 Cellular colocalization experiments showed that sKA511 can effectively bind to K81 within tumor cells. Figure 3 Cell scratch assays and Transwell assays showed that sKA511 could reduce the migration and invasion abilities of mouse breast cancer cells (4T1). Figure 4 and Figure 5 Simultaneously, sKA511 can specifically bind to phosphatidylserine (PS). Figure 6 Therefore, the protein sKA511 provided by this invention can reduce the migration and invasion capabilities of tumor cells, relieve immunosuppression, and thus improve the tumor development process. Attached Figure Description

[0013] Figure 1 This is an SDS-PAGE gel electrophoresis image of the sKA511 protein. The lanes in the image, from left to right, are: protein molecular marker, supernatant obtained by centrifugation after sonication of recombinant bacteria BL21(DE3)-pET-22b-sKA511, 30 mM imidazole elution solution after passing the supernatant through a nickel column, first 250 mM imidazole elution solution after passing the supernatant through a nickel column, and second 250 mM imidazole elution solution after passing the supernatant through a nickel column.

[0014] Figure 2This is a transmission electron microscope image of the in vitro self-assembly of sKA511 protein and cytokeratin 81 (K81); where A represents K81 and B represents sKA511.

[0015] Figure 3 The results of the immunofluorescence co-localization experiment of sKA511 protein and intracellular cytokeratin 81 (K81) are shown in Figure A. A is a fluorescence microscope image, with the top row of images at the same magnification and from left to right: the first image shows the cell nucleus (blue fluorescence), the second image shows K81 (red fluorescence), the third image shows sKA511 (green fluorescence), and the fourth image is a combined image of the three images on the left, showing the positional relationship between the cell nucleus, K81, and sKA511. The bottom row of images is a magnified view of a portion of the top row images, at the same magnification. Figure B is a fluorescence analysis graph of sKA511 protein and K81, with the vertical axis representing fluorescence intensity (au) and the horizontal axis representing the distance (μm) of a straight line passing through the cell, examining the changes in red and green fluorescence along this line.

[0016] Figure 4 The results of the cell scratch assay show the inhibition of mouse breast cancer cell migration by sKA511 protein; A is a microscopic photograph (showing scratches) of mouse breast cancer cells (4T1) at different time points (0h, 12h and 24h) after scratching, with each photograph magnified at 100x (10x eyepiece and 10x objective lens), and the scale bar (200μm) in the figure is applicable to all photographs; B is a statistical graph of the scratch area at different time points (12h and 24h) after scratching.

[0017] Figure 5 The results of the Transwell experiment on the inhibition of mouse breast cancer cell migration by sKA511 protein are shown. A is a microscopic photograph (stained with crystal violet) of the Transwell experiment results of mouse breast cancer cells (4T1). The magnification of each photograph is 40x (10x eyepiece, 4x objective). The scale bar (500μm) in the figure applies to all photographs. B is the average number of migrating cells per field of view under the microscope.

[0018] Figure 6 The results of the binding experiment of sKA511 protein to phosphatidylserine are shown. From left to right, the first image shows FITC-labeled sKA511 protein (green fluorescence); the second image shows the cell nucleus (blue fluorescence); the third image is a combined image of the first and second images, showing the positional relationship between the cell nucleus and the sKA511 protein, indicating that the sKA511 protein successfully binds to phosphatidylserine (PS) on the cell membrane. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to the embodiments. It should be noted that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0020] cell

[0021] Escherichia coli BL21(DE3) competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0022] Mouse breast cancer cells (4T1) were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-502, cell name: mouse breast cancer cells, animal species: mouse, tissue source: mammary tissue.

[0023] plasmid

[0024] pET-22b(+) plasmid: Escherichia coli expression vector, provided by Sangon Biotech (Shanghai) Co., Ltd. The vector is protected against ampicillin.

[0025] Reagents and Consumables

[0026] Ampicillin and IPTG were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0027] His-tag protein purification resin (nickel column): purchased from Shanghai Lianmai Biotechnology Co., Ltd., product number LM-616.

[0028] FITC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0029] The cytokeratin 81 (K81) used in the following examples was expressed in our laboratory. The amino acid sequence of the K81 protein is shown in SEQ ID NO: 4, and the gene coding sequence is shown in SEQ ID NO: 5. Our laboratory obtained the K81 protein by synthesizing the gene coding sequence of the K81 protein, using the pET-28a(+) plasmid as an expression vector, and expressing it in prokaryotes using Escherichia coli BL21(DE3).

[0030] K81 antibody: purchased from Wuhan Sanying Biotechnology Co., Ltd., product number: 1132-1-AP.

[0031] Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L): purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: A0453.

[0032] 1640 culture medium was purchased from Invitrogen, USA.

[0033] Anti-fluorescence quenching mounting solution (containing DAPI): purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0034] Crystal violet was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0035] The Transwell cell was purchased from Corning, Inc. in the United States.

[0036] culture medium

[0037] Each liter of LB medium contains: 5g yeast extract, 10g tryptone, 10g sodium chloride, and pH adjusted to 7.0.

[0038] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950ml double-distilled water. Adjust the pH to 7.0 with sodium hydroxide solution, and then bring the volume to 1L with double-distilled water. If preparing solid culture medium, add agar at a ratio of 1.5g / 100ml. Autoclave at 121℃ for 30 minutes.

[0039] Unless otherwise specified, all experimental reagents used in this invention are conventional reagents in the art and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers. Unless otherwise specified, all experimental methods used in this invention are conventional methods in the art and can be found in relevant experimental manuals, such as the Molecular Cloning Experimental Manual or the instructions of relevant reagent manufacturers.

[0040] Example 1. Obtaining sKA511, a protein used to reduce tumor invasiveness and relieve immunosuppression.

[0041] 1. Gene design and synthesis

[0042] The inventors designed a gene named sKA511, whose nucleotide sequence is shown in SEQ ID NO: 2, with a full length of 1470 bp. This gene encodes the sKA511 protein, whose amino acid sequence is shown in SEQ ID NO: 1, with a full length of 489 amino acids. NdeI restriction enzyme sites (CA / TATG) and XhoI restriction enzyme sites (C / TCGAG) were added to the 5' and 3' ends of the sKA511 gene sequence, respectively, to obtain the target gene sequence, as shown in SEQ ID NO: 3. The target gene was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized target gene was sequenced for verification. The target gene with the correct sequence was used for subsequent vector construction.

[0043] The amino acid sequence of the sKA511 protein (489aa):

[0044]

[0045] The nucleotide sequence of the sKA511 gene (1470 bp):

[0046]

[0047] The target gene (1479bp) used for vector construction:

[0048]

[0049]

[0050] 2. Carrier Construction

[0051] The pET-22b(+) plasmid was used as the expression vector. The pET-22b(+) plasmid and the synthesized target gene were double-digested with restriction endonucleases NdeI and Xho1, respectively. The target gene was then ligated into the pET-22b(+) plasmid to obtain the ligation product. Vector construction was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0052] 3. Transformation

[0053] (1) Take Escherichia coli BL21(DE3) competent cells out of the -80℃ freezer and incubate on ice for 5 min.

[0054] (2) After the glycerol for storing BL21(DE3) competent cells is melted, add the competent cells to the above ligation product, mix by pipetting 3-4 times with the pipette tip, and let stand in an ice bath for 30 minutes.

[0055] (3) Quickly wipe the water off the tube wall with absorbent paper, then heat shock at 42°C for 90 seconds, and immediately ice bath for 2 minutes.

[0056] (4) Add 800 μl of LB liquid medium under sterile conditions, and incubate at 37°C and 150 rpm for 45 min.

[0057] (5) Centrifuge at 8000 rpm for 5 min to collect the bacterial cells, discard part of the supernatant, resuspend the E. coli in the remaining 100 μl of supernatant, and then spread it evenly on LB solid medium containing 100 μg / ml ampicillin. Place it in a 37℃ incubator and invert it for 10-16 h.

[0058] (6) Select a single clone and inoculate it into LB liquid medium containing 100 μg / ml ampicillin. After culturing at 37℃ for 10-16 h, the positive clone was identified by Sangon Biotech (Shanghai) Co., Ltd., and the recombinant bacteria BL21(DE3)-pET-22b-sKA511 was obtained.

[0059] 4. Protein expression and purification

[0060] (1) Inoculation: Prepare and sterilize LB liquid medium. Place the sterilized LB liquid medium in a clean bench and allow it to cool to room temperature. Add ampicillin and mix well to achieve a final concentration of 100 μg / mL. Inoculate the above recombinant bacteria BL21(DE3)-pET-22b-sKA511 into the medium at a concentration of 200 μL / L. Place the medium in a shaker and incubate at 170 rpm and 37°C for 8-10 h.

[0061] (2) Induction: After culturing in a shaker for 8-10 hours, take out 2 mL of bacterial culture and measure its OD using a spectrophotometer. 600 Value. When the OD of the bacterial culture... 600 When the value reaches 0.6-0.8, add IPTG to the bacterial culture to a final concentration of 200 μL / mL, and then incubate on a shaker at 37℃ and 170 rpm for 8 h.

[0062] (3) Purification: After adding IPTG and culturing on a shaker for 8 hours, the bacterial culture was removed and centrifuged at 8000 rpm for 5 minutes at 4°C. After centrifugation, the supernatant was removed, and the precipitate was retained. The precipitate was the recombinant bacteria BL21(DE3)-pET-22b-sKA511.

[0063] (4) Ultrasonic disruption: The recombinant bacteria BL21(DE3)-pET-22b-sKA511 was ultrasonically disrupted and then centrifuged. The resulting supernatant contained the target protein sKA511.

[0064] (5) The supernatant solution was purified by His-tag protein purification resin (nickel column, Shanghai Lianmai, LM-616) to obtain 30mM imidazole elution solution, 250mM imidazole first elution solution and 250mM imidazole second elution solution respectively. The protein purification effect was detected by polyacrylamide gel electrophoresis (SDS-PAGE).

[0065] (6) The 250 mM imidazole first elution solution was dialyzed twice with PBS (0.01 M, pH 7.4) to obtain sKA511 protein solution. The sKA511 protein solution was freeze-dried and the resulting powder was stored at -20 °C.

[0066] The results are as follows Figure 1 As shown, both the first elution solution with 250 mM imidazole and the second elution solution with 250 mM imidazole contained a large amount of the target protein sKA511 (a band between 55 kDa and 70 kDa).

[0067] Example 2. In vitro self-assembly experiment of sKA511 protein and cytokeratin 81

[0068] The sKA511 protein solution and cytokeratin 81 solution (K81 keratin solution) were mixed in different proportions, and then dialyzed sequentially with 6M urea buffer, 4M urea buffer, and 2M urea buffer at 25°C. Each dialyze lasted approximately 3 hours, and the experiment was conducted in a sealed container filled with N2. The mixture was then dialyzed with assembly buffer in a chromatography cabinet at 4°C for 24 hours. The reaction was terminated by adding 0.2% glutaraldehyde solution.

[0069] Using transmission electron microscope (TEM) tweezers, hold a 300-mesh carbon-plated copper mesh support film on a clean sealing film. With both hands suspended, drop 20 μL of sample vertically downwards. After 2-5 minutes, use pointed filter paper to absorb excess solution from the edges, and allow the sample to air dry for approximately 10 minutes. Then, place the dried copper mesh support film on a clean sealing film and use a pipette to drop 10 μL of 3% uranium acetate staining solution. Stain for 90 seconds, absorb excess staining solution with pointed filter paper, and then place the copper mesh support film on the filter paper to dry for 3 minutes. Finally, observe the sample using a field emission transmission electron microscope (TAlos F200S) under 120 kV electron conditions.

[0070] The results are as follows Figure 2 As shown, A represents cytokeratin 81 (K81), and B represents sKA511 protein. It can be seen that even a low concentration (0.1 mg / ml) of sKA511 protein can exhibit good assembly-binding ability with K81 in vitro.

[0071] The formulation of the above buffer solution is as follows:

[0072] 6M urea buffer: 10mM Tris (pH 7.4), 2.5mM EDTA, and 10mM dithiothreitol (DTT) or 5mM tris(2-carboxyethyl)phosphine (TCEP), 6M urea.

[0073] 4M urea buffer: 10mM Tris (pH 7.4), 2.5mM EDTA, and 10mM dithiothreitol (DTT) or 5mM tris(2-carboxyethyl)phosphine (TCEP), 4M urea.

[0074] 2M urea buffer: 10mM Tris (pH 7.4), 2.5mM EDTA, and 10mM dithiothreitol (DTT) or 5mM tris(2-carboxyethyl)phosphine (TCEP), 2M urea.

[0075] Assembly buffer: 10 mM Tris (pH 7.4), 2.5 mM EDTA, and 10 mM dithiothreitol (DTT) or 5 mM tris(2-carboxyethyl)phosphine (TCEP), 175 mM NaCl, 5 mM MgCl2.

[0076] Example 3. Immunofluorescence co-localization experiment of sKA511 protein and intracellular cytokeratin 81

[0077] The sKA511 protein was dissolved in Na2CO3-NaHCO3 buffer (pH 9.5). FITC (fluorescein isothiocyanate) was slowly added to the resulting sKA511 solution. After stirring at room temperature for 2 hours, the free FITC was removed through a dialysis bag, and the protein fraction was collected to obtain FITC-labeled sKA511, which was stored at 4°C protected from light for later use.

[0078] The formula for the Na2CO3-NaHCO3 buffer solution is as follows: Take 13 mL of 200 mM Na2CO3 solution and 37 mL of 200 mM NaHCO3 solution and mix them evenly to obtain the Na2CO3-NaHCO3 buffer solution.

[0079] Mouse breast cancer cells 4T1 were seeded into 24-well plates with cell spreaders attached, with 3 × 10⁶ cells per well. 4 Cells were cultured at 37°C for 12 hours, followed by two 5-minute washes with 0.01M PBS (pH 7.4). Cells were then fixed with 4% paraformaldehyde for 15 minutes, followed by three 5-minute washes with PBST. Cells were permeabilized with 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 minutes, followed by three 5-minute washes with PBST. Cells were blocked with 3% BSA at room temperature for 1 hour, then incubated with K81 antibody (Wuhan Sanying, catalog number: 1132-1-AP) overnight at 4°C, followed by one hour at room temperature. Cells were washed three times with PBST for 5 minutes each time. Secondary antibody Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L) (Shanghai Beyotime, catalog number: A0453) was added, and the cells were incubated at room temperature for 2 hours. Cells were washed three times with PBST for 5 minutes each time. Add DAPI (4′, 6-diamidinyl-2-phenylindole), and take a picture using a fluorescence microscope after 15 minutes.

[0080] The results are as follows Figure 3 As shown, sKA511 can effectively bind to cytokeratin 81 (K81) intracellularly.

[0081] Example 4. Experiment on the inhibition of mouse breast cancer cell migration by sKA511 protein

[0082] 1. Cell scratch assay

[0083] Mouse breast cancer cells 4T1 were seeded into 6-well plates, with 3 × 10⁶ cells per well. 5 Cells were cultured in wells at 37°C for 12 hours and 24 hours, respectively. A yellow pipette tip was used to scribble a line in the central area of ​​the cell growth zone, removing cells from the central portion to create a scratch. The cells were then washed twice with PBS (0.01M, pH 7.4). Two experimental groups and one control group were set up. 1 μM and 3 μM sKA511 protein solutions were prepared using PBS (0.01M, pH 7.4). 50 μL of 1 μM and 3 μM sKA511 protein solutions were added to each well of the two experimental groups. 50 μL of PBS (0.01M, pH 7.4) was added to each well of the control group. Cells from both groups were cultured at 37°C for 12 hours and 24 hours, respectively, and then photographed under a microscope. The scratch area was statistically analyzed using ImageJ software, with the scratch area of ​​the control group set as 100%. Cell growth and migration ability was assessed based on the ability of cells at the scratch edge to gradually enter the blank area, thus healing the "scratch."

[0084] The results are as follows Figure 4 As shown, sKA511 can effectively inhibit the metastasis of mouse breast cancer cells 4T1.

[0085] 2. Transwell transfer experiment

[0086] 100 μL of mouse breast cancer cell suspension (4T1) was added to a Transwell chamber (Corning, USA). Experimental group: 700 μL of 1640 medium (Invitrogen) containing 1 μM sKA511 was added to the lower chamber of a 24-well culture plate; Control group: 700 μL of 1640 medium (Invitrogen) was added to the lower chamber of a 24-well culture plate. The Transwell chambers containing 4T1 cells were placed into the 24-well culture plates, avoiding air bubble formation. After 18 h of culture, the Transwell chambers were removed, the culture medium in the wells was discarded, and the cells were washed twice with PBS (0.01 M, pH 7.4), then fixed with methanol for 30 minutes. The Transwell chambers were then allowed to air dry appropriately. Cells were stained with 0.1% crystal violet for 20 minutes, and the unmigrated cells on the upper layer were gently wiped away with a cotton swab. The cells were washed three times with PBS (0.01 M, pH 7.4), and then observed and counted under a 40x microscope in five randomly selected fields of view.

[0087] The results are as follows Figure 5 As shown, sKA511 can effectively reduce the metastatic ability of 4T1 cells.

[0088] Example 5. Binding experiment of sKA511 protein with phosphatidylserine (PS)

[0089] Mouse breast cancer cells (4T1) were seeded into 24-well plates with cell spreaders attached, with 3 × 10⁶ cells per well. 4 Cells were collected. After cell adhesion, they were treated with 100 mM H2O2 for 12 h to induce apoptosis. Then, FITC-labeled sKA511 was added, and the cells were incubated at room temperature in the dark for 2 h, followed by washing three times with PBS (0.01 M, pH 7.4). Finally, DAPI was added, and the cells were photographed using a fluorescence microscope after 15 min.

[0090] The results are as follows Figure 6 As shown, after treatment with H2O2, phosphatidylserine (PS) is everted, and FITC-labeled sKA511 can specifically bind to the everted PS.

Claims

1. A protein for reducing the invasiveness of breast cancer, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The gene encoding the protein of claim 1.

3. An expression cassette containing the gene of claim 2.

4. A vector containing the gene of claim 2.

5. Bacteria or fungi containing the gene described in claim 2.

6. Use of the protein of claim 1 in the preparation of a medicament for inhibiting breast cancer metastasis.

7. A drug for inhibiting breast cancer metastasis, comprising the protein of claim 1.

8. The drug according to claim 7, wherein the dosage form is an injection.

9. A method for preparing the protein of claim 1, comprising: The gene described in claim 2 is introduced into an expression vector to obtain a recombinant vector; the recombinant vector is introduced into an expression host bacterium to obtain a recombinant bacterium; The recombinant bacteria were cultured and protein expression was induced; the protein was extracted and purified.

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