Application of bifidobacterium breve secretory protein pflB in preparation of malignant tumor treatment medicine

Abstract

The invention belongs to the technical field of biological medicine, discloses application of bifidobacterium breve secretory protein pflB in preparation of malignant tumor treatment medicine, and provides that the pflB protein (which is probiotic bifidobacterium breve specific bioactive protein) can effectively inhibit proliferation of malignant tumors (such as colorectal cancer, melanoma, lung cancer, prostatic cancer and breast cancer) for the first time. The apoptosis of malignant tumors is induced, and normal cells are not obviously influenced. The pflB protein can achieve the anti-tumor purpose by increasing the apoptosis of tumor cells and adjusting the tumor immune microenvironment (promoting the expression of cell factors such as IL-1beta, IL-6 and / or TNF-alpha).

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of the secreted protein pflB of Bifidobacterium breve in the preparation of drugs for the treatment of malignant tumors. Background Art

[0002] Currently, the treatment of malignant tumors mainly includes surgical treatment, radiotherapy, chemotherapy, immunotherapy and other methods. In addition, in recent years, significant progress has been made in the research of specific bioactive proteins in the field of tumor treatment. Some polypeptide drugs with anti-tumor activity, such as Aplidin, a cyclic depsipeptide extracted from the marine organism Aplidium albicans, can block the tumor cell cycle, induce apoptosis, and inhibit the secretion of vascular endothelial growth factor and the expression of its receptor; certain intestinal probiotics such as Akkermansia mucophila can regulate the intestinal immune response by producing short-chain fatty acids and generating specific bioactive proteins such as Amuc_1100 and Amuc_2172, thereby maintaining the balance of the intestinal internal environment and targeting the treatment of colorectal cancer, showing a unique anti-tumor mechanism.

[0003] Bifidobacterium breve is an important probiotic and has been widely used in the fields of food, health products and pharmaceuticals. It is widely used in fermented dairy products, beverages and health foods, and can provide unique protection for the intestinal health of infants, effectively reducing the incidence of intestinal infections. In addition, Bifidobacterium breve can also be used to prepare drugs for the treatment of intestinal mucosal damage, alcohol-induced liver damage and constipation. At the same time, it also has probiotic functions such as regulating the immune system, improving allergic reactions, and promoting normal intestinal development. Especially in the health of infants, Bifidobacterium breve helps to promote the growth and development of premature infants, reduce the infection rate, and prevent the occurrence of allergic diseases, and has a wide range of application scenarios.

[0004] The pflB protein is a formate acetyltransferase, and its main function is to convert pyruvate produced during glucose metabolism into formate. Existing studies have shown that the pflB protein is related to the nutrient release associated with apoptosis. For example, in Salmonella, the expression of the pflB gene is significantly increased in the apoptotic supernatant, while the pflB mutant is significantly reduced in these supernatants, indicating that the pflB protein may play an important role in the bacteria's utilization of nutrients released by host cell death.

[0005] Bacteria exert their anti - malignant tumor effects through specific bioactive proteins. However, up to now, there has been no report that specific bioactive proteins in Bifidobacterium breve (such as Bifidobacterium breve ATCC 15700, B.bre) have anti - malignant tumor effects. Summary of the Invention

[0006] An object of the first aspect of the present invention is to provide the use of pflB protein in the preparation of drugs for preventing and / or treating tumors.

[0007] An object of the second aspect of the present invention is to provide the use of pflB protein in inhibiting the proliferation of tumor cells.

[0008] An object of the third aspect of the present invention is to provide the use of a vector, expression cassette, transgenic cell line, recombinant bacterium and / or recombinant virus containing a nucleic acid molecule encoding pflB protein.

[0009] An object of the fourth aspect of the present invention is to provide a product.

[0010] An object of the fourth aspect of the present invention is to provide a method for inhibiting the proliferation of tumor cells in vitro and inducing apoptosis of tumor cells.

[0011] In order to achieve the above - mentioned objects, the technical solutions adopted by the present invention are as follows:

[0012] The first aspect of the present invention provides the use of pflB protein in the preparation of drugs for preventing and / or treating tumors.

[0013] In some embodiments of the present invention, the amino acid sequence of the pflB protein is as shown in SEQ ID NO:2, or has at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with SEQ ID NO:2, and has the same function as the protein shown in SEQ ID NO:2.

[0014] In some embodiments of the present invention, the tumor includes malignant tumors.

[0015] In some embodiments of the present invention, the malignant tumors include at least one of colorectal cancer, melanoma, lung cancer, bladder cancer, breast cancer, testicular cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, cholangiocarcinoma, gallbladder cancer; preferably, the malignant tumors include colorectal cancer, melanoma, lung cancer, prostate cancer and breast cancer.

[0016] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient / carrier.

[0017] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizing agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption promoters, surfactants, preservatives, pigments, flavors, and solvents.

[0018] In some embodiments of the present invention, the fillers are selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binders are selected from cellulose derivatives, alginates, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrants are selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, or cross-linked sodium carboxymethyl cellulose; the lubricants are selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, colloidal silicon dioxide, talc, or magnesium stearate; the suspending agents are selected from colloidal silicon dioxide, beeswax, cellulose, solid polyethylene glycol; the wetting agents are selected from glycerol, Tween-80, hydrogenated castor oil, or lecithin; the solvents are selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol, or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactants are selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, sorbitan fatty acid ester, or polysorbate (Tween), etc.; the flavoring agents are selected from aspartame, sucralose, flavor, steviol glycoside, acesulfame potassium, citric acid, or sodium saccharin; the preservatives are selected from at least one of methyl paraben or propyl paraben.

[0019] In some embodiments of the present invention, the drug may further contain other active pharmaceutical ingredients, and combination therapy can be achieved by combining with other drugs.

[0020] In some embodiments of the present invention, the drug can be administered by local administration, and the administered dose is a pharmaceutically acceptable dose.

[0021] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, bilayer tablets, multilayer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing pellets or tablets, pH-dependent capsules containing pellets or tablets, gastrointestinal compound capsules), suppositories, injections, sprays, and injections.

[0022] In some embodiments of the present invention, the effective dose of the pflB protein is 15-50 μg; preferably 20-40 μg.

[0023] The pflB protein in the present invention can be derived from, including but not limited to: Enterobacter, Salmonella, Shigella, Klebsiella, Pseudomonas, Proteus, Citrobacter, Vibrio, Clostridium, Bacillus, Bifidobacterium, etc., such as Bifidobacterium breve ATCC 15700. It can also be obtained by in vitro induced expression.

[0024] The second aspect of the present invention provides the use of the pflB protein in at least one of (a1)-(a6):

[0025] (a1) Inhibiting the proliferation of tumor cells;

[0026] (a2) Preparing a product for inhibiting the proliferation of tumor cells;

[0027] (a3) Inducing apoptosis of tumor cells;

[0028] (a4) Preparing a product for inducing apoptosis of tumor cells;

[0029] (a5) Promoting the expression levels of IL-1β, IL-6 and TNF-α;

[0030] (a6) Preparing a product for promoting the expression levels of IL-1β, IL-6 and TNF-α.

[0031] In some embodiments of the present invention, the above uses are all for non-disease diagnosis and treatment purposes.

[0032] In some embodiments of the present invention, the effective dose of the pflB protein is 0.1-15 μg; preferably 0.1-10 μg; more preferably 0.8-10 μg.

[0033] In some embodiments of the present invention, the product includes reagents and kits.

[0034] In some embodiments of the present invention, the tumor includes malignant tumors.

[0035] In some embodiments of the present invention, the malignant tumor includes at least one of colorectal cancer, melanoma, lung cancer, bladder cancer, breast cancer, testicular cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cavity cancer, pancreatic cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, cholangiocarcinoma, and gallbladder cancer; preferably, the malignant tumor includes colorectal cancer, melanoma, lung cancer, prostate cancer, and breast cancer.

[0036] In some embodiments of the present invention, the amino acid sequence of the pflB protein is as shown in SEQ ID NO: 2, or has at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with SEQ ID NO: 2, and has the same function as the protein shown in SEQ ID NO: 2.

[0037] The third aspect of the present invention provides the use of a vector, expression cassette, transgenic cell line, recombinant bacterium, and / or recombinant virus containing a nucleic acid molecule encoding the pflB protein in at least one of (b1) to (b7):

[0038] (b1) Inhibiting the proliferation of tumor cells;

[0039] (b2) Preparing a product for inhibiting the proliferation of tumor cells;

[0040] (b3) Inducing apoptosis of tumor cells;

[0041] (b4) Preparing a product for inducing apoptosis of tumor cells;

[0042] (b5) Promoting the expression levels of IL-1β, IL-6, and / or TNF-α;

[0043] (b6) Preparing a product for promoting the expression levels of IL-1β, IL-6, and / or TNF-α;

[0044] (b7) Preparing a drug for preventing and / or treating tumors;

[0045] The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1.

[0046] In some embodiments of the present invention, the product includes reagents and kits.

[0047] In some embodiments of the present invention, (b1) to (b6) are all for non-disease diagnosis and treatment purposes, such as inhibiting the proliferation of tumor cells in vitro and inducing apoptosis of tumor cells in vitro.

[0048] In some embodiments of the present invention, the tumor includes a malignant tumor.

[0049] In some embodiments of the present invention, the malignant tumor includes at least one of colorectal cancer, melanoma, lung cancer, bladder cancer, breast cancer, testicular cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cavity cancer, pancreatic cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, cholangiocarcinoma, and gallbladder cancer; preferably, the malignant tumor includes colorectal cancer, melanoma, lung cancer, prostate cancer, and breast cancer.

[0050] In some embodiments of the present invention, the transgenic cell line does not contain propagation materials.

[0051] In some embodiments of the present invention, the vector includes a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0052] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0053] In some embodiments of the present invention, the viral vector includes at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxviral vector, a Sendai viral vector, and a herpes simplex viral vector.

[0054] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0055] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, an F cosmid, or an artificial chromosome.

[0056] The fourth aspect of the present invention provides a product including a PflB protein and a pharmaceutically acceptable excipient / carrier.

[0057] In some embodiments of the present invention, the product includes a reagent, a kit, and a drug.

[0058] In some embodiments of the present invention, the pharmaceutically acceptable excipient includes at least one of a filler, a disintegrant, a diluent, a dispersant, an excipient, a stabilizer, a lubricant, a binder, a wetting agent, a flavoring agent, a solubilizer, a suspending agent, a solvent, a sustained-release agent, an emulsifier, an absorption enhancer, a surfactant, a preservative, a pigment, a fragrance, and a solvent.

[0059] In some embodiments of the present invention, the product has at least one of the following effects: inhibiting the proliferation of tumor cells, inducing apoptosis of tumor cells, promoting the expression levels of IL-1β, IL-6, and / or TNF-α, and having an anti-tumor effect.

[0060] In some embodiments of the present invention, the effective dose of the pflB protein is 0.1 - 15 μg; preferably 0.1 - 10 μg; more preferably 0.8 - 10 μg.

[0061] The fifth aspect of the present invention provides a method for inhibiting the proliferation of tumor cells and inducing apoptosis of tumor cells in vitro, including the step of using the pflB protein to act on tumor cells. The amino acid sequence of the pflB protein is as shown in SEQ ID NO:2, or has at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with SEQ ID NO:2 and has the same function as the protein shown in SEQ ID NO:2.

[0062] The beneficial effects of the present invention are:

[0063] The present invention firstly proposes that the pflB protein (a bioactive protein of the probiotic Bifidobacterium breve) can effectively inhibit the proliferation of malignant tumors (such as colorectal cancer, melanoma, lung cancer, prostate cancer, breast cancer), induce apoptosis of malignant tumors, and has no significant effect on normal cells. The pflB protein can achieve the anti-tumor purpose by increasing the apoptosis of tumor cells and regulating the tumor immune microenvironment (promoting the expression of cytokines such as IL-1β, IL-6, and / or TNF-α).

[0064] Bifidobacterium breve is a probiotic coexisting in the human body, with characteristics such as high safety and wide application. The present invention is of great significance for finding new active substances in Bifidobacterium breve that can effectively inhibit intestinal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is the spectrum diagram of the pET28a(+) - pflB plasmid.

[0066] Figure 2 It is the electrophoretic detection diagram of the pflB protein. In the figure, 1 is before induction; 2 is after induction; 3 is the lysis supernatant; 4 is the lysis precipitate; 5 is the flow-through; 6 is the 20 mM eluate; 7 is the 50 mM eluate; 8 is the 100 mM eluate; 9 is the 200 mM eluate; 10 is the 300 mM eluate; 11 is the 500 mM eluate; 12 is the concentrate.

[0067] Figure 3To study the effect of the specific bioactive protein pflB of Bifidobacterium breve on the proliferation of normal and malignant tumor cells. In the figure, ns represents no significant difference, * represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.005, and **** represents P ≤ 0.001.

[0068] Figure 4 To study the effect of the specific bioactive protein pflB of Bifidobacterium breve on the proliferation of malignant tumor cells such as melanoma, lung cancer, prostate cancer or breast cancer. In the figure, ns represents no significant difference, * represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.005, and **** represents P ≤ 0.001.

[0069] Figure 5 To study the effect of the specific bioactive protein pflB of Bifidobacterium breve on the apoptosis of normal and malignant tumor cells; among them, A is the result diagram of the apoptosis of normal intestinal epithelial cells NCM460 detected by flow cytometry for pflB protein, and B is the statistical result diagram of A; C is the result diagram of the apoptosis of colorectal cancer cell line MC38 detected by flow cytometry for pflB protein, and D is the statistical result diagram of C; E is the result diagram of the apoptosis of colorectal cancer cell line CT26 detected by flow cytometry for pflB protein, and F is the statistical result diagram of E. In the figure, ns represents no significant difference, *, $ or # represents P ≤ 0.05, **, $$ or ## represents P ≤ 0.01, ***, $$$ or represents P ≤ 0.005, and ****, $$$$ or represents P ≤ 0.001.

[0070] Figure 6 To study the promotion of the expression of inflammatory factors in RAW264.7 cells by the specific bioactive protein pflB of Bifidobacterium breve. In the figure, ns represents no significant difference, * represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.005, and **** represents P ≤ 0.001.

[0071] Figure 7 To study the inhibition of the growth of malignant tumors in mice by the specific bioactive protein pflB of Bifidobacterium breve; among them, A is the actual tumor diagram of mice in each treatment group; B is the statistical result of the body weight of mice in each treatment group; C is the statistical result of the tumor volume of mice in each treatment group; D is the statistical result of the tumor weight of mice in each treatment group; E is the tumor volume result of mice in the PBS group; F is the statistical result of the tumor volume of mice in the pflB protein group. In the figure, ns represents no significant difference, ** represents P ≤ 0.01, and **** represents P ≤ 0.001.

[0072] Figure 8For the short Bifidobacterium - specific bioactive protein PflB to promote the expression of tumor - related inflammatory factors in mice, in the figure, ns represents no significant difference, ** represents P≤0.01, and **** represents P≤0.001.

[0073] Figure 9 The PflB protein was obtained by proteomic screening of B.bre EVs. Detailed implementation manners

[0074] The content of the present invention will be further described in detail through specific embodiments below.

[0075] It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0077] The features and performance of the present invention will be further described in detail below in combination with the embodiments.

[0078] The specific bioactive protein PflB of Bifidobacterium breve (ATCC 15700) mentioned in the present invention is based on the previous experimental basis of the laboratory, that is, preliminary identification was obtained through proteomic analysis of the extracellular membrane vesicles (Bifidobacterium breve Extracellular vesicles, B.bre EVs) secreted by Bifidobacterium breve (ATCC 15700) ( Figure 9 ), and then the specific bioactive protein PflB of Bifidobacterium breve (ATCC 15700) was constructed in vitro and expressed and purified on a large scale using bioengineering technology to verify its effect on inhibiting malignant tumors.

[0079] Example 1 Expression and purification of the specific bioactive protein PflB of Bifidobacterium breve (ATCC 15700)

[0080] (1) Preparation of Escherichia coli BL21(DE3) competent cells

[0081] The successfully constructed pET28a(+)-pflB plasmid containing the bacteriophage T7 promoter and the pflB gene will be introduced into Escherichia coli BL21(DE3) competent cells and grown overnight on an LB solid medium plate containing KanR resistance. Single colonies that have been successfully cultured will be picked and transferred to an LB liquid medium containing KanR resistance for continued culture for 12 - 16 h to obtain recombinant bacteria.

[0082] The nucleotide sequence of the pflB gene is as follows:

[0083] ATGACCGCAGTAGAAAATGCAGCTGTCTCCCAAGAGGAGCTCGACGCCAAGG

[0084] CGTGGGCCGGCTTCACCGAGGGCAACTGGCAGAAGGATATCGATGTCCGCGACTT

[0085] CATCCAGAAGAACTACACCCCGTACGAGGGTGATGAGAGCTTCCTGGCTCCTGCC

[0086] ACTGAGAAGACGAAGCACCTGTGGAAGTACCTCGACGACAACTACCTGGCTGTAG

[0087] AGCGCAAGCAGCGCGTCTACGATGTGGACACCCACACCCCGGCTGGCATCGACGC

[0088] CTTCCCGGCTGGCTACATCGACTCCCCGGAAGTCGACAATGTGATCGTTGGTCTGC

[0089] AGACCGACGTGCCCTGCAAGCGCGCTATGATGCCGAACGGCGGCTGGCGTATGGT

[0090] CGAGCAGGCCATCAAGGAAGCCGGCAAGGAGCCGGATCCGGAGATCAAGAAGAT

[0091] CTTCACCAAGTACCGCAAGACCCACAATGACGGCGTGTTCGGTGTGTACACCAAG

[0092] AACATCAAGATCGCCCGCCACAACAAGATTCTGACCGGTCTGCCGGATGCTTACG

[0093] GCCGTGGCCGCATCATCGGCGACTACCGTCGTGTGGCTCTGTACGGTGTCAACAAG

[0094] CTGATCGCCTTCAAGAAGCGCGACAAGGACTCCATCCCGTACCGCAACGACTTCA

[0095] CCGAGCCGGAGATCGAGCACTGGATCCGCTTCCGTGAAGAGCACGACGAGCAGAT

[0096] CAAGGCCCTCAAGCAGCTGATCAACCTCGGCAACGAGTATGGCCTGGATCTGACC

[0097] CGCCCGGCTCAGACCGCTCAGGAAGCCGTGCAGTGGACCTACATGGGCTACCTGG

[0098] CCTCCATCAAGTCCCAGGACGGCGCCGCAATGTCCTTCGGCCGTAACTCCGCGTTC

[0099] CTCGATGTCTTCATCGAACGCGACCTCAAGGCCGGCAAGATTACCGAGACCGATG

[0100] CTCAGGAGCTCATCGACAACATCGTCATGAAGCTGCGCATCGTCCGCTTCCTGCGC

[0101] ACCAAGGATTACGACAACATCTTCTCTGGTGACCCGTACTGGGCGACCTGGTCTGA

[0102] TGCCGGCTTCGGCGACGACGGCCGTCCGCTGGTCACCAAGACCTCTTTCCGTCTG

[0103] CTCAACACCCTGACCCTTGAGCACCTCGGACCCGGCCCTGAGCCGAACATCACCA

[0104] TCTTCTGGGATCCGAAGCTGCCGGAAGGCTACAAGCGCTTCTGCGCCAAGATCTC

[0105] CATCGACACCTCGGCTATTCAGTACGAGTCCGACAAGGAAATCCGCTCCCACTGGG

[0106] GCGACGACGCCGCCATTGCCTGCTGCGTCTCCCCGATGCGCGTGGGCAAGCAGAT

[0107] GCAGTTCTTCGCTGCTCGTGTGAACTCCGCCAAGGCTCTGCTGTACGCCATCAACG

[0108] GCGGCCGCGATGAGATGACCGGCATGCAGGTCATCGACAAGGGCATCATCGACCC

[0109] GATCACCCCGGAAGCCGATGGCACTCTGGACTACGCGAAGGTCAAGGACAACTAT

[0110] GAGAAGGCTCTCGAGTGGCTGTCCGAGACTTACGTCGAGGCTCTGAACATCATCC

[0111] ACTACATGCACGATAAGTACGCCTACGAGTCCATCGAGATGGCCCTGCACGACAAG

[0112] GAAGTATACCGTACCCTTGGTTGCGGTATGTCCGGTCTGTCCATCGCCGCCGACTCC

[0113] CTGGCTGCCATCAAGTATGCCAAGGTCTACCCGATCTACAACAAGGATGCCAAGAA

[0114] CCTCGAAGGCCACGAGTACGAGTACGTCGAGGGCGCTGACGACGATTTGGTCGTC

[0115] GGCTACCGCACTGAGGGCGACTTCCCGGTCTACGGCAACGATGACGATCGCGCCG

[0116] ATGACATCGCCAAGTGGGTTGTCTCCACTGTGATGGGCCAGGTCAAGCGCCTGCC

[0117] CGTCTACCGTGGCGCCGTCCCGACCCAGTCCATCCTGACCATCACCTCCAACGTGG

[0118] AGTACGGCAAGAACACCGGTTCCTTCCCGTCCCGCCACAAGAAGGGCACTCCGTA

[0119] CGCTCCGGGCGCCAACCCAGAGAACGGCATGGACTCCCACGGCATGCTGCCGTCC

[0120] ATGTTCTCCGTCGGCAAGATCGACTACAACGACGCTCTTGACGGCATCTCGCTGAC

[0121] CAACACCATCACTCCTGATGGCTTGGGCCGCGATGAGGACGAGCGTATCGGCAAC

[0122] CTGGTCGGCATCCTGGATGCCGGCAACGGCCACGGCCTCTACCACGCGAACATCA

[0123] ACGTTCTGCGCAAGGAGACCATGGAGGACGCCGTCGAGCACCCCGAAAAGTACC

[0124] CGCACCTGACCGTGCGCGTCTCCGGCTACGCGGTGAACTTCGTCAAGCTCACCAA

[0125] GGAGCAGCAGCTCGACGTCATCTCCCGTACCTTCCACCAGGGCGCCGTCGTCGAC

[0126] TGA(SEQ ID NO:1).

[0127] (2) Induced expression of specific bioactive protein pflB

[0128] When inducing the expression of specific bioactive protein pflB, the recombinant bacteria in step (1) are transferred to LB liquid medium with KanR resistance at a ratio of 1:100, and cultured with shaking at 37 °C for 2 - 3 h until the OD of the bacterial liquid 600The value is 0.5 to 1.0. Add 1.0 mM inducer IPTG and culture with shaking at 25 °C for 20 h. After induction, centrifuge the bacterial solution at 8500 rpm and 4 °C for 10 min. Discard the supernatant, wash the bacterial pellet with PBS, and repeat the above centrifugation step. Finally, store the bacterial pellet at -80 °C.

[0129] (3) Add cell lysate (Cellapy Biotech, PRO202) to the bacteria in step (2), resuspend the bacteria completely on ice, and lyse by ultrasonic disruption until the solution is completely clear. Centrifuge the lysate at 8500 rpm and 4 °C for 10 min, separate the supernatant and precipitate on ice, and store them separately for subsequent detection. At this time, the PflB protein is in the supernatant.

[0130] (4) Purify the PflB protein using a Ni NTA Beads gravity column (TianDiRenHe, SA004GC05). Equilibrate with 5 column volumes of Lysis Buffer to keep the packing material in the same buffer system as the target protein, and repeat 2 - 3 times. Add the sample to the equilibrated gravity column and let the sample stay for at least 2 min. Wash away non-specifically adsorbed miscellaneous proteins with 10 - 15 column volumes of Wash Buffer and collect the wash solution. Elute with 5 - 10 column volumes of Elution Buffer, collect in fractions, collect one tube for each column volume, and detect separately.

[0131] (5) Purify and concentrate the protein using a Milipore 5000 KDa ultrafiltration tube. Centrifuge at 4000 rpm and 4 °C for 30 min. The liquid above the ultrafiltration tube is the purified and concentrated PflB protein.

[0132] The results showed that in this example, the plasmid pET28a(+)-pflB expressing PflB was successfully constructed ( Figure 1 ), and the PflB protein was successfully induced ( Figure 2 ). Figure 2 In, 1 is the bacterial supernatant before induction, 2 is the bacterial supernatant after induction, demonstrating the successful induction expression of the PflB protein. 3 is the bacterial lysis supernatant, 4 is the bacterial lysis precipitate, proving that PflB can be highly expressed in the bacterial supernatant. 5 is the purified flow-through, 6 - 11 are purified elution solutions with different concentrations (20, 50, 100, 200, 300, 500 mM respectively), indicating that the 300 mM elution solution can elute all the PflB protein. 12 is the purified and concentrated PflB protein, with a single protein band and a protein purity above 95%, meeting the standards for subsequent experiments.

[0133] The amino acid sequence of the PflB protein is as follows:

[0134] MTAVENAAVSQEELDAKAWAGFTEGNWQKDIDVRDFIQKNYTPYEGDESFLAPA

[0135] TEKTKHLWKYLDDNYLAVERKQRVYDVDTHTPAGIDAFPAGYIDSPEVDNVIVGLQT

[0136] DVPCKRAMMPNGGWRMVEQAIKEAGKEPDPEIKKIFTKYRKTHNDGVFGVYTKNIK

[0137] IARHNKILTGLPDAYGRGRIIGDYRRVALYGVNKLIAFKKRDKDSIPYRNDFTEPEIEH

[0138] WIRFREEHDEQIKALKQLINLGNEYGLDLTRPAQTAQEAVQWTYMGYLASIKSQDGA

[0139] AMSFGRNSAFLDVFIERDLKAGKITETDAQELIDNIVMKLRIVRFLRTKDYDNIFSGDP

[0140] YWATWSDAGFGDDGRPLVTKTSFRLLNTLTLEHLGPGPEPNITIFWDPKLPEGYKRFC

[0141] AKISIDTSAIQYESDKEIRSHWGDDAAIACCVSPMRVGKQMQFFAARVNSAKALLYAI

[0142] NGGRDEMTGMQVIDKGIIDPITPEADGTLDYAKVKDNYEKALEWLSETYVEALNIIH

[0143] YMHDKYAYESIEMALHDKEVYRTLGCGMSGLSIAADSLAAIKYAKVYPIYNKDAKNL

[0144] EGHEYEYVEGADDDLVVGYRTEGDFPVYGNDDDRADDIAKWVVSTVMGQVKRLPV

[0145] YRGAVPTQSILTITSNVEYGKNTGSFPSRHKKGTPYAPGANPENGMDSHGMLPSMFSV

[0146] GKIDYNDALDGISLTNTITPDGLGRDEDERIGNLVGILDAGNGHGLYHANINVLRKET

[0147] MEDAVEHPEKYPHLTVRVSGYAVNFVKLTKEQQLDVISRTFHQGAVVD(SEQ ID NO:2).

[0148] Example 2: Experiment on the in vitro inhibition of tumor cells by the specific bioactive protein pflB of Bifidobacterium breve

[0149] (1) Detection of the effect of the specific bioactive protein pflB of Bifidobacterium breve on the normal intestinal epithelial cell line NCM460 and colorectal cancer cell lines MC38 and CT26 by CCK-8 method

[0150] Resuscitate the normal intestinal epithelial cell line NCM460 and colorectal cancer cell lines MC38 and CT26 and passage them for more than three generations. Seed 96-well plates at 5000 cells / well and culture them in a 5% CO2, 37 °C incubator. After 24 h, add 100 μL of the specific bioactive protein pflB of Bifidobacterium breve (prepared in Example 1) filtered through a 0.22 μm filter membrane at a concentration of 0, 1, 8, 16, 32, 64 μg / mL per well. After 24 h, aspirate the culture medium of each well, add 100 μL of CCK-8 reagent at a concentration of 10% to each well, incubate at 37 °C in the dark for 30 min, and use a microplate reader to detect the absorbance value at 450 nm and calculate the cell survival rate according to the kit instructions.

[0151] (2) Detection of the effect of the specific bioactive protein pflB of Bifidobacterium breve on the apoptosis of tumor cells by flow cytometry

[0152] Seed NCM460, MC38, and CT26 in 12-well plates. Set up a normal control group: cells + RPMI 1640 medium (Gibco, C11875500BT), a positive control group: cells + medium + 1‰ (V / V) Apoptosis Inducer A (Abbikine, KTA0002), and an experimental group: cells + medium + 10, 20, 40 μg / mL of the specific bioactive protein pflB of Bifidobacterium breve filtered through a 0.22 μm filter membrane, 100 μL each. After 24 h, aspirate the culture medium of each well and collect the cells. Stain different groups according to the experimental steps of the cell apoptosis kit (Abbikine, KTA0002), analyze the cells by flow cytometry, and use FlowJo to analyze the results after the detection.

[0153] (3) Detection of the effect of the specific bioactive protein pflB of Bifidobacterium breve on macrophage inflammatory factors by real-time fluorescence quantitative PCR

[0154] Resuscitate and passage mouse RAW264.7 cells for more than three generations, seed 12-well plates at 10,000 cells / well, and culture them in an incubator at 5% CO2 and 37°C. After 24 h, add 2000 μL of specific bioactive protein pflB of Bifidobacterium breve filtered through a 0.22 μm filter membrane at concentrations of 1, 8, 16, and 64 μg / mL, as well as 500 ng / mL of LPS. After 24 h, remove the supernatant, collect the treated RAW264.7 cells, and extract cellular RNA using the Trizol method. Detect the extracted RNA using Nanodrop, and an OD 260 / OD 280 ratio in the range of 1.8 - 2.0 is considered qualified. According to the III All-in-one RT SuperMix Perfectfor qPCR kit instructions, reverse transcribe the RNA into cDNA. Prepare the qPCR system using upstream and downstream primers, SYBR Green qPCR MasterMix, DEPC water, and cDNA, and perform the reaction according to the three-step method. After the reaction, calculate the relative expression level of the target gene according to the 2 -ΔΔCt -ΔΔCt method, and normalize it using the expression level of the internal reference gene β-Actin. The amplification system, primer sequences, and qPCR program are shown in Table 1, Table 2, and Table 3, respectively.

[0155] Table 1 qPCR amplification system

[0156]

[0157] Table 2 qPCR amplification primer sequences

[0158]

[0159] Table 3 qPCR amplification program

[0160]

[0161] Detect whether the specific bioactive protein pflB of Bifidobacterium breve can inhibit the proliferation of normal intestinal epithelial cells NCM460 and colorectal cancer cells MC38 and CT26 by the CCK-8 method. The results show that the specific bioactive protein pflB of Bifidobacterium breve has a certain inhibitory effect on the proliferation of both malignant tumor cells and has no inhibitory effect on normal intestinal epithelial cells NCM460 ( Figure 3 ), that is, the specific bioactive protein pflB of Bifidobacterium breve can inhibit malignant tumor cells without affecting normal cells.

[0162] Using the same method, the effects of the specific bioactive protein pflB of Bifidobacterium breve on the proliferation of malignant tumor cells such as melanoma cells (B16-F10 cells), lung cancer cells (A459 cells), prostate cancer cells (Du145 cells), or breast cancer cells (4T1 cells) were further investigated. The results are as Figure 4 shown. The specific bioactive protein pflB of Bifidobacterium breve has an inhibitory effect on the proliferation of malignant tumor cells such as melanoma, lung cancer, prostate cancer, or breast cancer.

[0163] After 24 hours of administering the specific bioactive protein pflB of Bifidobacterium breve to different cells, the apoptosis rates of two malignant cells, MC38 and CT26, increased, while the apoptosis rate of normal cells, NCM460, decreased significantly ( Figure 5 ), indicating that the specific bioactive protein pflB of Bifidobacterium breve can kill cancer cells by promoting apoptosis of cancer cells and can inhibit apoptosis of normal cells.

[0164] RAW264.7 cells were induced to polarize into the M1 type using LPS. After extracting RNA, it was reverse transcribed into cDNA, and then the expression levels of three pro-inflammatory factors (IL-1β, IL-6, and TNF-α) were detected by qPCR. The results showed that the specific bioactive protein pflB of Bifidobacterium breve could promote the expression levels of the three cytokines ( Figure 6 ), indicating that the specific bioactive protein pflB of Bifidobacterium breve can promote inflammation and kill cancer cells by promoting inflammation.

[0165] Example 3 In vivo experiment on the inhibition of malignant tumor growth by the specific bioactive protein pflB of Bifidobacterium breve

[0166] After one week of adaptive feeding of C57 / BL6J male mice, MC38 colorectal cancer cells in the logarithmic growth phase with good growth status were taken at a concentration of 2×10 5 cells / mL. 0.1 mL was subcutaneously inoculated at the right lumbar and dorsal region of each mouse. When the tumor volume grew to 50 - 100 mm 3 , the mice were randomly divided into a PBS group and a pflB group, and 100 μL of PBS and pflB (40 μg) were respectively intravenously injected into the mice once every two days for a total of 5 times. During the experiment, the longest and shortest diameters of the tumors were measured before each administration, and the tumor volume of the mice was calculated using the formula V = 1 / 2 × a × b × b (a is the longest diameter and b is the shortest diameter), and a tumor volume change curve was plotted. At the same time, the body weight change of the mice was measured and a body weight change curve was plotted. After the experiment, the tumors of the mice were collected, and cell RNA was extracted using the Trizol method. The extracted RNA was detected using Nanodrop, and the OD 260 / OD 280 ratio within the range of 1.8 - 2.0 was considered qualified. According to III All-in-one RT SuperMix Perfect for qPCR Kit Instruction Manual Reverse transcribes RNA into cDNA, prepares the qPCR system using forward and reverse primers, SYBR Green qPCR Master Mix, DEPC water, and cDNA, and performs the reaction according to the three-step method. After the reaction, calculate the relative expression level of the target gene according to the 2 -ΔΔCt method, and normalize it using the expression level of the internal reference gene β-Actin. The amplification system, primer sequences, and qPCR program are the same as shown in Tables 1 to 3.

[0167] Compared with the mice in the PBS group, the tumor weight and volume of the mice given the pflB protein were significantly reduced, and the pflB protein did not significantly affect the body weight change of the mice ( Figure 7 ). qPCR was used to measure the expression levels of pro-inflammatory factors in the tumor tissues of mice in different treatment groups. The results showed that the specific bioactive protein pflB of Bifidobacterium breve could promote inflammation and by promoting the expression levels of pro-inflammatory factors ( Figure 8 ), which indicated that the pflB protein could inhibit the growth of malignant tumors by regulating the changes in the tumor immune microenvironment of mice.

[0168] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of pflB protein in the preparation of a drug for preventing and / or treating tumors.

2. The application according to claim 1, wherein The amino acid sequence of the pflB protein is as shown in SEQ ID NO:2, or an amino acid sequence having at least 90% homology with SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that The tumors include malignant tumors; Preferably, the malignant tumors include at least one of colorectal cancer, melanoma, lung cancer, bladder cancer, breast cancer, testicular cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, cholangiocarcinoma, gallbladder cancer.

4. The application according to any one of claims 1 to 3, characterized in that, The drug further comprises a pharmaceutically acceptable excipient / carrier.

5. Use of pflB protein in at least one of (a1) to (a6): (a1) Inhibiting the proliferation of tumor cells; (a2) Preparing a product for inhibiting the proliferation of tumor cells; (a3) Inducing apoptosis of tumor cells; (a4) Preparing a product for inducing apoptosis of tumor cells; (a5) Promoting the expression levels of IL-1β, IL-6 and TNF-α; (a6) Preparing a product for promoting the expression levels of IL-1β, IL-6 and TNF-α; Preferably, the product includes reagents and kits.

6. The application according to claim 5, characterized in that, The effective dose of the pflB protein is 0.1 - 15 μg.

7. Use of a vector, expression cassette, transgenic cell line, recombinant bacterium and / or recombinant virus containing a nucleic acid molecule encoding pflB protein in at least one of (b1) to (b7): (b1) Inhibiting the proliferation of tumor cells; (b2) Preparing a product for inhibiting the proliferation of tumor cells; (b3) Inducing apoptosis of tumor cells; (b4) Preparing a product for inducing apoptosis of tumor cells; (b5) Promoting the expression levels of IL-1β, IL-6 and / or TNF-α; (b6) Preparing a product for promoting the expression levels of IL-1β, IL-6 and / or TNF-α; (b7) Preparing a drug for preventing and / or treating tumors; The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:1; Preferably, the product includes reagents and kits.

8. The application according to any one of claims 5 to 7, characterized in that The tumors include malignant tumors; Preferably, the malignant tumors include at least one of colorectal cancer, melanoma, lung cancer, bladder cancer, breast cancer, testicular cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, cholangiocarcinoma, gallbladder cancer.

9. A product, comprising pflB protein and a pharmaceutically acceptable excipient / carrier.

10. A method for inhibiting the proliferation of tumor cells in vitro and inducing apoptosis of tumor cells, comprising the step of using PflB protein to act on tumor cells, wherein the amino acid sequence of the PflB protein is as shown in SEQ ID NO:2, or an amino acid sequence having at least 90% homology with SEQ ID NO:2 and having the same function as the protein shown in SEQ ID NO:2.

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