Application of CYB5 protein in preparation of medicine for preventing and / or treating liver injury
By constructing the CYB5 protein as a recombinant plasmid and transferring it into probiotics to form engineered probiotics, and using the probiotics to secrete exosomes to make the CYB5 protein work in the body, the problem of acute liver injury treatment is solved and an efficient and safe treatment is achieved. The oral administration form significantly improved liver function.
Patent Information
- Application Number
- CN202310910794.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-24
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art is difficult to effectively treat acute liver injury, and the oral bioavailability of protein drugs is low and the half-life is short, which affects patient compliance and treatment effect.
Use CYB5 protein to prepare reorganized plasmids, and transfer it to probiotics, secrete the exoscover through probiotics to make CYB5 protein play a role in the body, form engineering probiotics, and treat acute liver damage in the form of oral administration.
Significantly reduce the activity of serum valley and cornea, improve liver tissue necrosis, improve patient compliance, reduce production costs, solve the lack of protein drugs, and achieve significant treatment effects.
Smart Images
Figure CN119345332A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biopharmaceuticals, and in particular to the application of CYB5 protein in the preparation of drugs for preventing and / or treating liver damage. Background Art
[0002] Cytochrome B5 (CYB5) is a membrane-bound cytochrome that can reduce iron hemoglobin (methemoglobin) to ferrous hemoglobin. The cytochrome B5 family includes the heme binding domains of various proteins. The structure of a series of oxidoreductases consists of a heme binding domain homologous to the heme binding domain of B5 and a flavin dehydrogenase or a molybdenum pterin domain in parallel. Two members have been found in mammals: cytochrome B5 and sulfite oxidase. Cytochrome B5 is found in mitochondria and endoplasmic reticulum in membrane-bound form and in soluble form in red blood cells. Peptide drugs and protein drugs have the characteristics of high specificity and high safety, and have been widely used to treat various diseases. However, due to the structural organization and physiological function of the gastrointestinal tract, protein drugs have low bioavailability and short half-life after oral administration. Therefore, in order to improve the bioavailability of protein drugs, protein drugs are mostly administered by injection in clinical practice, which reduces patient compliance.
[0003] Acute liver injury refers to liver cell damage caused by various causes in patients without chronic liver disease. In most patients, the liver can maintain the normal operation of its main functions. Clinically, mild cases are manifested by elevated serum transaminases and bilirubin; severe cases may develop liver failure, coagulation dysfunction, hepatic encephalopathy, etc. There are many causes of acute liver injury, including viral infection, drug-induced, excessive ethanol intake, ischemia, radiation damage, shock, and accidental ingestion of toxic food. At present, there are three main strategies for the treatment of acute liver injury: liver protection, enzyme reduction, and jaundice treatment. Mild cases of acute liver injury can recover in the short term, but if they are not treated in time or the treatment effect is poor, they can develop into chronic liver disease, and eventually liver fibrosis and cirrhosis may occur, requiring liver transplantation if necessary. Therefore, the development of a new drug for the treatment of acute liver injury is a technical problem that needs to be solved urgently.
[0004] The inventor unexpectedly discovered during the research that CYB5 protein has the effect of treating acute liver injury, and the CYB5 protein is prepared into probiotics, and the probiotics secrete exosomes to make the protein work in the body. If it can be applied, it will improve patient compliance and reduce production costs. In addition, the recombinant probiotics can solve the problem of easy degradation during oral administration, play a role in treating acute liver injury, and achieve good therapeutic effects. Summary of the invention
[0005] In view of the above technical problems, the primary purpose of the present invention is to provide the use of CYB5 protein in the preparation of a drug for preventing and / or treating liver injury, wherein the amino acid sequence of the CYB5 protein is shown in SEQ ID No.1.
[0006] Preferably, the CYB5 protein comprises a basic protein formed by any CYB5 protein sequence of humans, chimpanzees, rhesus monkeys, green monkeys, and white-cheeked gibbons.
[0007] Preferably, the nucleotide sequence of the CYB5 protein is shown as SEQ ID No.2.
[0008] Preferably, the liver injury is acute liver injury.
[0009] The second object of the present invention is to provide a recombinant plasmid expressing CYB5 protein. The recombinant plasmid is obtained by inserting a gene encoding CYB5 protein into an expression vector. The amino acid sequence of the CYB5 protein is shown in SEQ ID No.1.
[0010] Preferably, the expression vector includes a eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR / Cas gene editing technology and other medically available gene editing technologies or viral vectors.
[0011] Preferably, the eukaryotic plasmid expression vector is selected from one or more of pcDNA3.1+ / -, pcDNA4 / HisMax B, pSecTag2A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-) / myc-HisA, pcDNA6-Myc / His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-myc, pCMV-HA, pIRES-2puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1hygro, pSilencer3.1-H1 neo, and pSilencer4.1-CMVneo.
[0012] Preferably, the viral expression vector is selected from pLKO.1, pLVXIRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCVMCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper or pAAV-LacZ.
[0013] Preferably, the expression vector includes pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.
[0014] Preferably, the expression vector comprises pET-28a, the recombinant plasmid is named pET-28a-CYB5, and the gene sequence is shown in SEQ ID No.3.
[0015] The third object of the present invention is to provide an engineered probiotic expressing CYB5 protein, wherein the engineered probiotic is obtained by transforming the prepared recombinant plasmid, or by integrating the gene encoding CYB5 protein into the probiotic.
[0016] Preferably, the probiotics are selected from Escherichia coli, probiotic Bacillus, Lactococcus, Butyric acid Bacillus, Lactobacillus, Bifidobacterium, Actinomycetes, and Pichia pastoris.
[0017] The fourth object of the present invention is to provide the use of the engineered probiotics in the preparation of drugs for preventing and / or treating liver damage.
[0018] Preferably, the drug is an oral preparation, specifically including any one of oral liquid, tablet, capsule, granule, powder, suspension, emulsion, pill, and powder.
[0019] The fifth object of the present invention is to provide a probiotic tablet having an auxiliary protective effect on liver damage, wherein the probiotic tablet is prepared by adding the engineered probiotics to a pharmaceutically acceptable excipient.
[0020] Preferably, the content of the recombinant probiotics is 2×10 9 CFU.
[0021] The beneficial effects of the present invention are as follows: the present invention provides a new use of CYB5 protein for treating acute liver injury; it can significantly reduce serum alanine aminotransferase activity and improve liver tissue necrosis; and the CYB5 gene is constructed into a recombinant plasmid, and the plasmid is transferred into probiotics to obtain engineered probiotics, and the CYB5 gene is efficiently expressed in the engineered probiotics; the present invention changes the conventional administration mode of protein drugs (muscular injection or intravenous injection), and applies protein drugs to the treatment of acute liver injury by oral administration, which solves the long-standing unresolved technical problems in the field and achieves remarkable results. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 PCR reaction results of EcN transformed with pET-28a-CYB5 plasmid and pET-28a-EGFP
[0023] Figure 2 Effect of engineered probiotics on protecting the liver of mice with acute liver injury
[0024] Figure 3 Effect of engineered probiotics on reducing serum ALT activity in mice with acute liver injury DETAILED DESCRIPTION
[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the following examples are further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents and materials used in the following examples are commercially available unless otherwise specified.
[0026] The Escherichia coli DH5α in the following examples was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and is hereinafter abbreviated as Escherichia coli DH5α; Escherichia coli Nissle 1917 was purchased from Germany (trademark name Mutaflor), and is hereinafter abbreviated as Escherichia coli EcN.
[0027] Example 1 Preparation of recombinant plasmid and recombinant probiotics
[0028] 1. Experimental Reagents
[0029] The PCR primers used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and the corresponding restriction sites were introduced respectively.
[0030] EGFP (enhanced green fluorescent protein) is a mutant of GFP. The fluorescence intensity it emits is more than 6 times greater than that of GFP. It is more suitable than GFP as a reporter gene to study gene expression, regulation, cell differentiation, and protein localization and transport in organisms.
[0031] The bacterial plasmid DNA extraction kit was purchased from AXYGEN.
[0032] PrimeSTAR HS (Premix, 2X) and T4 DNA ligase were purchased from Takara.
[0033] DNA endonucleases BamHI and EcoRII were purchased from Beijing NEB Company.
[0034] The formula of LB (Luria-Bertani) liquid culture medium is: 1% tryptone, 0.5% yeast extract, and 0.5% NaCl.
[0035] The formula of LB solid culture medium is: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 2% agar.
[0036] The recombinant plasmid pET-28a-CYB5 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the recombinant plasmid pET-28a-EGFP was purchased from Wuhan Miaoling Biotechnology Co., Ltd.
[0037] 2. Primer information synthesis
[0038] T7 and T7ter primers were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. The primers were dissolved in sterile deionized water to a final concentration of 10 μmol / L.
[0039] 3. Construction of recombinant strains
[0040] (1) Extraction of recombinant plasmid DNA: E. coli DH5α containing pET-28a-CYB5 plasmid and pET-28a-EGFP was transferred to LB liquid medium containing 30 μg / mL kanamycin for amplification and culture at a speed of 200 rpm and a temperature of 37° C. The plasmid was extracted using a plasmid extraction kit from AXYGEN.
[0041] (2) Preparation of EcN chemically competent cells: Take out the E. coli EcN from the -80°C refrigerator, pick up a small amount of the preservation solution with a sterilized pipette tip, streak it on LB solid medium, and culture it in a 37°C incubator overnight: Pick up a moist, smooth single colony from the LB solid medium, put it into LB culture solution, and culture it at 37°C for 12 hours; when the OD600 value of the bacterial solution is 0.3-0.5, transfer the bacterial solution to a sterilized centrifuge tube precooled on ice, place it on ice for 30 minutes, and incubate it at 4 ℃, centrifuge at 1520g for 10min; discard the supernatant, gently suspend the cells with 500μL pre-cooled 0.1mol / LCaCl2 solution (filtered and sterilized), centrifuge at 1520g for 10min at 4℃; repeat the previous step once; discard the supernatant, add 500μL pre-cooled 0.1mol / LCaCl2 solution (filtered and sterilized), carefully suspend the cells, and the competent cell suspension is prepared; the prepared competent cell suspension is directly used for transformation experiments. Add an equal volume of sterilized 20% glycerol to the unused competent cells, mix well, and dispense into 1.5mL centrifuge tubes (100μL per tube), and store in a -80℃ refrigerator.
[0042] (3) Transfer the recombinant plasmids pET-28a-CYB5 and pET-28a-EGFP into EcN respectively: Take out the EcN chemical competent cells from the -80℃ refrigerator and place them on an ice box for 10-20 minutes to thaw. Take out 50μL of competent cells and add 5μL of plasmid, gently stir with your fingers several times to mix, and place the mixture on ice for 25 minutes; after heat shock at 42℃ for 45s, quickly put it back on ice and let it stand for 2 minutes, add 0.5mL of room temperature LB liquid culture medium (without antibiotics), and culture at 37℃, 200rpm for 1h. Take 200μL of the above bacterial solution and spread it on an agar plate containing 30μg / mL kanamycin. After the bacterial solution is completely absorbed by the culture medium, invert the culture dish and culture it at 37℃ for 12h.
[0043] (4) Verification of successful colonies after heat shock transformation: Single clones were picked and cultured in liquid culture medium containing 30 μg / mL kanamycin.
[0044] (5) PCR verification: Using the monoclonal bacterial solution as a template, PCR amplification was performed using pET-28a universal primers T7 and T7ter. The PCR amplification system was:
[0045]
[0046] The PCR reaction conditions are: 98.0℃, 5min pre-denaturation, 30 cycles of denaturation, annealing, and extension, where the denaturation conditions are 98.0℃, 10s, the annealing conditions are 58.0℃, 5s, the extension I conditions are 72.0℃, 30s; the extension II conditions are 72.0℃, 10min. The PCR product was subjected to agarose gel electrophoresis (1%), 50V electrophoresis for 45min, and then stained with 3X Gel Green dye. The PCR verification results are as follows Figure 1 As shown, the PCR of the bacterial solution of the successful transformation of pET-28a-CYB5 should contain the target band of CYB5 (765bp), and the arrow in the figure points to the target band, indicating that the pET-28a-CYB5 plasmid was successfully transformed into EcN. The PCR of the bacterial solution of the successful transformation of the pET-28a-EGFP plasmid should contain the target band of EGFP (1080bp), and the target band in the figure indicates that the pET-28a-EGFP plasmid was successfully transformed into EcN.
[0047] Example: Engineered probiotics improve symptoms of acute liver injury in mice
[0048] 1. Animal Experiments
[0049] SPF healthy male BALB / c mice, weighing 18-22 g, had not used any drugs before the experiment and were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences with a license number of SCXK(Gan)-2020-0002. They were adapted to feeding for one week, given animal diet and free access to water, and then grouped for experiments.
[0050] Drugs and reagents: Probiotics containing the recombinant plasmid pET-28a-CYB5 and the probiotics containing the recombinant plasmid pET-28a-EGFP were obtained according to the method described in Example 1. Acetaminophen (APAP) was purchased from McLean Biotechnology Co., Ltd.; β-lactose (containing 70% β-lactose and 30% α-lactose) was purchased from McLean Biotechnology Co., Ltd.
[0051] Reagent preparation: Weigh a certain amount of acetaminophen (APAP) and prepare it into 30 mg / mL. APAP is poorly soluble in water, so it needs to be prepared in warm water (55-60°C). During the administration process, the modeling reagent must also be kept in a warm water bath.
[0052] The 24 BALB / c mice were randomly divided into 4 groups:
[0053] Normal control group (Control): gavage 0.2 mL of normal saline every day for two weeks before modeling;
[0054] Model group: gavage 0.2 mL of normal saline every day for two weeks before modeling;
[0055] EGFP group: 2×10 9 cfu of engineered probiotics containing the recombinant plasmid pET-28a-EGFP;
[0056] CYB5 group: 2×10 9 cfu of engineered probiotics containing the recombinant plasmid pET-28a-CYB5;
[0057] Model induction: The mice were fasted overnight (about 15 hours), and 300 mg / kg of APAP (prepared in 55-60°C saline) was intraperitoneally injected. Food was given immediately after the intraperitoneal injection. Blood samples were collected 24 hours later, and after standing at room temperature for 30 minutes, centrifuged at 2000g, 4°C for 10 minutes, and the supernatant was taken to measure serum aspartate aminotransferase and alanine aminotransferase. After blood collection, the liver was dissected and removed for pathological observation.
[0058] 2. Experimental results
[0059] like Figure 2 As shown in the figure, the liver of the normal group mice was pink and soft, while the liver of the model group and EGFP group mice had a large number of bleeding spots on the surface and the entire liver showed fibrosis. Based on this, we believe that the acute liver injury model was successfully established. The liver of the CYB5 group mice also had some bleeding spots and fibrosis symptoms, but the bleeding spots were less than those of the model group and EGFP group. Therefore, the engineered probiotics expressing CYB5 can improve the liver damage caused by acetaminophen.
[0060] like Figure 3 As shown, the serum alanine aminotransferase activity of the Model group and the EGFP group was significantly higher than that of the normal group, indicating that the liver function was impaired. After the engineered probiotics expressing CYB5 were given in advance, the serum alanine aminotransferase activity of the mice decreased significantly, indicating that the engineered probiotics expressing CYB5 have a protective effect on the liver and can be used to prevent or treat acute liver injury.
[0061] In summary, the present invention provides a new use of CYB5 protein for treating acute liver injury; it can significantly reduce serum alanine aminotransferase activity and improve liver tissue necrosis; and the CYB5 gene is constructed into a recombinant plasmid, and the plasmid is transferred into probiotics to obtain engineered probiotics, and the CYB5 gene is efficiently expressed in the engineered probiotics; the present invention changes the conventional administration method of protein drugs (muscular injection or intravenous injection), and applies protein drugs to the treatment of acute liver injury by oral administration, which solves the long-standing unresolved technical problems in the field and achieves remarkable results.
[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. Use of CYB5 protein in the preparation of a drug for preventing and / or treating liver damage, characterized in that: The amino acid sequence of the CYB5 protein is shown in SEQ ID No.
1.
2. A recombinant plasmid expressing CYB5 protein, characterized in that: The recombinant plasmid is obtained by inserting the gene encoding the CYB5 protein into an expression vector. The amino acid sequence of the CYB5 protein is shown in SEQ ID No.
1.
3. The recombinant plasmid according to claim 2, characterized in that The expression vectors include eukaryotic expression plasmid vectors, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR / Cas gene editing technology and other medically available gene editing technologies or viral vectors.
4. The recombinant plasmid according to claim 3, characterized in that The eukaryotic plasmid expression vector is selected from one or more of pcDNA3.1+ / -, pcDNA4 / HisMaxB, pSecTag2A, pVAX1, pBudCE4.1, pTracerCMV2, pcDNA3.1(-) / myc-HisA, pcDNA6-Myc / HisB, pCEP4, pIRES, pIRESneo, pIREShyg3, pCMV-myc, pCMV-HA, pIRES-2puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6hygro, pSilencer3.1-H1hygro, pSilencer3.1-H1neo, and pSilencer4.1-CMVneo.
5. The recombinant plasmid according to claim 3, characterized in that The viral expression vector is selected from pLKO.1, pLVXIRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCVMCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper or pAAV-LacZ.
6. The recombinant plasmid according to claim 3, characterized in that The expression vectors include pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.
7. An engineered probiotic expressing CYB5 protein, characterized in that: The engineered probiotics are obtained by transforming the recombinant plasmid prepared according to claim 3, or by integrating the gene encoding the CYB5 protein into the probiotics.
8. The engineered probiotic according to claim 7, characterized in that The probiotics are selected from Escherichia coli, probiotic bacillus, lactococcus, butyric acid bacteria, lactobacillus, bifidobacterium, actinomycetes, and Pichia pastoris.
9. Use of the engineered probiotics according to claim 7 in the preparation of a drug for preventing and / or treating liver damage.
10. A probiotic tablet having an auxiliary protective effect on liver damage, characterized in that: The probiotic tablet is prepared by adding the engineered probiotics described in claim 7 to pharmaceutically acceptable excipients.