Gene editing engineering bacterium for treating cholestatic diseases
By screening and integrating the highly efficient 7β-hydroxysteroid dehydrogenase gene sequence into E. coli Nissle 1917, gene editing engineering bacteria were constructed, and the problems of expensive existing drugs and difficulty in predicting the activity of intestinal microbial enzymes were solved, and CDCA was effectively transformed into UDCA, improving the course of cholestatic disease and reducing treatment costs.
Patent Information
- Application Number
- CN202411656552.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-03-11
AI Technical Summary
Existing drugs used to treat primary sclerotic cholangitis (PSC) such as ursodeoxycholic acid (UDCA) are expensive and require long-term treatment, which is difficult to meet the patient's economic affordability. At the same time, the 7α-hydroxysteroid dehydrogenase annotation accuracy in intestinal microorganisms is low, making it difficult to effectively predict its activity.
Sequences with a similarity of more than 30% to 7β-HSDH were screened through BlastP homologous search, and the Clean comparison learning model was used for numbering and annotation. AlphaFold2 homologous model was constructed. Combined with molecular docking, the 7β-hydroxysteroid dehydrogenase gene sequence efficiently transformed CDCA into UDCA was screened, and it was integrated into the phage attb site of Nissle 1917 in E. coli to construct gene editing engineering bacteria.
The conversion of CDCA into UDCA is achieved, thereby improving the course of cholestatic disease, reducing the treatment cost of patients, and laying the foundation for the development of probiotic products that improve the prognosis of patients with PBC.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology, and specifically relates to an Escherichia coli Nissle 1917 gene-edited engineering bacterium that can be used to treat cholestatic diseases and an application thereof. Technical Background
[0002] Cholestatic disease refers to the accumulation of bile in the liver or bile duct system, which leads to excessive accumulation of bile components (such as bile acid, cholesterol, bilirubin, etc.) in the liver and systemic circulation, thus causing damage to liver cells and the body. It is common in common bile duct stones, benign common bile duct stenosis, pancreatitis, pancreaticobiliary tumors, primary or secondary sclerosing cholangitis, etc.
[0003] Primary sclerosing cholangitis (PSC) is a type of cholestatic disease with a global prevalence of about 2% (Trivedi PJ, Hirschfield GM. Recent advances in clinical practice: epidemiology of autoimmune liver diseases. [J] Gut. 2021; 70(10)). Ursodeoxycholic acid (UDCA) is the only drug currently approved for the treatment of PSC, which is effective in treating PSC patients. However, this drug is very expensive (Clinical Review Report: Obeticholic Acid (Ocaliva): (Intercept Pharmaceuticals Canada, Inc.). Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; August [M] 2017.), and the disease requires continuous treatment, making it difficult for ordinary people to afford the full cost of treatment.
[0004] A large number of studies have shown that an increase in the ratio of chenodeoxycholic acid (CDCA) to cholic acid (CA) can lead to cholestasis, and early UDCA intervention can significantly delay the progression of the disease (Tang R, Wei Y, Li Y, et al. Gut microbial profile is altered in primary biliary cholangitis and partially restored after UDCA therapy. [J] Gut. 2018; 67 (3): 534-541.). Among them, intestinal microorganisms are closely related to changes in the bile acid pool. It is currently known that the enzymes responsible for converting CDCA to UDCA in intestinal bacteria are 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). However, the annotation accuracy of hydroxysteroid dehydrogenase is very low, and conventional prediction methods often cannot predict its correct activity. Summary of the invention
[0005] In view of this, the present invention uses BlastP to perform homology search in the HMP and MGYG databases to screen sequences with a similarity of more than 30% to the known 7β-HSDH. The candidate sequences obtained by screening are numbered and annotated using the Clean comparative learning model, and 136 sequences are predicted from more than 63,000 candidates for AlphaFold2 homology modeling. The modeled protein is molecularly docked and screened with 7-ketolithocholic acid, and then 6 sequences are finally selected based on the abundance of candidate genes in the population. The 6 candidate sequences are designed into complete expression frames and integrated into the bacteriophage attb site of Escherichia coli Nissle 1917, and the 7α-HSDH activity of Escherichia coli Nissle 1917 itself is added. A strain of Escherichia coli Nissle 1917 gene-edited engineering bacteria that efficiently converts CDCA into UDCA to improve cholestatic diseases is screened, and its sequence is derived from Ruminococcus gnavus.
[0006] The specific technical solutions of the present invention are as follows:
[0007] The invention discloses an application of 7β-hydroxysteroid dehydrogenase derived from Ruminococcus gnavus in preparing a medicine for treating cholestatic diseases. The nucleotide sequence of the 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO.1.
[0008] In a specific application of the present invention, the drug is a gene-edited engineered bacterium.
[0009] Preferably, the gene-edited engineered bacteria are prokaryotic cells. Further, the prokaryotic cells are selected from Escherichia coli.
[0010] In a specific example of the present invention, the gene-edited engineered bacteria is Escherichia coli Nissle 1917.
[0011] Specifically, the 7β-hydroxysteroid dehydrogenase gene sequence was integrated into the genome of Escherichia coli Nissle 1917 to construct a gene-edited engineered bacterium that can convert chenodeoxycholic acid into ursodeoxycholic acid.
[0012] In a specific example of the present invention, the integration site is attb as shown in SEQ ID NO.2.
[0013] The gene editing engineered bacteria are obtained by the following method:
[0014] 1) Prepare competent E. coli Nissle1917 containing pCas9 plasmid.
[0015] 2) Preparation of attb-pTargetF plasmid and homologous repair fragment;
[0016] 3) electroporating pTarget and homologous repair fragments into the competent cells prepared in step 1) and selecting positive clones;
[0017] 4) Eliminate the pCas9 plasmid and pTargetF plasmid of the positive clones.
[0018] A specific example of the present invention, the steps are as follows:
[0019] 1) pCas9 was transferred into E. coli Nissle1917, and positive clones were selected. The obtained positive clones were placed in a 30°C shaker at 200 rpm to expand the culture until the OD was close to 0.6, and arabinose with a final concentration of 10 mM was added for induction, and the cells were harvested by centrifugation at 4°C and 8000g for 10 min to prepare E. coli Nissle1917 competent cells containing pCas9.
[0020] 2) Use primers p1 and p2 to reversely amplify the pTargetF plasmid, transfer the amplified fragment into DH5α, sequence and verify that the sgRNA site of the pTargetF plasmid in the clone has been changed to attb, expand the culture of positive clones to extract attb-pTargetF plasmid. Use E. coli Nissle1917 as a template and use primers p3, p4, p5, and p6 to amplify the upstream and downstream homology arms respectively, use the synthetic 7β-HSDH gene expression frame as a template and use primers p7 and p8 to amplify its sequence, and then use primers p3 and p6 to perform overlap extension PCR on the upstream and downstream homology arms and the 7β-HSDH gene expression frame to obtain the homology repair fragment.
[0021] 3) The attb-pTargetF plasmid and homologous repair fragment obtained in step 2) were transformed into the competent cells prepared in step 1) by electroporation, and spread on LB plates containing kanamycin (50 μg / mL) and spectinomycin (50 μg / mL) and cultured at 30°C for 12 hours. Primers p9 and p10 were used to verify positive clones.
[0022] 4) The obtained positive clones were inoculated into LB containing IPTG (0.5 mM), cultured at 37°C with a shaker at 200 rpm for 12 h, and then spread onto LB plates. After the plates were cultured at 37°C for 12 h, clones were selected and inoculated onto LB plates containing kanamycin (50 μg / mL) and spectinomycin (50 μg / mL) to verify the disappearance of the plasmid.
[0023] The primers in steps 2) and 3) are:
[0024] p1: 5'-TCGTCCGTACACTAGTATTATACCTAGGAC-3',
[0025] p2: 5'-AATACTAGTGTACGGACGATCGCGTAACGGTTTTAGAGCTAGAAATAGCA-3',
[0026] p3: 5'-TTACGATGGCGATAATATTTCACC-3',
[0027] p4: 5'-ACAACGTCGTGACTGGGATACTAACTTGAGCGAAACGGGA-3',
[0028] p5: 5'-GGAAAGCCCAATCTTCACATCAATC-3',
[0029] p6: 5'-CTTGAGGGGTTTTTTGCTGAAAGTAAAAAAGCAGGCTTCAAC-3',
[0030] p7: 5'-CGCTCAAGTTAGTATGATCTCTCC-3',
[0031] p8: 5'-CTTTGTTAGCAGCCGGATCTCA-3',
[0032] p9: 5'-GGCCGAGGTTTCCCGGAGGC-3',
[0033] p10: 5'-CCACGCCGGTTTACCTGGC-3'.
[0034] Advantages of the present invention:
[0035] Escherichia coli Nissle 1917 (Escherichia coli Nissle 1917), referred to as EcN, is one of the most studied probiotics. EcN is different from other Escherichia coli. It does not carry pathogenic factors and therefore has no pathogenicity. At the same time, it can colonize in the intestine and prevent pathogens from invading the intestinal mucosa, and has a protective and repairing effect on the intestinal mucosal barrier; EcN also participates in the body's immune regulation, balances the secretion of immune factors, enhances the host's immune ability, and then relieves and treats inflammation. It is the preferred chassis for designing engineered probiotics. Therefore, the use of EcN that can convert CDCA into UDCA can not only improve the course of cholestatic diseases, but also play a probiotic role on the host.
[0036] UDCA is a drug approved for cholestatic diseases, and the treatment cost ranges from US$1,500 to US$4,500 per month, which is a heavy financial burden for many patients.
[0037] The present invention uses Nissle1917 as the base bacteria and constructs a gene-edited bacterium expressing 7β-HSDH by gene editing. The strain can effectively convert CDCA in the intestine into UDCA, improve the course of cholestatic diseases, and reduce the treatment cost of patients. This lays the foundation for further development of probiotic products that improve the prognosis of PBC patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 The abundance information of 7βHSDH in normal people.
[0039] Figure 2 The docking results of the candidate with 7-ketolithocholic acid and NADPH molecules.
[0040] Figure 3 PCR agarose was used to verify positive clones.
[0041] Figure 4This is the expression status of 7β-HSDH protein in the knock-in positive clone.
[0042] Figure 5 Mass spectrum for the activity verification of the conversion of CDCA into UDCA by the engineered Escherichia coli Nissle 1917.
[0043] Figure 6 This is the improvement of liver tissue in cholestasis model mice by oral administration of Rg sequence EcN-7β-HSDH.
[0044] Figure 7 These are the biochemical indicators of liver function in each group of mice, including serum ALT, AST and ALP levels. DETAILED DESCRIPTION
[0045] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.
[0046] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.
[0047] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.
[0048] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.
[0049] The reagents involved in the experiment are as follows:
[0050] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, dilute to 1 L with tap water, natural pH. Sterilize at 121°C for 20 min.
[0051] SOC liquid medium: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM D-glucose, dilute to 1 L with tap water. Sterilize with high pressure steam at 115°C for 20 minutes. After cooling to room temperature, add 5 mL of 1 M glucose solution (pre-prepared and sterilized by filtration with a 0.2 μm filter membrane).
[0052] The plasmid extraction kit was purchased from TIANGEN, the gel recovery kit was purchased from OMEGA, the seamless cloning kit was purchased from Beyotime, DNA restriction endonucleases were purchased from NEB, PCR enzymes were purchased from Novazonics, DH5α competent cells were purchased from Qingke, and antibodies were purchased from Abcam.
[0053] Unless otherwise specified, reagents or drugs can be purchased through commercial channels. Unless otherwise specified, the methods involved, such as gene amplification, are carried out in accordance with conventional molecular biology test procedures or in accordance with the reagent product instructions.
[0054] The nucleotide sequence of the 7β-HSDH gene expression cassette in the following examples is shown in SEQ ID NO.1.
[0055] Example 1 Screening of 7β-HSDH candidate sequences
[0056] 1) Using BlastP, homology search was performed in the HMP and MGYG databases to screen sequences with a similarity of more than 30% to 7β-HSDH, and more than 63,000 sequences were obtained.
[0057] 2) The candidate sequences obtained by screening were predicted by using the CLEAN comparative learning model to obtain the 7β-HSDH (EC: 1.1.1.201) candidate sequence and calculate the abundance of the population to screen out the candidate sequences in intestinal bacteria. Figure 1 , a total of 6 candidate sequences exist in the human population. The top 3 bacterial abundance sequences were selected for subsequent experiments, and the candidate sequences were derived from Ruminococcus gnavus (SEQ ID NO.1), Mediterraneibacter faecis (SEQ ID NO.3), and Oscillospiraceae (SEQ ID NO.4).
[0058] 3) In addition, in order to screen out possible high-activity sequences, AlphaFold2 was used to perform structural simulation on the predictions and then molecular docking was performed with NADPH and 7-keto-lithocholic acid. Based on the molecular docking results, three candidate sequences were screened out: from Prevotella jejuni (SEQ ID NO.5), Vibrio marisflavi (SEQ ID NO.6), and Eggerthellaceae (SEQ ID NO.7). The molecular docking results are shown in Figure 2. Figure 2 As shown, the top 3 non-repetitive sequences with docking energy were selected for subsequent experiments.
[0059] 4) Based on 2) and 3), a total of 6 candidate sequences were obtained for further research.
[0060] Example 2 Purification and Activity Assay of Candidate 7β-HSDH Protein
[0061] The specific steps are as follows:
[0062] 1) Transform the expression plasmid into BL21(DE3) competent cells:
[0063] Take 3 μL of pET28a(+)-RgHSDH, pET28a(+)-MfHSDH, pET28a(+)-OsHSDH, pET28a(+)-PjHSDH, pET28a(+)-VmHSDH, pET28a(+)-EgHSDH, and plasmid (commissioned by Shanghai Sangon Biotechnology Co., Ltd. to synthesize Rg: Ruminococcus gnavus, Mf: Mediterraneibacter faecis, Os: Oscillospiraceae, Pj: Prevotella jejuni, Vm: Vibrio marisflavi, Eg: Eggerthellaceae) and transfer it into the thawed BL21(DE3) competent cells. After standing on ice for 30 min, heat shock in a 42°C water bath for 45-60 s, quickly transfer to an ice bath and stand for 2 min. Add 700 μL SOC medium, mix well and resuscitate at 37°C, 200 rpm for 60 min. Take 50 μL of bacterial solution and spread it on SOB medium containing ampicillin (100 μg / mL), and place the plate upside down in a 37°C incubator for overnight culture.
[0064] 2) Verify positive transformants:
[0065] Transformants were selected for verification using the following primers: T7: 5′-TAATACGACTCACTATAGGG-3′, T7t: 5′-GCTAGTTATTGCTCAGCGG-3′.
[0066] 3) Inducible expression:
[0067] Select one of the positive transformants for resuscitation and activation. Take 500 μL of the activated transformant and add it to 50 mL of LB liquid medium, add kanamycin to a final concentration of 100 μg / mL, and culture it in a shaking incubator at 37°C until OD600 = 0.5, then add IPTG to a final concentration of 0.5 mM for induction, the induction temperature is 37°C, and the time is 4 h.
[0068] 4) Nickel column purification:
[0069] After the induction of expression, the bacteria were ultrasonically disrupted to obtain intracellular proteins. After the nickel column was regenerated, the protein was eluted with 20mM, 100mM, 200mM and 400mM imidazole. The eluted protein was desalted, freeze-dried and dissolved in PBS for activity determination.
[0070] 5) Determination of activity and kinetic parameters:
[0071] 1mM 7-keto-LCA, 2mM NADPH and 100μL enzyme were dissolved in 3ml 100mM Tris-HCl buffer (pH8.0). At 25°C, the change of NADPH at 340nm was detected, and one activity unit was defined as the amount of enzyme that produces 1μM NADP+ per minute. The kinetic parameters of the purified candidate protein were measured at different concentrations of substrate 7-keto-LCA (0.0125, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0mM), and the results are shown in Table 1.
[0072] Table 1
[0073]
[0074] The results showed that the Rg-derived sequence had the highest specific activity of -21.9 U / mg, followed by the Mf-derived sequence, while the Eg and Os-derived sequences had only trace activity, and the Pj and Vm sequences had no activity.
[0075] Example 3 Construction of Escherichia coli Nissle 1917 gene editing engineering bacteria
[0076] 1) pCas9 was transferred into E. coli Nissle1917, and positive clones were selected. The obtained positive clones were placed in a 30°C shaker at 200 rpm to expand the culture until the OD was close to 0.6, and arabinose with a final concentration of 10 mM was added for induction, and the cells were harvested by centrifugation at 4°C and 8000g for 10 min to prepare E. coli Nissle1917 competent cells containing pCas9.
[0077] 2) Use primers p1 and p2 to reversely amplify the pTargetF plasmid, transfer the amplified fragment into DH5α, sequence and verify that the sgRNA site of the pTargetF plasmid in the clone has been changed to attb, expand the culture of positive clones to extract attb-pTargetF plasmid. Use E. coli Nissle1917 as a template and use primers p3, p4, p5, and p6 to amplify the upstream and downstream homology arms respectively, use the synthetic 7β-HSDH gene expression frame as a template and use primers p7 and p8 to amplify its sequence, and then use primers p3 and p6 to perform overlap extension PCR on the upstream and downstream homology arms and the 7β-HSDH gene expression frame to obtain the homology repair fragment.
[0078] 3) The attb-pTargetF plasmid and homologous repair fragment obtained in step 2) were transformed into the competent cells prepared in step 1) by electroporation, and spread on LB plates containing kanamycin (50 μg / mL) and spectinomycin (50 μg / mL) and cultured at 30°C for 12 hours. Primers p9 and p10 were used to verify positive clones.
[0079] 4) The obtained positive clones were inoculated into LB containing IPTG (0.5 mM), cultured at 37°C with a shaker at 200 rpm for 12 h, and then spread onto LB plates. After the plates were cultured at 37°C for 12 h, clones were selected and inoculated onto LB plates containing kanamycin (50 μg / mL) and spectinomycin (50 μg / mL) to verify the disappearance of the plasmid.
[0080] The primers in steps 2) and 3) are:
[0081] p1: 5'-TCGTCCGTACACTAGTATTATACCTAGGAC-3',
[0082] p2: 5'-AATACTAGTGTACGGACGATCGCGTAACGGTTTTAGAGCTAGAAATAGCA-3',
[0083] p3: 5'-TTACGATGGCGATAATATTTCACC-3',
[0084] p4: 5'-ACAACGTCGTGACTGGGATACTAACTTGAGCGAAACGGGA-3',
[0085] p5: 5'-GGAAAGCCCAATCTTCACATCAATC-3',
[0086] p6: 5'-CTTGAGGGGTTTTTTGCTGAAAGTAAAAAAGCAGGCTTCAAC-3',
[0087] p7: 5'-CGCTCAAGTTAGTATGATCTCTCC-3',
[0088] p8: 5'-CTTTGTTAGCAGCCGGATCTCA-3',
[0089] p9: 5'-GGCCGAGGTTTCCCGGAGGC-3',
[0090] p10: 5'-CCACGCCGGTTTACCTGGC-3'.
[0091] Example 4 Verification of protein expression of Escherichia coli Nissle 1917 7β-HSDH engineered bacteria
[0092] 1) The activated EcN attb::7β-HSDH engineered bacteria were inoculated into LB medium, OD600 was adjusted to 0.1, and cultured in a 37°C shaker at 200 rpm for 36 h.
[0093] 2) Prepare western blot samples from bacterial culture after culture completion and use 6×His tag antibody as primary antibody for incubation. Figure 4 The results showed that the six candidate sequence proteins were all expressed normally.
[0094] Example 5: Verification of the activity of engineering bacteria transforming CDCA into UDCA
[0095] 1.LC-QQQ-MS Sample Preparation
[0096] 1) Take the EcN attb::7β-RgHSDH engineered bacteria cultured overnight and put them into LB medium. When the OD600 is grown to 0.1, add CDCA with a final concentration of 0.5 mM and culture at 37°C and 200 rpm for 36 h.
[0097] 2) Take 150 μL of bacterial solution for sample preparation, add 600 μL of ice-cold methanol, vortex for 15 min, and incubate at -20°C for 1 h.
[0098] 3) Centrifuge at 14000 rpm and 4°C for 30 min, collect the supernatant and filter it with a 0.22 μm filter membrane.
[0099] 4) Take 100 μL of the filtered supernatant, add 100 μL of CDCA-d4 internal standard, vortex for 1 min, centrifuge at 14000 rpm, 4°C for 30 min, and aspirate the supernatant.
[0100] 5) Centrifuge at 14000 rpm, 4°C for 30 min, take 100 μL of the supernatant, filter with a 0.22 μm filter membrane, and place in a sample injection vial for testing. 2. LC-QQQ-MS chromatography / mass spectrometry conditions
[0101] 1) Chromatographic / mass spectrometry conditions: The chromatographic column was Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); mobile phase A was water (containing 0.1% formic acid), mobile phase B was acetonitrile (containing 0.1% formic acid), and the mobile phase gradient was 0-1 min, 5% B-25% B; 1-9 min, 25%-30% B; 9-10 min, 30%-40% B; 10-17 min, 40%-45% B; 17-18.5 min, 45%-95% B; 18.5-20.5 min, 95% B. The running time for each sample was 20.5 min, the column equilibration time was 3 min, and the post-column effluent was directly introduced into the mass spectrometry system for detection; flow rate: 0.3 mL·min-1; autosampler temperature: 4°C; column temperature: 50°C; injection volume: 5 μL. ESI-MRM mode was used for positive ion monitoring; capillary voltage: 4.5 kV; source temperature: 450 °C; cone gas flow rate: 40 L·h-1; desolvation gas flow rate: 40 L·h-1; curtain gas flow rate: 25 L·h-1. CDCA mass spectrometry conditions: m / z 391.3 / 391.3; DP 45V; EP 4V; CE 28V; CXP 5V. Mass spectrometry conditions: m / z 395.3 / 395.2; DP 31V; EP5V; CE 21V; CXP 3V. UDCA mass spectrometry conditions: m / z 391.3 / 391.3; DP 45V; EP 4V; CE 28V; CXP 5V.
[0102] 2) Analyze the test results. Figure 5 As shown, the peak time of CDCA standard is 20.879min ( Figure 5 A), UDCA peak time is 17.521min ( Figure 5 B); The concentration of UDCA detected in the sample co-incubated with CDCA was 0.324 mM ( Figure 5 C), the conversion efficiency was 64.7%, indicating that the EcN attb::Rg7β-HSDH engineered bacteria has the ability to convert CDCA into UDCA.
[0103] Example 6 Investigating the effect of engineered bacteria on improving cholestatic diseases
[0104] 1) Construction of mouse model: C57BL / 6 mice were adaptively fed in an SPF environment for 1 week and randomly divided into 4 groups, 6 mice in each group: model group (ANIT treatment), ECN treatment group (wild-type ECN gavage treatment), ECN-HSDH treatment group (EcN attb::7β-HSDH gavage treatment), UDCA treatment group (UDCA gavage). The gavage dose of UDCA was 15 mg kg -1 ·d -1The intragastric bacterial dose was 1×10 9 CFU·bacterial solution, 200μL / 25g per day. In order to construct a primary sclerosing cholangitis mouse model, ANIT was administered by gavage every day at a dose of 0.1mL / 10g. The experiment lasted for 4 weeks. After 4 weeks, the mice were killed and their livers and blood were collected.
[0105] 2) Liver section staining: Use frozen 4% paraformaldehyde to fix the tissue, then slice it and perform HE staining on the slices. Figure 6 As shown. In the model group, multiple focal necrosis of hepatocytes was observed under the microscope, and the portal area was obviously hyperplastic with more inflammatory cells infiltration. Interface hepatitis was observed in the hepatocytes around the portal area. Compared with the model group, the EcN treatment group showed almost no improvement, with hepatocyte necrosis and hyperplasia of fibrous tissue, interface hepatitis. The UDCA treatment group was accompanied by a small number of inflammatory cells, no hyperplasia of fibrous tissue, and almost no pathological conditions. In the EcN-HSDH treatment group, individual hepatocytes showed focal necrosis, no hyperplasia of fibrous tissue, and the pathological conditions were greatly improved. This shows that EcN-HSDH treatment can significantly improve liver damage caused by cholestasis and verification.
[0106] 3) Detection of serum liver function indicators: The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in mouse serum was measured using the Hisan liver function test kit, and the data were analyzed. The results are as follows: Figure 7 As shown in the figure, ANIT gavage induced an increase in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and there was no improvement in serum enzyme activity after EcN treatment. EcN-HSDH treatment and UDCA treatment downregulated the surge in serum enzyme activity caused by liver damage.
Claims
1. Use of 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus in the preparation of a drug for treating cholestatic diseases, wherein the nucleotide sequence of the 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The drug is a gene-edited engineered bacterium.
3. The use according to claim 1, characterized in that The gene-edited engineered bacteria are prokaryotic cells.
4. The use according to claim 3, characterized in that The prokaryotic cell is selected from Escherichia coli.
5. The use according to claim 4, characterized in that The Escherichia coli is Escherichia coli Nissle 1917.
6. The use according to claim 5, characterized in that The 7β-hydroxysteroid dehydrogenase gene sequence was integrated into the genome of Escherichia coli Nissle 1917.
7. The use according to claim 6, characterized in that The nucleotide sequence of the integration site is shown in SEQ ID NO.
2.
8. The use according to any one of claims 1 to 7, characterized in that The medicine is an oral preparation.
9. The use according to claim 1, characterized in that The cholestatic disease is selected from the group consisting of common bile duct stones, benign common bile duct strictures, pancreatitis, pancreaticobiliary tumors, and primary or secondary sclerosing cholangitis.
10. A gene-edited engineered bacterium of Escherichia coli, characterized in that The 7β-hydroxysteroid dehydrogenase gene is integrated into its genome, and the nucleotide sequence of the 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO.1.