Werococcus capable of promoting morbidity sex difference of autoimmune liver diseases and application of Werococcus

Abstract

The invention relates to isovirococcus particularis capable of promoting sex difference of autoimmune liver diseases and application of the isovirococcus particularis. The autoimmune liver diseases are complex in pathogenesis, and the morbidity has significant gender difference. The invention finds that isovirococcus particularis can degrade arginine through arginine decarboxylase coded by the speA gene of the isovirococcus particularis, thereby causing hypoargininemia and further causing apoptosis of testicular interstitial cells and reduction of testosterone synthesis. The decrease of the testosterone level promotes the proliferation of B cells and focuses in the liver, and biliary epithelial cells are damaged, so that the morbidity process of the autoimmune liver disease is accelerated. In addition, the invention also constructs a recombinant bacterium pET28a-speA / BL21, and the recombinant bacterium can degrade arginine in intestinal tracts and simulate the effect of iso-prosperococcus particularis. The invention provides a new perspective for the pathogenesis of autoimmune liver diseases, and provides a new target for early screening and intervention for patients with testosterone deficiency related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical preparations, and in particular to a Veillonella bacterium capable of promoting gender differences in the onset of autoimmune liver disease and its application Background Art

[0002] Autoimmune liver disease is a type of liver disease mediated by autoimmune reactions, and its pathogenesis is not yet fully understood. It is generally believed that in addition to environmental triggers, immunogenetics, innate immunity and adaptive immunity, bile acid metabolism mediated by the gut-liver axis, intestinal flora, sex hormone levels and other factors all play an important role in its pathogenesis. Among them, the gender difference in prevalence is one of the most significant clinical features of the disease. For example, the male-female prevalence of primary biliary cholangitis is 1:5-6, indicating that sex hormones may play a vital role in the pathogenesis.

[0003] In recent years, with the deepening of research on intestinal flora, people have gradually realized the important role of intestinal flora in the physiological and pathological functions of the human body. The intestinal flora participates in regulating the immune and metabolic processes of the human body through various pathways and has become a new target for disease prevention and treatment. The significant enrichment of Veillonella dispar in the intestines of patients with autoimmune diseases has attracted widespread attention. Studies have shown that ursodeoxycholic acid (UDCA), as a first-line treatment for primary biliary cholangitis, can significantly reduce the abundance of Veillonella dispar during treatment, and the abundance of this bacterium is significantly higher in patients with poor response to UDCA.

[0004] Arginine is a nutritionally semi-essential amino acid with a wide range of sources in the body, including food, endogenous pathways, and self-protein conversion. Abnormal arginine metabolism is closely related to the occurrence and development of a variety of diseases, especially in the reproductive system. Arginine has a positive effect on testicular health and male reproductive function by improving testicular blood flow, supporting sperm production, antioxidant protection, and androgen testosterone regulation. Studies have shown that there is an amino acid intestinal circulation system in the human body, and amino acids in the intestine can be reabsorbed back into the body through specific mechanisms, affecting the amino acid level throughout the body.

[0005] However, how intestinal flora affects the pathogenesis of autoimmune liver disease by regulating arginine metabolism, especially the mechanism related to gender differences, has not yet been fully elucidated. It is in this context that the present invention conducts in-depth research on the role of Veillonella dispar in the pathogenesis of autoimmune liver disease and its relationship with arginine metabolism and testosterone levels, aiming to provide a new perspective on the pathogenesis of autoimmune liver disease and provide new targets for early screening and intervention of related diseases.

[0006] In order to solve the above problems, the applicant proposed a Veillonella that promotes gender differences in the onset of autoimmune liver disease and its application. Summary of the invention

[0007] The purpose of the present invention is to provide a Veillonella capable of promoting gender differences in the onset of autoimmune liver disease and an application thereof, so as to solve the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solutions: a Veillonella that promotes gender differences in the onset of autoimmune liver disease and its application, wherein the Veillonella dispar has the ability to degrade arginine, and its speA gene can encode arginine decarboxylase to degrade arginine, further causing a decrease in testosterone synthesis, thereby promoting B cell proliferation and focusing in the liver, damaging bile duct epithelial cells.

[0009] The Veillonella dispar was extracted from the feces of patients with autoimmune liver disease and confirmed by pure culture and 16SrRNA sequencing.

[0010] A recombinant bacterium pET28a-speA / BL21 has an exogenous arginine decarboxylase encoding gene speA integrated into its genome and is capable of degrading arginine in the intestine, thereby reducing testosterone synthesis.

[0011] Optionally, the method for constructing the recombinant bacteria includes:

[0012] a. Extracting genomic DNA of Veillonella dispar;

[0013] b. Designing primers based on the speA gene sequence of Veillonella dispar, and performing PCR amplification to obtain the speA gene fragment;

[0014] c. The speA gene fragment obtained by PCR amplification was connected to the pGEM-T vector to construct the recombinant plasmid pGEM-T-speA, and then sequenced and verified;

[0015] d. Double-digest the correctly sequenced recombinant plasmid pGEM-T-speA and the expression vector pET28a, respectively, and connect the speA gene fragment after digestion with the pET28a vector to construct the recombinant plasmid pET28a-speA;

[0016] e. The recombinant plasmid pET28a-speA was transformed into Escherichia coli BL21 competent cells to obtain the recombinant bacteria pET28a-speA / BL21.

[0017] Use of the recombinant bacteria pET28a-speA / BL21 according to claim 3 or 4 in the preparation of reagents for promoting the study of gender differences in the onset of autoimmune liver disease. Use of the recombinant bacteria pET28a-speA / BL21 according to claim 3 or 4 in the preparation of reagents for early screening and intervention of patients with a series of diseases caused by testosterone deficiency.

[0018] Optionally, the method includes administering the recombinant bacteria to experimental animals by oral gavage, and observing changes in arginine levels, testosterone levels, and liver B cell activation factor levels in the experimental animals.

[0019] Optionally, the experimental animals are C57BL / 6J mice.

[0020] Optionally, it also includes observing the apoptosis of Leydig cells in the testis of experimental animals and performing histological examination on the liver to evaluate the focusing of B cells in the liver and the damage of bile duct epithelial cells.

[0021] The method and application of any one of claims 1 to 4 is used for targeted elimination or inhibition of Veillonella dispar and its speA for the treatment of autoimmune liver disease.

[0022] Beneficial effects: First, the present invention discovered the important role of Veillonella dispar in the pathogenesis of autoimmune liver disease, especially its mechanism of causing hypoargininemia by degrading arginine, thereby causing Leydig cell apoptosis and reduced testosterone synthesis. This discovery not only reveals the connection between intestinal flora and autoimmune liver disease, but also provides a new idea for exploring the pathogenesis of autoimmune diseases from the perspective of pathogenic microorganisms.

[0023] Secondly, the recombinant bacteria pET28a-speA / BL21 constructed by the present invention can effectively degrade arginine in the intestine, simulating the effect of Veillonella dispar. The construction of this recombinant bacteria provides a powerful tool for experimental research on autoimmune liver disease and helps to further clarify the pathogenesis of the disease.

[0024] In addition, the present invention also found that the decline in testosterone levels promoted the proliferation of B cells and their accumulation in the liver, damaging bile duct epithelial cells, thereby accelerating the pathogenesis of autoimmune liver disease. This discovery provides new evidence for understanding the role of sex hormones in the pathogenesis of autoimmune liver disease, and also provides a new target for early screening and intervention of related diseases.

[0025] In summary, the present invention not only contributes to a deeper understanding of the pathogenesis of autoimmune liver disease, but also has important scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is a statistical chart of the target amino acid metabolism results in the serum of two groups of mice;

[0027] Figure 2 TUNEL staining images of Leydig cells in the two groups of mice;

[0028] Figure 3 This is a picture of apoptosis-related proteins in Leydig cells of two groups of mice detected by Western blot;

[0029] Figure 4 This is a graph of serum testosterone levels in two groups of mice detected by LC-MC / MC. DETAILED DESCRIPTION

[0030] The following describes the preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0031] The present invention aims to provide a new discovery on Veillonella dispar in the pathogenesis of autoimmune liver disease and its application, which specifically includes the following contents:

[0032] Screening and identification of Veillonella dispar: Veillonella dispar was extracted from the feces of patients with autoimmune liver disease, and its species was identified by 16S rRNA sequencing. It was amplified using Veillonella dispar-specific primers to further confirm its biochemical characteristics.

[0033] Mechanism of action of Veillonella dispar: It was found that the speA gene of Veillonella dispar can encode arginine decarboxylase (ADC), which degrades arginine and causes hypoargininemia, which in turn causes apoptosis of Leydig cells and leads to reduced testosterone synthesis. The decrease in testosterone levels promotes B cell proliferation and accumulation in the liver, damages bile duct epithelial cells, and accelerates the pathogenesis of autoimmune liver disease.

[0034] Construction and application of recombinant bacteria: The speA gene was cloned into the prokaryotic expression vector BL21 (DE3) to construct the recombinant bacteria pET28a-speA / BL21. The recombinant bacteria can colonize and degrade arginine in the intestine, simulating the effect of Veillonella dispar. Animal experiments confirmed that after oral administration of the recombinant bacteria, the arginine concentration in the blood of mice was significantly reduced, testosterone synthesis was reduced, and the level of B cell activation factor in the liver was increased, which promoted B cell proliferation and damaged bile duct epithelial cells.

[0035] The technical solution of the present invention involves the screening, identification, research on the mechanism of action of Veillonella dispar, and the construction and application of recombinant bacteria. The following is a detailed description of the technical solution:

[0036] (I) Screening and identification of Veillonella dispar

[0037] Sample collection

[0038] Collection subjects: Patients diagnosed with autoimmune liver disease were selected as sample collection subjects to ensure the representativeness and accuracy of the samples.

[0039] Collection method: Collect stool samples from patients in sterile containers and immediately refrigerate or freeze them to prevent changes in the microorganisms in the samples.

[0040] Strain isolation and purification

[0041] Culture medium selection: According to the growth characteristics of Veillonella dispar, select a suitable culture medium for strain isolation and purification. For example, a selective culture medium containing specific nutrients and antibiotics can be used to inhibit the growth of other microorganisms and promote the reproduction of Veillonella dispar.

[0042] Isolation method: Spread the stool sample evenly on the culture medium and obtain single colonies by streak separation or dilution spreading method. Then, select single colonies with morphology consistent with the characteristics of Veillonella dispar for further purification.

[0043] Strain identification

[0044] 16S rRNA sequencing: Extract the purified genomic DNA of Veillonella dispar and perform PCR amplification of the 16S rRNA gene. The amplified product is sequenced and compared with the sequence in the NCBI database to confirm the strain type and identification results.

[0045] Specific primer amplification: Design and synthesize specific primers for Veillonella dispar, and perform PCR amplification on the purified strain. Analyze the band size and position of the amplified product by electrophoresis to further confirm the biochemical characteristics of the strain.

[0046] (II) Study on the mechanism of action of Veillonella dispar

[0047] Cloning and expression of speA gene

[0048] Extraction of genomic DNA: Select a purified single colony of Veillonella dispar and inoculate it into liquid culture medium for overnight culture. Extract genomic DNA according to the kit instructions to ensure the purity and integrity of the DNA.

[0049] PCR amplification: According to the speA gene sequence of Veillonella dispar, synthetic primers were designed for PCR amplification. High-quality amplification products were obtained by optimizing the PCR reaction system and conditions.

[0050] Gene cloning: The PCR amplification product is purified by gel recovery and then connected to the expression vector. The positive clones are screened by transforming E. coli competent cells, and the plasmid PCR identification and sequencing verification are performed.

[0051] Protein expression: The constructed recombinant plasmid is transformed into the expression host bacteria for induction of expression. The bacteria are broken by ultrasound, the recombinant protein is extracted and purified, and its molecular weight and purity are detected by SDS-PAGE electrophoresis.

[0052] Arginine degradation ability assay

[0053] In vitro experiment: Add the purified recombinant arginine decarboxylase to a buffer containing arginine, and measure the concentration of the remaining arginine after a certain reaction time. Evaluate the degradation ability of the recombinant enzyme by comparing it with the control group (without enzyme).

[0054] In vivo experiment: Veillonella dispar or recombinant bacteria are administered to experimental animals (such as mice) by oral gavage, and serum and tissue samples are collected at different time points. The arginine concentration in serum and tissue is measured by high performance liquid chromatography (HPLC) to evaluate the degradation ability of the strain or recombinant bacteria in vivo.

[0055] Effects of hypoargininemia on Leydig cells and testosterone synthesis

[0056] Cell experiment: Purified Leydig cells were co-cultured with arginine solutions of different concentrations to observe cell morphology and proliferation. Cell apoptosis rate was detected by flow cytometry to evaluate the effect of hypoarginineemia on Leydig cells.

[0057] Animal experiment: Veillonella dispar or recombinant bacteria were administered to experimental animals by oral gavage, and testicular tissue samples were collected. The expression levels of apoptosis-related proteins in testicular interstitial cells were detected by TUNEL staining and Western blot to evaluate the effect of hypoargininemia on testosterone synthesis.

[0058] Effects of decreased testosterone levels on B cell proliferation and intrahepatic bile duct epithelial cell injury

[0059] Cell experiment: Purified B cells were co-cultured with testosterone solutions of different concentrations to observe cell proliferation and differentiation. The expression levels of B cell surface markers were detected by flow cytometry to evaluate the effect of testosterone levels on B cell proliferation.

[0060] Animal experiment: Veillonella dispar or recombinant bacteria were administered to experimental animals by oral gavage, and liver tissue samples were collected. Immunohistochemical staining and flow cytometry were used to detect the infiltration of B cells in the liver and the expression level of activation factors (such as BAFF) to evaluate the effect of decreased testosterone levels on the damage of intrahepatic bile duct epithelial cells.

[0061] (III) Construction and application of recombinant bacteria

[0062] Construction of recombinant bacteria

[0063] Gene cloning and vector selection: The speA gene of Veillonella dispar was cloned into the prokaryotic expression vector BL21 (DE3). The reason for choosing this vector is that it has an efficient expression system and stable genetic characteristics, and is suitable for the construction of recombinant bacteria.

[0064] Plasmid construction and transformation: The cloned speA gene was connected to the expression vector to form a recombinant plasmid. The positive clones were screened by transforming E. coli competent cells, and the plasmid was identified by PCR and sequenced.

[0065] Recombinant bacteria screening and identification: The constructed recombinant plasmid was transformed into the expression host bacteria BL21 (DE3) for induction of expression. The bacteria were broken by ultrasound, and the recombinant protein was extracted and purified. The molecular weight and purity were detected by SDS-PAGE electrophoresis. At the same time, the presence and correctness of the speA gene in the recombinant bacteria were verified by PCR amplification and sequencing.

[0066] Verification of the degradation ability of recombinant bacteria

[0067] In vitro experiment: The recombinant bacteria were inoculated into a liquid culture medium containing arginine, and the concentration of the remaining arginine was measured after a certain period of culture. The degradation ability of the recombinant bacteria was evaluated by comparing with the control group (without bacteria).

[0068] In vivo experiment: The recombinant bacteria were administered to experimental animals (such as mice) by gavage, and serum and tissue samples were collected at different time points. The arginine concentration in serum and tissues was measured by HPLC to evaluate the degradation ability of the recombinant bacteria in vivo.

[0069] Application of recombinant bacteria in autoimmune liver disease models

[0070] Establishment of animal model: Select a suitable animal model of autoimmune liver disease (such as autoimmune hepatitis model, primary biliary cholangitis model, etc.), and establish the model by injecting autoimmunogens or using gene knockout technology.

[0071] Administration of recombinant bacteria: The recombinant bacteria were administered to the experimental animal model by gavage, and different doses and time points were set for administration. At the same time, a control group (administered with physiological saline or empty vector plasmid transformed bacteria) was set up for comparison.

[0072] Index detection: Serum and tissue samples of experimental animals were collected at different time points after administration to detect relevant indicators (such as arginine concentration, testosterone level, B cell activation factor expression level, degree of intrahepatic bile duct epithelial cell damage, etc.). By comparing the differences between the experimental group and the control group, the effect and mechanism of the recombinant bacteria in the autoimmune liver disease model were evaluated.

[0073] (IV) Optimization and improvement of technical solutions

[0074] Optimization of strain screening and identification methods

[0075] High-throughput sequencing technology: Use high-throughput sequencing technology to quickly and accurately identify and classify a large number of samples. By constructing a 16S rRNA gene library, deep sequencing and data analysis can be performed to improve the accuracy and efficiency of strain identification.

[0076] Specific primer design and optimization: Based on the genome sequence and biochemical characteristics of Veillonella dispar, more specific and sensitive primers are designed for PCR amplification. By optimizing parameters such as primer sequence, reaction system and conditions, the specificity and efficiency of PCR amplification can be improved.

[0077] In-depth exploration of the mechanism of action

[0078] Multi-omics analysis: Combining genomics, transcriptomics, proteomics, and metabolomics, we conducted in-depth studies on the interaction between Veillonella dispar and host cells. By analyzing information such as gene expression profiles, protein interaction networks, and metabolic pathways, we revealed the specific mechanism of Veillonella dispar in the pathogenesis of autoimmune liver disease.

[0079] Animal model optimization: Select animal models that are closer to human autoimmune liver disease for research, such as humanized mouse models, etc. Improve the accuracy and reliability of the model by optimizing parameters such as model establishment methods and conditions.

[0080] Construction and application expansion of recombinant bacteria

[0081] Optimization of expression system: Optimize and transform prokaryotic expression vectors and host bacteria to improve the expression level and stability of recombinant proteins. For example, transform the vector through genetic engineering to enhance its replication ability and expression efficiency; or select host bacteria that are more suitable for the growth and expression of recombinant bacteria for transformation and expression.

[0082] Application expansion: Apply recombinant bacteria to the study of other autoimmune diseases or metabolic diseases. By exploring the effects and mechanisms of recombinant bacteria in different disease models, new strategies and methods can be provided for the prevention, diagnosis and treatment of related diseases.

[0083] In summary, the technical scheme of the present invention involves multiple aspects such as the screening, identification, and mechanism of action research of Veillonella dispar, as well as the construction and application of recombinant bacteria. By optimizing and improving each link and step in the technical scheme, the accuracy and reliability of the research can be further improved, providing strong support for revealing the pathogenesis of autoimmune liver disease and providing new treatment methods.

[0084] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0085] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A Veillonella bacteria having the ability to promote gender differences in the onset of autoimmune liver disease and its use, characterized in that: The Veillonella dispar has the ability to degrade arginine, and its speA gene can encode arginine decarboxylase to degrade arginine, further causing a decrease in testosterone synthesis, thereby promoting B cell proliferation and focusing in the liver and damaging bile duct epithelial cells.

2. The use of Veillonella dispar according to claim 1, characterized in that: The Veillonella dispar was extracted from the feces of patients with autoimmune liver disease and confirmed by pure culture and 16S rRNA sequencing.

3. A recombinant bacterium pET28a-speA / BL21, characterized in that: The recombinant bacteria has an exogenous arginine decarboxylase encoding gene speA integrated into its genome, which can degrade arginine in the intestine, thereby reducing testosterone synthesis.

4. The recombinant bacterium pET28a-speA / BL21 according to claim 3, characterized in that The construction method of the recombinant bacteria comprises: a. Extracting genomic DNA of Veillonella dispar; b. Designing primers based on the speA gene sequence of Veillonella dispar, and performing PCR amplification to obtain the speA gene fragment; c. The speA gene fragment obtained by PCR amplification was connected to the pGEM-T vector to construct the recombinant plasmid pGEM-T-speA, and then sequenced and verified; d. Double-digest the correctly sequenced recombinant plasmid pGEM-T-speA and the expression vector pET28a, respectively, and connect the speA gene fragment after digestion with the pET28a vector to construct the recombinant plasmid pET28a-speA; e. The recombinant plasmid pET28a-speA was transformed into Escherichia coli BL21 competent cells to obtain the recombinant bacteria pET28a-speA / BL21.

5. Use of the recombinant bacterium pET28a-speA / BL21 according to claim 3 or 4 in the preparation of a reagent for promoting the study of gender differences in the onset of autoimmune liver disease.

6. Use of the recombinant bacterium pET28a-speA / BL21 according to claim 3 or 4 in the preparation of an agent for early screening and intervention of patients with a series of diseases caused by testosterone deficiency.

7. A method for studying gender differences in the incidence of autoimmune liver disease using the recombinant bacterium pET28a-speA / BL21 according to claim 3 or 4, characterized in that: The method comprises administering the recombinant bacteria to experimental animals by oral gavage, and observing changes in arginine levels, testosterone levels and liver B cell activation factor levels in the experimental animals.

8. The research method according to claim 7, characterized in that: The experimental animals were C57BL / 6J mice.

9. The research method according to claim 7 or 8, characterized in that: It also includes observing the apoptosis of testicular interstitial cells in experimental animals and performing histological examination of the liver to evaluate the focusing of B cells in the liver and the damage of bile duct epithelial cells.

10. The method and application of any one of claims 1 to 4 for targeted elimination or inhibition of Veillonella dispar and its speA for the treatment of autoimmune liver disease.

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