A microbial anti-inflammatory molecule of clostridium prasrj and screening method and application thereof

Abstract

The application relates to the field of biotechnology, in particular to a Faecalibacterium prausnitzii microbial anti-inflammatory molecule and a screening method and application thereof. The amino acid sequence of the Faecalibacterium prausnitzii microbial anti-inflammatory molecule is shown in (a) or (b); (a) the amino acid sequence is shown in SEQ ID NO. 1; (b) a protein derived from (a) with anti-inflammatory activity obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a). Compared with the microbial anti-inflammatory molecule protein of a representative strain A2-165 of the Faecalibacterium genus, the inhibiting effects of the Faecalibacterium prausnitzii microbial anti-inflammatory molecule on the NF-kappa B signal pathway and the inflammatory factor TNF alpha are 50% and 56% respectively, and the anti-inflammatory effect (inhibiting the NF-kappa B signal pathway) of the Faecalibacterium prausnitzii microbial anti-inflammatory molecule screened in the application can reach more than 90%, and the Faecalibacterium prausnitzii microbial anti-inflammatory molecule has the advantages of small use dosage and strong anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a microbial anti-inflammatory molecule of Faecalibacterium prausnitzii and a screening method and application thereof. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is one of the stubborn diseases of the digestive tract, mainly including Crohn's disease (CD) and Ulcerative Colitis (UC). Compared with other digestive diseases, IBD has the characteristics of high treatment cost, difficult cure and high recurrence rate, which seriously affects the quality of life of patients.

[0003] In the immune response, NF-κB is the most critical transcription factor in the inflammatory response, which is involved in innate immunity and adaptive immune response. In the intestinal tissue of IBD patients or other acute and chronic enteritis patients, the NF-κB signaling pathway is activated, which induces the production of a large number of pro-inflammatory cytokines and induces the occurrence of inflammation. These cytokines are divided into two categories, one is the cytokine that promotes innate immune immunity, such as type I interferon (IFNβ), IL-12 and macrophage chemotactic factor, etc.; the other is the cytokine that promotes acquired immunity, such as IL-1, IL-2, IL-6, IL-8, IL-12, IL-18 and type II interferon (IFNγ). Tumor necrosis factor TNFα is one of the most important pro-inflammatory factors, which can be expressed in intestinal epithelial cells, endothelial cells and immune cells in the lamina propria. TNFα is an important regulatory factor in the pathogenesis of IBD. First, TNFα can induce the destruction of epithelial cell tight junction, change the function of intestinal epithelial barrier and affect intestinal permeability; second, TNFα can promote the expression of CEACAM6, mediate immune tissue damage and increase the invasion and infection of intestinal bacteria.

[0004] At present, the drugs commonly used in clinical treatment of IBD include aminosalicylic acid, glucocorticoids, antibiotics and immunosuppressants, which can all play a role by inhibiting the NF-κB signaling pathway and inflammatory response. Infliximab, adalimuma and certolizumab are also TNFα-targeted therapeutic drugs. However, these drugs all have obvious side effects, and the curative effect is not lasting and easy to relapse, and the treatment effect on different patients is very different. Therefore, it is urgent to find safer and more effective substances to inhibit the NF-κB signaling pathway and TNFα.

[0005] Studies have shown that the intestinal microbiome is essential for maintaining intestinal homeostasis and human health, and changes in the intestinal microbiome are closely related to diseases associated with chronic inflammation in the intestine. Fecal bacteria therapy, also known as fecal microbiota transplantation (FMT) or intestinal bacteria transplantation, is a method of transplanting functional flora from healthy human feces into the patient's intestine through a certain means to restore the diversity of healthy intestinal flora and achieve the treatment of intestinal and extra-intestinal diseases. Currently, fecal bacteria transplantation has been considered as a special organ transplantation for inflammatory bowel disease, irritable bowel syndrome, metabolic syndrome, neurodevelopmental and neurodegenerative diseases, and autoimmune enteropathy. However, fecal bacteria transplantation is limited by the screening of healthy donors, the preparation of fecal bacteria, the acceptance of patients, and the treatment effect. The screening of healthy donors for fecal bacteria transplantation is extremely strict, and the diet, sleep quality, and drug use of healthy donors can affect the composition and abundance of their flora, ultimately affecting the treatment effect. Therefore, it is important to screen effective strains and their components for the treatment of digestive system inflammatory reactions.

[0006] Faecalibacterium prausnitzii (F. prausnitzii) is an anaerobic bacterium that mainly exists in the terminal ileum and ileocecal region, accounting for about 5-15% of the total bacterial flora in healthy adult feces, and is one of the most abundant symbiotic bacteria in the intestinal flora of healthy adults. In previous studies, it was found that intestinal flora, especially F. prausnitzii, plays a very important role in the occurrence, development, and prognosis of UC. The degree of reduction of F. prausnitzii is closely related to the severity of IBD and prognosis. In addition, the abundance of F. prausnitzii in the feces of patients with NAFLD is also significantly reduced, and supplementing F. prausnitzii can improve lipid deposition in NAFLD mice. In recent years, scientists have found a 15kDa protein in the culture supernatant of F. prausnitzii, which not only can inhibit the NF-κB signaling pathway of intestinal epithelial cells, but also can significantly reduce intestinal inflammation in mice induced by DNBS or DSS, and is called microbial anti-inflammatory molecule (MAM). According to literature reports and strain classification database LPSN, Faecalibacterium prausnitzii genus is divided into 7 species. Exploring the anti-inflammatory effect of each strain is important for screening probiotic strains. And MAM as the most important anti-inflammatory protein of F. prausnitzii, can determine the effect of the strain by screening the anti-inflammatory effect of MAM.

[0007] Currently, there is no effective treatment for inflammatory bowel disease, irritable bowel syndrome, metabolic syndrome and autoimmune enteropathy. Fecal bacteria transplantation as a treatment is limited by the screening of healthy donors, preparation of fecal bacteria, diversity and uncertainty of components, and the therapeutic effect varies greatly. As one of the most abundant symbiotic bacteria in the intestinal flora of healthy adults, studies have reported that Prevotella can improve the progression of inflammatory bowel disease and non-alcoholic fatty liver disease. However, the effects of different strains of Prevotella are not consistent. SUMMARY

[0008] The present application aims to overcome the deficiencies of the prior art and provide a Prevotella microbial anti-inflammatory molecule and a screening method and application thereof. The anti-inflammatory effect of the Prevotella microbial anti-inflammatory molecule screened by the present application can reach more than 90%. It has the advantages of small dosage and strong anti-inflammatory effect.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] In a first aspect, the present application provides a Prevotella microbial anti-inflammatory molecule, the amino acid sequence of the Prevotella microbial anti-inflammatory molecule is as shown in (a) or (b);

[0011] (a) the amino acid sequence is as shown in SEQ ID NO. 1;

[0012] (b) a protein derived from (a) with anti-inflammatory activity, wherein the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids.

[0013] As a preferred embodiment of the Prevotella microbial anti-inflammatory molecule described in the present application, the nucleotide sequence of the Prevotella microbial anti-inflammatory molecule is as shown in SEQ ID NO. 2.

[0014] The Prevotella microbial anti-inflammatory molecule screened by the present application has strong anti-inflammatory effect under the premise of low dosage. Compared with the microbial anti-inflammatory molecule protein of the representative strain A2-165 of the Faecalibacterium genus, the inhibitory effects on the NF-κB signaling pathway and the inflammatory factor TNFα are 50% and 56% respectively, while the anti-inflammatory effect (inhibiting the NF-κB signaling pathway) of the Prevotella microbial anti-inflammatory molecule screened by the present application can reach more than 90%.

[0015] Preferably, the Prevotella microbial anti-inflammatory molecule is derived from the CNCM I 4644 strain (the nucleotide sequence can be obtained from the NCBI database).

[0016] In a second aspect, the present application provides a nucleic acid molecule encoding the above-mentioned Prevotella microbial anti-inflammatory molecule.

[0017] In a third aspect, the present application provides an expression vector, which carries the nucleic acid molecule.

[0018] As a preferred embodiment of the expression vector of the present application, the expression vector comprises a eukaryotic expression vector pcDNA3.1.

[0019] In a fourth aspect, the present application provides a host cell, which comprises the Faecalibacterium microbial anti-inflammatory molecule or the nucleic acid molecule or the expression vector.

[0020] As a preferred embodiment of the host cell of the present application, the NF-kB luciferase reporter vector or the luciferase reporter vector of the inflammatory cytokine TNF alpha promoter is introduced into the host cell.

[0021] In a fifth aspect, the present application provides a screening method of the Faecalibacterium microbial anti-inflammatory molecule, comprising the following steps:

[0022] S1, obtaining the genome sequences of different strains of Faecalibacterium from the NCBI database;

[0023] S2, cloning the microbial anti-inflammatory molecules of different strains of Faecalibacterium into a eukaryotic expression vector, and co-transferring the NF-kB luciferase reporter vector or the luciferase reporter vector of the inflammatory cytokine TNF alpha promoter into a host cell, to screen the Faecalibacterium microbial anti-inflammatory molecules that have inhibitory effect on the expression of NF-kB and TNF alpha.

[0024] The present application is designed for the common target of clinical intestinal inflammation treatment, i.e. the expression of NF-kB signal pathway and pro-inflammatory factor TNF alpha, introduces the luciferase reporter vector which is sensitive and can be accurately quantified, and screens the microbial anti-inflammatory molecules of Faecalibacterium genus on the intestinal epithelial cell line.

[0025] Preferably, the screening method of the Faecalibacterium microbial anti-inflammatory molecule comprises the following steps:

[0026] 1. Download 71 strains of Faecalibacterium genus genome sequences from the NCBI database, find out the gene sequences encoding microbial anti-inflammatory molecules and translate them into amino acid sequences through sequence alignment, and perform evolutionary analysis on the amino acid sequences of the above microbial anti-inflammatory molecules through MEGA-X software, and find that they can be divided into at least 7 lineages.

[0027] 2. From the 7 lineages of Faecalibacterium, representative strains were selected, and the sequences of the microbial anti-inflammatory molecules were cloned into eukaryotic expression vectors, and co-transfected into the intestinal epithelial cell line HCT116 with NF-κB luciferase reporter gene vectors or luciferase reporter gene vectors of inflammatory cytokine TNFα promoters, to screen MAM proteins that inhibit the expression of NF-κB and TNFα, and it was found that the strains of lineage A had stronger inhibitory effect on NF-κB and TNFα.

[0028] 3. Further screening of 5 strains of lineage A, the sequences of the microbial anti-inflammatory molecules were cloned into eukaryotic expression vectors, and co-transfected into the intestinal epithelial cell line HCT116 with NF-κB luciferase reporter gene vectors or luciferase reporter gene vectors of inflammatory cytokine TNFα promoters, to screen the MAM protein with the strongest inhibitory effect on the expression of NF-κB and TNFα, i.e. the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii CNCM I 4644 strain.

[0029] 4. Different doses of eukaryotic expression vectors of CNCM I 4644-MAM were co-transfected into the intestinal epithelial cell line HCT116 with NF-κB luciferase reporter gene vectors or luciferase reporter gene vectors of inflammatory cytokine TNFα promoters, and it was found that the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii still had a strong anti-inflammatory effect even at a very low expression level.

[0030] In a sixth aspect, the present application provides a method for grouping Faecalibacterium prausnitzii, which comprises phylogenetic analysis of the amino acid sequence of the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii.

[0031] The above grouping method performs phylogenetic analysis on the amino acid sequence of the microbial anti-inflammatory molecule MAM of Faecalibacterium prausnitzii, rather than the traditional 16sRNA sequence, and the method is more conducive to screening the anti-inflammatory function of MAM protein.

[0032] In a seventh aspect, the present application provides an anti-inflammatory drug, which comprises the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii.

[0033] In some embodiments, the mass concentration of the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii is 0.1-0.8 μg / well. Preferably, the mass concentration of the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii is 0.1 μg / well.

[0034] In an eighth aspect, the present application provides the use of the microbial anti-inflammatory molecule of Faecalibacterium prausnitzii in the preparation of an anti-inflammatory drug.

[0035] The microbial anti-inflammatory molecule of Faecalibacterium prausnitzii of the present application can be used to treat intestinal inflammation such as inflammatory bowel disease, irritable bowel syndrome, metabolic syndrome and autoimmune enteropathy.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application provides a Faecalibacterium prausnitzii microbial anti-inflammatory molecule and a screening method and application thereof. Compared with the microbial anti-inflammatory molecule protein of the representative strain A2-165 of the Faecalibacterium genus, the inhibition of the NF-κB signaling pathway and the inflammatory factor TNFα is 50% and 56%, respectively, while the anti-inflammatory effect (inhibition of the NF-κB signaling pathway) of the Faecalibacterium prausnitzii microbial anti-inflammatory molecule screened by the present application can reach more than 90%, which has the advantages of small dosage and strong anti-inflammatory effect. Moreover, the present application is designed for the common target for the treatment of clinical intestinal inflammation, that is, the expression of the NF-κB signaling pathway and the pro-inflammatory factor TNFα, and introduces the luciferase reporter gene vector, which is a sensitive and accurate quantitative method, to screen the microbial anti-inflammatory molecule of the Faecalibacterium genus on the intestinal epithelial cell line. Finally, the present application proves again in the dextran sulfate sodium salt-induced mouse intestinal inflammation model that, compared with the representative strain A2-165-MAM, the screened CNCM I 4644-MAM has a more obvious therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Evolutionary tree of microbial anti-inflammatory molecules of the Faecalibacterium genus;

[0039] Figure 2 Anti-inflammatory result graph of microbial anti-inflammatory molecules of the representative strain of each lineage;

[0040] Figure 3 Anti-inflammatory result graph of microbial anti-inflammatory molecules of the strain in the lineage A;

[0041] Figure 4 Anti-inflammatory result graph of microbial anti-inflammatory molecules of the CNCM I 4644 strain;

[0042] Figure 5 Anti-inflammatory result graph of the MAM protein of the CNCM I 4644 strain significantly improving the dextran sulfate sodium salt (DSS)-induced colitis Figure 5 A: expression of exogenous MAM protein; Figure 5 B is the animal experiment process scheme; Figure 5 C is the change of the body weight of the mice after modeling; Figure 5 D is the daily water consumption (milliliters) of each mouse; Figure 5 E is the colon HE staining of the mice in each group; Figure 5 F is the pathological score. DETAILED DESCRIPTION

[0043] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in conjunction with the drawings and specific embodiments.

[0044] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0045] In the following examples, the eukaryotic expression vector used is pcDNA3.1.

[0046] Example 1, screening method of microbial anti-inflammatory molecules of Faecalibacterium prausnitzii

[0047] The present example provides a screening method of microbial anti-inflammatory molecules of Faecalibacterium prausnitzii, comprising the following steps:

[0048] 1. Download 71 strains of Faecalibacterium from the NCBI database, find the gene sequences encoding microbial anti-inflammatory molecules by sequence alignment and translate them into amino acid sequences, and perform Maximum Likelihood evolution analysis on the amino acid sequences of the above 100 microbial anti-inflammatory molecules by MEGA-X software, and find that they can be divided into at least 7 lineages, respectively, lineage A to lineage G. The phylogenetic tree of microbial anti-inflammatory molecules of Faecalibacterium is shown in Figure 1 .

[0049] 2. Select one representative strain from each of the 7 lineages of Faecalibacterium, respectively, A, M21 / 2; B, A2-165; C, KLE1255; D, SG-1377; E, APC942 32-1; F, L2 / 6; G, CNCM I 4575.

[0050] After codon optimization of the nucleotide sequences of their microbial anti-inflammatory molecules (as shown in Table 1), they are cloned into the eukaryotic expression vector pcDNA3.1, and co-transfected into the intestinal epithelial cell line HCT116 with the NF-κB luciferase reporter gene vector or the luciferase reporter gene vector of the inflammatory cytokine TNFα promoter, to screen microbial anti-inflammatory molecules that have inhibitory effect on the expression of NF-κB and TNFα. The experimental results show that the microbial anti-inflammatory molecules of the M21 / 2 strain in the A lineage have stronger inhibitory effect on NF-κB and TNFα Figure 2 .

[0051] The specific experimental steps are as follows:

[0052] a) Codon optimization: Since the microbial anti-inflammatory molecules are expressed proteins of Faecalibacterium genus, there is a great difference in the codon usage frequency between Faecalibacterium genus and human. In order to improve the expression level of microbial anti-inflammatory molecules in the intestinal epithelial cell line HCT116, the codons in the nucleotide sequence of microbial anti-inflammatory molecules are adjusted to the codons with high frequency of use in human cells in combination with codon usage bias, ribosome binding, mRNA structure, etc. The optimized sequence is shown in Table 1.

[0053] b) Gene cloning: The optimized nucleotide sequence of microbial anti-inflammatory molecules synthesized by Shengong Bioengineering Co., Ltd. is double digested with HindIII and BamHI enzyme sites, and then the sequence of microbial anti-inflammatory molecules is connected into the pcDNA3.1 vector using ligase, transformed into Top10 competent cells for amplification, and identified.

[0054] c) Luciferase reporter gene screening experiment: pcDNA3.1-MAM, pNF-κB-luc (or pTNF-α-promoter-luc), pRL-TK (as an internal reference) are co-transfected into HCT116 cell line, and the cells are collected and lysed after 48 hours, and detected by Dual Luciferase Reporter Assay Kit (promega, #E1910). Among them, the NF-κB luciferase reporter gene vector (pNF-κB-luc, #D2206), the luciferase reporter gene vector of the inflammatory cytokine TNFα promoter (pTNF-α-promoter-luc, #D2480) and the sea anemone luciferase reporter gene vector (pRL-TK, #D2760) are purchased from Biyun Tian Biological Company.

[0055] 3, Further screening of 5 strains of A lineage, respectively: SL3 / 3, AM100 B16A, Fp137, CNCM I 4644 and Fp1160. The nucleotide sequence of microbial anti-inflammatory molecules is codon optimized and cloned into the eukaryotic expression vector pcDNA3.1, co-transfected into the intestinal epithelial cell line HCT116 with the NF-κB luciferase reporter gene vector or the luciferase reporter gene vector of the inflammatory cytokine TNFα promoter, so as to screen the microbial anti-inflammatory molecules with the strongest expression inhibition effect on NF-κB and TNFalpha, i.e. the Faecalibacterium prausnitzii microbial anti-inflammatory molecules of CNCM I 4644 strain (i.e. CNCM I 4644-MAM protein) Figure 3 ).

[0056] ​The amino acid sequence of the microbial anti-inflammatory molecule of the strain of Prevotella plancida CNCM I 4644 is shown in SEQ ID NO. 1.

[0057] SEQ ID NO. 1:

[0058] MMMPANFSAVSENEMTYVMGGSVADYLAPAMGAAQWQNFHKNLITIVGNKYVQGFLDN

[0059] TVNAVFSGTWTPGAGLTGFGGQFSNIWKKNYTDNVTDQSTGAQKFGYGALGVVNSILNVAGNLAAIYNLGFGTAKNIVNENKFGF.

[0060] 4. The eukaryotic expression vector (pcDNA3.1-CNCM I 4644-MAM) of different doses of CNCM I 4644-MAM protein was co-transfected into the intestinal epithelial cell line HCT116 with the NF-κB luciferase reporter gene vector or the luciferase reporter gene vector of the inflammatory cytokine TNFα promoter, respectively, and it was found that the microbial anti-inflammatory molecule of Prevotella plancida still had a strong anti-inflammatory effect under the condition of very low expression amount. Figure 4

[0061] Table 1

[0062]

[0063]

[0064]

[0065]

[0066] Example 2, animal experiment

[0067] In order to further explore the anti-inflammatory effect of CNCM I 4644-MAM protein in vivo, the results Figure 5 .

[0068] ​The present application extracts the protein of representative strain A2-165-MAM of Prevotella plara and the protein of CNCM I 4644-MAM through a conventional E. coli exogenous expression and purification system, and the specific steps are as follows: the nucleotide sequence of A2-165-MAM and CNCM I 4644-MAM is inserted into a His tag CATCATCACCATCACCAT at the C terminal, cloned into a pET-28a(+) vector, expanded using a DH5a competent cell, and then the two vectors are respectively transferred into a BL21 competent cell, induced for expression at 18°C in a shaking bed using 1 mg / ml IPTG, and then the two MAM proteins are respectively extracted and purified using a His tag protein purification kit (purchased from Biyun Tian, #P2229S). The expression of the MAM protein is detected by a Western method as shown in Figure 5 -A.

[0069] Then, 8-week-old C57 male mice are selected, and the mice are given 0.5 mg per mouse per day by gavage, and the control group is given the same volume of PBS by gavage. After 6 days, 3% DSS water is freely drunk to model intestinal inflammation, and the control group drinks normal sterile water. The modeling is stopped after 7 days. The mice are sacrificed on the 9th day, and the intestinal tissues are taken for HE staining Figure 5 -B). The results show that the normal control group of mice grows well, and the body weight gradually increases; while the body weight of the model group of mice decreases significantly from the 4th day of the experiment, and the body weight decreases by about 20% on the 8th day. Compared with the model group and the A2-165 strain MAM protein gavage group, the body weight of the mice gavaged with CNCM I 4644-MAM protein decreases more gently, less than 10% Figure 5 -C). The daily water consumption (ml) of each mouse is shown in Figure 5 -D.

[0070] The colon HE staining shows that the colon tissue mucosa of the normal group of mice is complete, the content of goblet cells is rich, the gland structure is arranged in order, and there is no obvious neutrophil infiltration in the lamina propria. The colon pathology of the intestinal inflammation model group shows that the epithelial integrity is destroyed, the goblet cells are absent, the glands are deformed, and the crypts are twisted and irregular; after the intervention of A2-165-MAM protein, the destruction of the epithelium can be relieved to a certain extent. However, the pathological relief effect of the CNCM I 4644-MAM protein intervention group is more obvious, which is manifested as an increase in the number of goblet cells, a recovery of part of the glands, a certain degree of reduction in the infiltration of inflammatory cells in the mucosa lamina propria, and a significant difference in the histopathology score compared with the A2-165 group (P<0.05) Figure 5 -E and Figure 5 -F).

[0071] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. The application of a Clostridium praosporum anti-inflammatory molecule in the preparation of anti-inflammatory drugs, characterized in that, The amino acid sequence of the Clostridium praosporum anti-inflammatory molecule is shown in SEQ ID NO.1.

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