A human bacterial flora mouse model of IgA vasculitis and its construction method

Abstract

The present invention discloses a human-derived bacterial flora mouse model of IgA vasculitis and a construction method thereof, and belongs to the field of biotechnology. The model is divided into an initial IgA vasculitis model and an IgA vasculitis recurrence model, and the intestinal flora of the model has intestinal microorganisms characteristic of donor IgA vasculitis patients. The construction method adopts a fecal microbiota transplantation method by gavage of mice. The present invention is easy to operate and has a high success rate of modeling, and provides a research model for exploring the mechanism of IgA vasculitis occurrence related to intestinal microorganisms.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an IgA vasculitis human bacterial flora mouse model and a construction method thereof. Background Art

[0002] IgA vasculitis (Immunoglobulin A vasculitis, IgAV), also known as Henoch–Scholein Purpura (HSP), is an immune vasculitis mediated by IgA. It often occurs in school-age children, most often involving the skin, and may also be accompanied by joint, gastrointestinal and kidney involvement. At present, the pathogenesis of IgAV has not been elucidated. Studies have shown that intestinal microorganisms are closely related to IgAV and play an important role in the pathogenesis of IgAV. Studies have found that the clinical manifestations of children with IgAV are related to specific intestinal microorganisms. However, the specific mechanism of intestinal microorganisms in the pathogenesis of children with IgAV is still unclear. Given that the relevant mechanism research is subject to ethical restrictions in humans, it is necessary to select appropriate animals to colonize the characteristic intestinal microorganisms of children with IgAV for mechanism research. At present, there is no classic IgAV animal disease model that can be used for the study of the correlation between intestinal microorganisms and IgAV. Human flora-associated (HFA) mice are mouse models constructed by transplanting human intestinal microorganisms into the mouse intestine, which can be used for the mechanism study of intestinal microorganisms through intestinal mucosal immune-mediated diseases. Summary of the invention

[0003] Therefore, it is necessary to provide an animal model that is simple to operate and truly reflects the IgAV disease condition for subsequent research.

[0004] To achieve the above purpose, the inventors provide the following technical solutions:

[0005] A mouse model of humanized flora of IgA vasculitis, including an initial IgA vasculitis model and a recurrent IgA vasculitis model, wherein the intestinal flora of the initial model has intestinal microorganisms characteristic of the initial IgA vasculitis patient of the donor, including one or more of the genera Prevotella, Paraprevotella, and Bifidobacterium; and the intestinal flora of the recurrent model has IgA-degrading intestinal commensal bacteria, including Alcaligenes bacteria.

[0006] Furthermore, the intestinal microorganisms of the primary model also include one or more of Staphylococcus, Enterobacteriaceae, Burkholderia, Sutterella, Collinsella, Catenibacterium, and Fusobacterium; the intestinal microorganisms of the relapse model also include one or more of Clostridium, Staphylococcus, Exiguobacterium, Paenisporosarcina, Akkermansia, Acinetobacter, Brachybacterium, F16 family, and Fusobacterium.

[0007] The method for constructing the IgA vasculitis human flora mouse model comprises the following steps:

[0008] Preparation of bacterial solution: Fecal samples from patients with initial or recurrent IgA vasculitis were collected, and normal saline (1 g feces: 15 mL normal saline) was added and mixed evenly to obtain a suspension; the suspension was filtered to remove the residue, and then centrifuged (5000 r / min, 3 min), and the supernatant bacterial solution was collected for later use;

[0009] Fecal microbiota transplantation (FMT): The bacterial solution prepared in the previous step was administered intragastricly into the stomach of mice for 3 to 4 consecutive days.

[0010] Model establishment: After the first oral gavage, mice were kept for 7 to 21 days in a 12 / 12 h light / dark cycle with alternating day and night light and dark, and were allowed to eat and drink freely.

[0011] Furthermore, the method for constructing the IgA vasculitis human flora mouse model comprises the following steps:

[0012] Preparation of fecal bacterial suspension: 80-100 g of feces from patients with primary or recurrent IgA vasculitis were collected, 180-220 ml of normal saline was added, and the fecal bacterial suspension was obtained after filtration; the fecal bacterial suspension was mixed with glycerol at a volume ratio of (4.5-5.5):1, centrifuged, and the supernatant was discarded; the precipitate was subjected to high-speed refrigerated centrifugation again, and the supernatant was discarded;

[0013] Preparation of fecal bacteria capsule: Place the sediment into the capsule and seal it;

[0014] Fecal microbiota transplantation: The capsules prepared in the previous step were injected into the stomach of mice by gavage for 3-4 consecutive days;

[0015] Model establishment: After the first oral administration, mice were fed continuously for 7 to 21 days.

[0016] Furthermore, the method for preparing the fecal bacteria capsule is to place the precipitate in an acid-resistant No. 2 capsule (volume of about 0.37 ml) and seal it; or place the precipitate in a No. 1 gelatin capsule (volume of about 0.50 ml), and then reseal it twice using a No. 0 capsule (volume of about 0.68 ml) and a No. 00 capsule (volume of about 0.93 ml) respectively; or place the precipitate in a No. 0 gelatin capsule and seal it, and then use a No. 00 acid-resistant hydroxypropyl methylcellulose capsule for a second sealing.

[0017] Furthermore, the feeding time after oral administration was determined according to the colonization time of different bacterial genera. In the humanized bacterial flora mouse model of patients with initial IgA vasculitis, Staphylococcus colonized 0 to 7 days after FMT, Enterobacteriaceae colonized 7 to 14 days after FMT, and Borreliales (including Burkholderia and Sutterella), Collinsella, Catenibacterium, and Fusobacterium colonized 14 to 21 days after FMT; in the humanized bacterial flora mouse model of patients with recurrent IgA vasculitis, Exiguobacterium, Paenisporosarcina, and Akkermansia colonized 0 to 7 days after FMT, Acinetobacter colonized 7 to 14 days after FMT, and Brevibacterium, F16 family, and Fusobacterium colonized 14 to 21 days after FMT.

[0018] Furthermore, the inclusion criteria for patients with IgA vasculitis are: age < 18 years old, regardless of gender; palpable rash accompanied by any of the following: ① abdominal pain, ② arthritis / arthralgia, ③ hematuria and / or proteinuria, ④ biopsy of any site indicating IgA deposition.

[0019] Furthermore, the mice are selected from SPF grade C57BL / 6J mice.

[0020] Furthermore, the intragastric volume of the mice is 200-300 μL / time, and the frequency of intragastric administration is at least once a day.

[0021] Furthermore, the bacterial flora structure of the stool samples of patients with recurrent IgA vasculitis is that the abundance of Burkholderiaceae family is 1.2-1.5%, the abundance of Rhodococcidae family is 0.1-0.3%, the abundance of Veillonellaceae family is 0.1-0.4%, and the abundance of Enterobacteriaceae family is 13.5-15%.

[0022] Furthermore, the relapse model serum IL-17A ≥ 25.10 pg / ml, IL-21 ≥ 89.95 pg / ml.

[0023] The method for judging the success of modeling is as follows: characteristic bacterial genera (Prevotella and Paraprevotella) are detected in the humanized bacterial flora mice of IgAV patients at the onset, and characteristic bacterial genera (Clostridium or Alcaligenes) of IgAV relapse patients are detected in the humanized bacterial flora mice of IgAV relapse patients; and the above characteristic bacterial genera are the different bacterial genera between IgAV patients at the onset and IgAV relapse patients, and between the humanized bacterial flora mice of IgAV patients at the onset and IgAV relapse patients, which means that the different bacterial genera between IgAV patients at the onset and relapse are successfully transferred to the mouse intestine. In the serum of mice in the relapse model, it can be detected that IgA and B-cell activating factor (BAFF), interleukin (IL)-17A, and IL-21 are elevated, among which IL-17A and IL-21 are significantly elevated.

[0024] Furthermore, the human bacterial flora mouse model of IgA vasculitis is used to screen drugs for the treatment of IgA vasculitis.

[0025] Furthermore, a detection kit for the IgA vasculitis human flora mouse model includes detection reagents for the content of Prevotella, Paraprevotella, Bifidobacterium, Clostridium and Staphylococcus.

[0026] Furthermore, the detection kit for the IgA vasculitis human flora mouse model also includes detection reagents for the abundance of Alcaligenesaceae bacteria, detection reagents for IL-17A, and detection reagents for IL-21.

[0027] Furthermore, the kit also includes one or more content detection reagents of Enterobacteriaceae, Burkholderia, Sutterella, Collinsella, Catenibacterium, Fusobacterium, Exiguobacterium, Paenisporosarcina, Akkermansia, Acinetobacter, Brevibacterium or F16 family.

[0028] Furthermore, the IgAV human bacterial flora mouse model is used in screening drugs for treating IgA vasculitis.

[0029] Different from the prior art, the above technical solution has the following beneficial effects:

[0030] (1) The present invention provides an animal model for exploring the mechanism of intestinal microorganisms involved in the occurrence and development of IgAV, and also provides a screening model for IgAV drug research.

[0031] (2) The present invention is easy to operate, has a high modeling success rate, and truly reflects the intestinal microbial characteristics of IgAV patients in the animal model. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a taxonomic branch diagram for the comparison of differential bacterial genera between children with IgAV initial onset and children with IgAV relapse as described in the specific implementation method. In the figure, from the inner circle to the outer circle, the taxonomic hierarchy relationship of the main bacterial groups in the sample from phylum to genus is represented. The size of the node corresponds to the relative abundance; the red node represents the differential bacteria with higher abundance in the microorganisms in the feces of children with IgAV relapse, and the blue node represents the differential bacteria with higher abundance in the feces of children with IgAV initial onset; the hollow node represents that the taxonomic unit has no significant difference between the groups.

[0033] Figure 2 Comparison of differential bacterial genera in mice with human flora of children with IgAV initial onset and relapse as described in the specific implementation method. Among them, (A) Taxonomic branch diagram: from the inner circle to the outer circle, it represents the taxonomic hierarchy relationship of the main flora in the sample from phylum to genus. The size of the node corresponds to the relative abundance; the red node represents the differential bacteria with higher abundance in the intestinal microorganisms of mice with human flora of children with IgAV initial onset, and the blue node represents the differential bacteria with higher abundance in the intestinal microorganisms of mice with human flora of children with IgAV relapse; the hollow node represents that the taxonomic unit has no significant difference between the groups; (B) Bar graph: Set the LDA threshold to 2, and the length of the bar graph represents the significance of the difference in the flora. PT represents mice with human flora of children with IgAV initial onset, and RT represents mice with human flora of children with IgAV relapse.

[0034] Figure 3 This is a Sankey diagram described in a specific embodiment. In the figure, Human represents the ASV specific to children with IgAV. PT represents the fecal sample of FMT mice in the IgAV initial group, RT represents the fecal sample of FMT mice in the IgAV relapse group, and HT represents the fecal sample of FMT mice in the healthy children group. D7, D14, and D21 represent the days for collecting mouse fecal samples (interval time after the first vaccination).

[0035] Figure 4 This is the intestinal microbial composition at the genus level before and after fecal microbiota transplantation in mice as described in the specific implementation method.

[0036] Figure 5 Different intestinal microorganisms of mice at the family level described in the specific implementation method. (a) Random forest analysis identified ten bacterial species as markers to distinguish between rIMb and rHMb groups; (b) The relative abundance of ten bacterial species in the rIMb and rHMb groups on day 7 after fecal microbiota transplantation. rIMb refers to mice with human flora from children with IgAV, and rHMb refers to mice with human flora from healthy children. P < 0.05 indicates statistical significance. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates that the difference is not significant.

[0037] Figure 6 The differential intestinal microorganisms of mice at the genus level described in the specific implementation method. (a) Random forest analysis identified 10 bacterial genera as markers to distinguish between rIMb and rHMb groups; (b) The relative abundance of the 10 bacterial genera in the rIMb and rHMb groups on day 7 after fecal microbiota transplantation. rIMb refers to mice with humanized microbiota from children with IgAV, and rHMb refers to mice with humanized microbiota from healthy children.

[0038] Figure 7 The relative abundance of differential intestinal microorganisms of mice in the family level described in the specific embodiment in human feces. IgAV refers to children with IgAV, and HC refers to healthy children.

[0039] Figure 8 Detailed description of the ROC curve for Burkholderiaceae.

[0040] Fig. 9 The composition of Burkholderiaceae in the feces of children with IgAV described in a specific implementation manner.

[0041] Fig.10 The composition of Burkholderiaceae in feces of healthy children described in a specific embodiment.

[0042] Fig.11 The relative abundance of Sutterella and Parasutterella in human feces according to a specific embodiment.

[0043] Fig.12 The serum IgA and cytokine levels of the model mice described in the specific implementation manner.

[0044] Fig.13 The figure is the ROC curve of serum IL-17A of model mice described in the specific implementation manner.

[0045] Fig.14 The figure is the ROC curve of serum IL-21 of the model mice described in the specific implementation manner. DETAILED DESCRIPTION

[0046] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0047] Example 1

[0048] 1. Stool donor screening and sample collection

[0049] The specimen collection process was as follows: the stool samples of children with IgAV initial onset and IgAV relapse were collected using a sterilized EP tube, and the stool samples of healthy children were collected using a test tube containing buffer. The subjects were asked to empty their bladders before defecation, and 2-3 g of inner feces were picked up with a sterile spoon. Two samples were collected at the same time, placed on ice for transportation, and stored in a -80℃ refrigerator as soon as possible. Five stool samples from children with IgAV initial onset, children with IgAV relapse, and healthy children with the same clinical manifestations were selected as FMT donor samples. All five children with IgAV initial onset had rash as the clinical manifestation alone, including 4 males and 1 female, aged 2 to 8 years old; all five children with IgAV relapse had rash and kidney involvement as the clinical manifestations, including 3 males and 2 females, aged 5 to 11 years old.

[0050] Inclusion criteria for children with IgAV: (1) age < 18 years, regardless of gender; (2) palpable rash (essential condition) and any of the following: ① abdominal pain; ② arthritis / arthralgia; ③ hematuria and / or proteinuria; ④ biopsy of any part showing IgA deposition. IgAV relapse group: patients who were previously diagnosed with IgAV and were asymptomatic for at least 4 weeks but developed symptoms again.

[0051] Exclusion criteria: (1) use of antibiotics, proton pump inhibitors, immunosuppressants, chemotherapy drugs, etc. within 2 weeks before collecting stool specimens; (2) suffering from immunodeficiency diseases; (3) comorbidities such as autism, inflammatory bowel disease, Clostridium difficile infection, epilepsy, constipation, etc.

[0052] Inclusion criteria for healthy children: (1) age <18 years, regardless of gender; (2) no history of allergic diseases such as allergic cough, allergic rhinitis, and allergic asthma; (3) no use of antibiotics, immunosuppressants, proton pump inhibitors, etc. within 2 weeks before sample collection.

[0053] 2 Experimental animals

[0054] 64 SPF-grade C57BL / 6J mice, half male and half female, aged 3-5 weeks, were selected and purchased from Shanghai Slake Experimental Animal Co., Ltd. They were adaptively fed in an SPF-grade barrier environment for 1 week and then transferred to a normal environment for breeding. The animal breeding center maintains good ventilation, and the breeding room is regularly cleaned and disinfected with disinfectant. A 12 / 12-hour light / dark cycle is maintained according to the alternation of light and dark during the day and night. All mice are placed in independent ventilation cages and raised in separate cages, with 2 mice in each cage (same sex), free to eat and drink, and drinking sterilized water is replaced regularly. The mouse cages and bedding are sterilized in advance with high-pressure steam and replaced regularly.

[0055] C57BL / 6J mice were randomly divided into the following four groups, with 16 mice in each group:

[0056] (1) Normal control group (group N);

[0057] (2) IgAV first-onset FMT group (PT group);

[0058] (3) IgAV recurrence FMT group (RT group);

[0059] (4) Healthy children FMT group (HT group).

[0060] 3 Construction of human microbiota mice

[0061] 3.1 Preparation of bacterial culture

[0062] (1) Stool samples of 5 children with IgAV first onset, 5 children with IgAV relapse, and 5 healthy children were collected and mixed with equal weight of samples from each group;

[0063] (2) Take 1 g of the mixed feces, add 10 mL of normal saline, gently stir or use a vortex oscillator to mix them evenly, and finally obtain a suspension;

[0064] (3) Prepare a filter, filter the suspension obtained in the previous step, remove the filtered residue, and collect the filtered suspension in a 10 mL sterilized centrifuge tube;

[0065] (4) Place the suspension in a centrifuge and centrifuge at 5000 r / min for 3 min;

[0066] (5) Collect the supernatant for later use.

[0067] 3.2 Fecal microbiota transplantation procedure

[0068] Each mouse was housed individually, and the prepared bacterial solution was gavaged into the mouse stomach by intragastric administration. The gavage volume of fecal bacterial suspension for each mouse was 200 μL / time. The mice in the experimental group were observed for 30 minutes after no abnormalities were found. The mice in the experimental group were housed together, and the gavage frequency was once / day for 3 consecutive days.

[0069] 4 Sample collection method

[0070] Fresh feces of mice were collected at the following time points before and after FMT (interval time after the first FMT): 7d, 14d, and 21d. Feces were collected by stress defecation method. The lower abdomen of the mouse was gently pressed and the fresh feces were promptly placed in a sterile centrifuge tube. Each tube was a collection of feces from all mice in the target group; all fecal samples were stored at -80°C.

[0071] 516s rDNA sequencing method

[0072] 5.1 Microbiome total DNA extraction and detection

[0073] OMEGA Soil DNA Kit (D5625-01) was used for nucleic acid extraction.

[0074] 5.2 PCR amplification of different regions of bacterial 16S rDNA gene functional genes

[0075] The highly variable V3-V4 region of the bacterial 16SrDNA gene, which is about 468 bp in length, was selected for sequencing. PCR amplification used primers specific to the bacterial 16SrDNA V3-V4 region, 338F (5'-barcode+ACTCCTACGGGAGGCAGCA-3'), 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The barcode in the front primer is a 7-base oligonucleotide sequence used to distinguish different samples in the same library.

[0076] The PCR reaction system is as follows:

[0077]

[0078] PCR program: pre-denaturation at 98°C for 30 s; 98°C for 15 s, 50°C for 30 s, 72°C for 30 s, amplification cycles for 25 to 27 times; maintain at 72°C for 5 min; store at 4°C.

[0079] The PCR products were separated by 2% agarose gel electrophoresis, and the fragments containing the target DNA were cut out and recovered using the Axygen gel recovery kit.

[0080] 5.3 PCR product quantification and sample mixing

[0081] The PCR products were quantified on a Microplate reader (BioTek, FLx800) using the Quant-iT PicoGreen dsDNA Assay Kit, and the samples were mixed according to the required data volume for each sample.

[0082] 5.4 Library construction

[0083] The library was constructed using Illumina's TruSeq Nano DNA LT Library Prep Kit.

[0084] 5.5 Library quality inspection and sequencing

[0085] After the library quality inspection was qualified, 2×250bp paired-end sequencing was performed on the Illumina NovaSeq 6000 instrument.

[0086] 6. Information Analysis

[0087] 6.1 Species composition analysis

[0088] The relative abundance of each sample at the six classification levels of phylum, class, order, family, genus and species, as well as the species composition of the samples were counted.

[0089] 6.2 Diversity Analysis

[0090] Diversity analysis was performed on each sample and group, including α diversity analysis and β diversity analysis.

[0091] 6.3 Species differences and landmark species analysis

[0092] (1) Venn diagram: The Venn diagram can be used to analyze the microbial community and the common or unique species among different samples (groups).

[0093] (2) Sankey diagram: Sankey diagram is also known as Sankey energy flow diagram, which can be used to show the changing distribution of microorganisms in different time periods.

[0094] (3) Streamgraph: Also called “Theme River”, it is a variation of the stacked area graph. It uses “flowing” shapes to show how different categories of data change over time.

[0095] 7 Statistical methods

[0096] SPSS24.0 software was used to analyze the data in this study. The measurement data that obeyed the normal distribution were expressed as mean ± standard deviation (x ± s); those that did not obey the normal distribution were expressed as median (lower quartile to upper quartile) [M (P25 ~ P75)]. The differences between the two groups were compared using the two independent sample nonparametric test (Mann-Whitney U test); the differences between three groups or more were compared using the Kruskal-Wallis test; P < 0.05 indicated that the difference was statistically significant.

[0097] 8 Results

[0098] 8.1 Establishment of the unique intestinal microbiota of IgAV patients in mice after FMT

[0099] Venn diagrams were used to analyze whether the intestinal microorganisms specific to IgAV children colonized in the mouse intestine after FMT. The results suggested that ASVs specific to IgAV children could colonize in the mouse intestine: 96 ASVs specific to IgAV children were detected in the intestine of mice with humanized flora from IgAV children, of which 49 belonged to Bacteroidetes, 31 belonged to Firmicutes, 9 belonged to Proteobacteria, 6 belonged to Actinobacteria, and 1 belonged to Fusobacteria; 116 ASVs specific to IgAV relapse were detected in the intestine of mice with humanized flora from IgAV relapse children, of which 44 belonged to Bacteroidetes, 46 belonged to Firmicutes, 10 belonged to Proteobacteria, 11 belonged to Actinobacteria, 1 belonged to Fusobacteria, 1 belonged to TM7, 1 belonged to Verrucomicrobia, and 2 belonged to unclassified phyla.

[0100] The analysis of species composition at the phylum level suggested (Table 1): Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria were the dominant phyla in both the mice with human microbiota from children with initial IgAV infection and the mice with human microbiota from children with recurrent IgAV infection. Among them, the abundance of Bacteroidetes and Proteobacteria in the mice with human microbiota from children with initial IgAV infection was higher than that before FMT, while the abundance of Firmicutes and Actinobacteria was lower than that before FMT; the abundance of Bacteroidetes and Proteobacteria in the mice with human microbiota from children with recurrent IgAV infection was higher than that before FMT, while the abundance of Firmicutes and Actinobacteria was lower than that before FMT.

[0101] Table 1 Statistics of the abundance percentage of human bacterial flora in children with IgAV before and after FMT in mice

[0102]

[0103] Note: P_before indicates mice with human flora from children with primary IgAV before FMT, PT indicates mice with human flora from children with primary IgAV after FMT; R_before indicates mice with human flora from children with recurrent IgAV before FMT, RT indicates mice with human flora from children with recurrent IgAV after FMT.

[0104] 8.2 Differential microbial analysis of mice after FMT

[0105] The microbial composition of mixed feces from children with IgAV initial onset and those from children with IgAV relapse was compared using LEfse analysis (see Figure 1The results showed that at the phylum level, Bacteroidetes, Fusobacterium, and Proteobacteria were enriched in the feces of children with IgAV initial onset, and Actinobacteria, Firmicutes, and TM7 were enriched in the feces of children with IgAV relapse. At the genus level, 25 genera and 21 genera were enriched in the feces of children with IgAV initial onset and IgAV relapse, respectively, and Clostridium was enriched in the feces of children with IgAV relapse.

[0106] The LEfse analysis was used to compare the differences in intestinal microbial composition between mice with humanized flora from children with IgAV initial onset and mice with humanized flora from children with IgAV relapse (see Figure 2 ). The results showed that at the phylum level, TM7 was enriched in the mice with human microbiota from children with IgAV initial onset, but not in the mice with human microbiota from children with IgAV relapse; at the genus level, Bifidobacterium, Olsenella, Paraprevotella, and Prevotella were enriched in the mice with human microbiota from children with IgAV initial onset, and Staphylococcus and Clostridium were enriched in the mice with human microbiota from children with IgAV relapse. The genera with significant differences (P < 0.05) included Bifidobacterium, Paraprevotella, Prevotella, and Staphylococcus (see Table 2).

[0107] Table 2 Comparison of the abundance of different bacterial genera in the human microbiota of children with primary and recurrent IgAV

[0108]

[0109] 8.3 Analysis of the duration of intestinal microbial colonization in mice with human microbiota after FMT

[0110] 8.3.1 Sankey diagram analysis

[0111] In order to evaluate the distribution of ASVs specific to IgAV patients in different time periods, a Sankey diagram was drawn. Figure 3 As shown in the figure, over time, the number of microbial colonization showed an overall decreasing trend, and there were also differences in the time of microbial colonization. Some microorganisms in the Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria were first colonized on the 7th day, some on the 14th day, and some on the 21st day.

[0112] 8.3.2 Changes in the colonization of ASVs specific to IgAV patients in recipient mice over time after FMT

[0113] In the intestines of mice with human flora from children with initial IgAV infection, 6 ASVs (23.08%) could persist in colonization, of which 3 belonged to the phylum Bacteroidetes, 2 belonged to the phylum Firmicutes, and 1 belonged to the phylum Proteobacteria; in the intestines of mice with human flora from children with recurrent IgAV infection, 17 ASVs (30.91%) could persist in colonization, of which 7 belonged to the phylum Bacteroidetes, 7 belonged to the phylum Firmicutes, and 3 belonged to the phylum Actinobacteria.

[0114] 8.3.3 NMDS analysis

[0115] NMDS analysis was performed on mice after FMT, and the results showed that the intestinal microbial communities of mice with human flora from children with initial IgAV disease (stress=0.0463) and mice with human flora from children with recurrent IgAV disease (stress=0.138) tended to be stable 14 to 21 days after FMT.

[0116] 8.3.4 River Chart Analysis

[0117] A river chart was used to show the changes in the abundance of Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria in the intestines of IgAV human microbiota mice over time. The results showed that the human microbiota mice of children with initial IgAV were mainly composed of Bacteroidetes and Firmicutes. The abundance of Bacteroidetes and Firmicutes decreased significantly 7 days after FMT, and remained stable after 14 days. Similarly, the human microbiota mice of children with recurrent IgAV were mainly composed of Bacteroidetes and Firmicutes. The abundance of Bacteroidetes decreased 7 days after FMT, and increased after 14 days, and remained stable. The abundance of Firmicutes increased 7 days after FMT, decreased after 14 days, and remained stable.

[0118] 8.3.5 Species composition analysis

[0119] To compare the changes in intestinal biocomposition over time between mice harboring humanized microbiota from children with initial IgAV infection and mice harboring humanized microbiota from children with recurrent IgAV infection, taxonomic composition analyses were performed at the phylum and genus levels, respectively.

[0120] The results of the intestinal microbial composition of mice with human flora from children with initial IgAV are shown in Tables 3 and 4. At the phylum level, the abundance of microorganisms in fecal samples at each time point was mainly concentrated in the Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria, among which the Bacteroidetes had the highest abundance value in each group, followed by Firmicutes; compared with before FMT, the abundance of Bacteroidetes decreased and the abundance of Firmicutes increased on the 7th day after FMT, that is, the Firmicutes / Bacteroidetes (F / B) ratio increased; 14 days after FMT, the abundance of Bacteroidetes rebounded and the abundance of Firmicutes decreased, and then the abundance of the two tended to stabilize.

[0121] Table 3 Statistics of the abundance percentage of intestinal microorganisms in human flora of children with initial IgAV

[0122]

[0123]

[0124] Note: P0 represents mice with intestinal microbiota of children with IgAV infection before FMT; PT represents mice with human microbiota of children with IgAV infection. D7, D14, and D21 represent the days when fecal samples of mice were collected (interval between the first inoculation and the first vaccination).

[0125] At the genus level, except for Others, the dominant genera in fecal microorganisms before and after FMT at different time points were Lactobacillus, Prevotella, and Flexispira. Compared with before FMT, the abundance of Lactobacillus, Flexispira, Desulfovibrio, Adlercreutzia, and Coprococcus increased on the 7th day after FMT. In addition, the abundance of Bacteroides, Oscillospira, Bifidobacterium, and Odoribacter decreased on the 7th day after FMT.

[0126] Table 4 Statistics of the abundance percentage of intestinal microorganisms in mice with human flora in children with initial IgAV

[0127]

[0128]

[0129] Note: P0 represents mice with intestinal microbiota of children with IgAV infection before FMT; PT represents mice with human microbiota of children with IgAV infection. D7, D14, and D21 represent the days when fecal samples of mice were collected (interval between the first inoculation and the first vaccination).

[0130] The results of the intestinal microbial composition of the human flora in children with IgAV relapse are shown in Tables 5 and 6. At the phylum level, Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria were the main components of the fecal samples in each group, and the abundance of Bacteroidetes was the highest in each group, followed by Firmicutes. Compared with before FMT, the abundance of Bacteroidetes and Actinobacteria decreased on the 7th day after FMT, while the abundance of Firmicutes and Proteobacteria increased; on the 14th day after FMT, the abundance of Bacteroidetes and Actinobacteria rebounded, while the abundance of Firmicutes and Actinobacteria decreased, and then the abundance of the two tended to stabilize.

[0131] Table 5 Statistics of the abundance percentage of intestinal microorganisms in mice with human flora in children with IgAV relapse

[0132]

[0133]

[0134] Note: R0 represents mice with intestinal microbiota of children with IgAV relapse before FMT; RT represents mice with human microbiota of children with IgAV relapse. D7, D14, and D21 represent the days when fecal samples of mice were collected (interval between the first inoculation and the day after the first inoculation).

[0135] At the genus level, except for Others, Lactobacillus and [Prevotella] were the main dominant genera in all groups. Compared with before FMT, the abundance of Lactobacillus, Flexispira, Prevotella, Desulfovibrio, Helicobacter, Adlercreutzia, Rikenella, Staphylococcaceae_Staphylococcus, and Akkermansia increased on the 7th day after FMT, while the abundance of [Prevotella], Oscillospira, Bacteroides, Odoribacter, Allobaculum, Bifidobacterium, Ruminococcaceae_Ruminococcus, Alistipes, Sphingomonas, Coprococcus, and [Ruminococcus] decreased on the 7th day after FMT.

[0136] Table 6 Statistics of the abundance percentage of intestinal microorganisms in mice with human flora in children with IgAV relapse

[0137]

[0138]

[0139] Note: R0 represents mice with intestinal microbiota from children with IgAV relapse before FMT; RT represents mice with human microbiota from children with IgAV relapse.

[0140] D7, D14, and D21 indicate the days when mouse fecal samples were collected (intervals after the first inoculation).

[0141] 8.3.6 Analysis of the colonization time of intestinal microorganisms specific to IgAV children in the mouse intestine

[0142] According to the results of the Sankey diagram, a table was drawn to show the time of colonization of the intestinal microorganisms specific to IgAV children in the mouse intestine. The results are shown in Tables 7 and 8. In the mice with humanized flora from children with initial IgAV, Staphylococcus, Coprococcus, and Psychrobacter were colonized 0 to 7 days after FMT, Enterobacteriaceae were colonized 7 to 14 days after FMT, and the order of Borrelia (including Burkholderia and Sutterella), Collinsella, Catenella, and Pseudomonas were colonized 1 to 2 days after FMT. In the mice harboring human microbiota from children with recurrent IgAV, Exiguobacterium, Paenisporosarcina, and Akkermansia were colonized 0 to 7 days after FMT, Acinetobacter was colonized 7 to 14 days after FMT, and Brachybacterium, F16 family, and Fusobacterium were colonized 14 to 21 days after FMT.

[0143] Table 7 Timeline of colonization of intestinal microorganisms specific to children with IgAV in the intestines of mice

[0144]

[0145]

[0146] Note: 0-7d, 7-14d, 14-21d indicate the time periods of microbial colonization.

[0147] Table 8 Colonization timeline of intestinal microorganisms specific to children with IgAV relapse in mice

[0148]

[0149]

[0150] Note: 0-7d, 7-14d, 14-21d indicate the time periods of microbial colonization.

[0151] 9 Conclusion

[0152] By transplanting the characteristic intestinal microorganisms of IgAV children into the intestines of C57BL / 6J (SPF grade) mice, IgAV humanized microbiota mice can be constructed, and Prevotella and Paraprevotella are the main characteristic colonization genera. The time when the unique intestinal microorganisms of IgAV children are successfully colonized in the intestines of mice varies. From 7 to 21 days after FMT, the unique intestinal microorganisms of IgAV children are colonized for the first time at different time points, some microorganisms are colonized in a specific time period, and Bacteroidetes microorganisms are colonized at different time points. The establishment of this model lays the foundation for subsequent research on the mechanism of intestinal microorganisms involved in the occurrence and development of IgAV.

[0153] Example 2

[0154] 1. Human fecal specimen collection

[0155] Human stool samples were obtained from 5 children with recurrent IgAV and 5 healthy children. All 5 children with recurrent IgAV showed purpuric rash and renal involvement, including 3 males and 2 females, aged 5 to 11 years.

[0156] The fecal specimen sampling process is as follows: use a sterile spoon to scrape the inside of fresh feces and place it in a sterilized EP tube, the amount is about 2 to 3 g. The collected samples are immediately placed on ice for transportation and finally stored in a -80°C refrigerator.

[0157] Inclusion criteria for children with recurrent IgAV were: (1) age <18 years, regardless of gender; (2) previously diagnosed with IgAV (meeting the diagnostic criteria for IgAV established by EULAR / PRINTO / PRES in 2010), but with clinical symptoms disappearing after treatment and with the reappearance of new purpuric rash, new or recurring manifestations of gastrointestinal, joint, kidney, and other systemic involvement at least 4 weeks later.

[0158] 2. Preparation of fecal bacteria suspension

[0159] Fecal specimens from 5 children with recurrent IgAV and 5 healthy children were collected, mixed by equal weight, and diluted with normal saline (1 g: 10 mL). The suspension was passed through a filter to remove the residue, and the remaining liquid was centrifuged (5000 r / min, 3 min) and the supernatant was used for oral administration to mice.

[0160] 3 Animal Grouping

[0161] SPF C57BL / 6J mice were adaptively fed for 7 days, and male and female mice were randomly divided into 2 groups in equal proportions. The specific groups are as follows:

[0162] (1) Human bacteria group of children with IgAV (rIMb group): 16 mice, half male and half female;

[0163] (2) Healthy children human bacteria group (rHMb group): 16 mice, half male and half female.

[0164] 4. Fecal microbiota transplantation

[0165] The prepared fecal bacterial suspensions from recurrent IgAV children and healthy children were gavaged into the mice with humanized flora from IgAV children and mice with humanized flora from healthy children at a dose of 200 μL / mouse, once a day for 3 consecutive days.

[0166] 5. Specimen Collection

[0167] The feces of mice were collected with sterile EP tubes before and 7 days after gavage, and immediately placed on ice for transportation and stored in a -80°C refrigerator.

[0168] Blood samples were collected from mice by cardiac blood sampling, and serum was collected by centrifugation (3000r / min, 15min) after static stratification. The levels of IgA, BAFF, IL-1β, IL-6, IL-17A, IL-21, and TNF-α in mouse serum were detected by ELISA.

[0169] 616s rDNA sequencing method

[0170] Same as 5 in Example 1.

[0171] 7. Detection of IgA and cytokines in serum

[0172] Mouse IgA, BAFF, IL-1β, IL-6, IL-17A, IL-21, and TNF-α ELISA detection kits were used to detect IgA, BAFF, IL-1β, IL-6, IL-17A, IL-21, and TNF-α in mouse serum, respectively.

[0173] 8 Statistical analysis

[0174] All data in this study were statistically analyzed using SPSS 24.0 and R language 4.1.2. The measurement data that followed the normal distribution were expressed as mean ± standard deviation, and the independent sample t test (homogeneity of variance, both groups of values ​​followed the normal distribution) was used for comparison between the two groups. For those with unequal variance or one group of values ​​that did not follow the normal distribution, the Mann-Whitney U test was used for comparison between the two groups; the measurement data that did not follow the normal distribution were expressed as median (lower quartile to upper quartile) [M (P25~P75)], and the Mann-Whitney U test was used for comparison between the two groups; the chi-square test was used for the count data; P < 0.05 indicated that the difference was statistically significant.

[0175] 9 Results

[0176] 9.1 Analysis of intestinal microbial colonization

[0177] A Venn diagram was drawn to analyze whether human-specific intestinal microbes can colonize in mice. The results showed that 7 days after fecal microbiota transplantation, intestinal microbes specific to IgAV children and healthy children could colonize in the intestines of recipient mice: 55 amplicon sequence variants (ASVs) specific to IgAV children were detected in the intestines of mice with humanized microbiota from IgAV children, and 35 ASVs specific to healthy children were detected in the intestines of mice with humanized microbiota from healthy children.

[0178] 9.2 Species composition analysis

[0179] The composition of intestinal microorganisms in mice with humanized microbiota from IgAV children and mice with humanized microbiota from healthy children before and after fecal microbiota transplantation was different at the phylum, family, and genus levels: at the phylum level, the abundance of Firmicutes (rIMb: 39.50% → 45.60%, rHMb: 33.59% → 34.61%) and Proteobacteria (rIMb: 3.33% → 4.61%, rHMb: 2.56% → 5.78%) increased in the mice after fecal microbiota transplantation, and the abundance of Bacteroidetes (rIMb: 53.59% → 46.99%, rHMb: 60.53% → 54.2 9%), the abundance of Actinobacteria in the intestines of mice with human microbiota from children with IgAV decreased (1.30%→0.81%), while the abundance of Actinobacteria in the intestines of mice with human microbiota from healthy children increased (1.52%→4.61%). At the family level, mice after fecal microbiota transplantation showed intestinal S24-7 (rIMb: 46.65%→40.77%, rHMb: 49.41%→46.04%), Lachnospiraceae (rIMb: 3.34%→1.81%, rHMb: 4.59%→4.04%), [Paraprevotellaceae] ae] (rIMb: 1.34% → 0.75%, rHMb: 3.76% → 2.11%), and the abundance of Lactobacillaceae increased (rIMb: 28.29% → 36.99%, rHMb: 20.14% → 22.53%). The abundance of Rikenellaceae in the intestines of mice with humanized microbiota from children with IgAV decreased (2.42% → 1.88%), while the abundance of Rikenellaceae in the intestines of mice with humanized microbiota from healthy children increased (2.49% → 2.71%). At the genus level, the mice after fecal microbiota transplantation all showed intestinal Lactobacillus (rIMb: 27.86% → 3 The abundance of [Prevotella] in the intestinal tract of mice with human microbiota from children with IgAV increased (0.79%→0.94%), while the abundance of [Prevotella] in the intestinal tract of mice with human microbiota from healthy children decreased (0.96%→0.81%) (see Figure 4 ).

[0180] After fecal microbiota transplantation, the composition of intestinal microorganisms in the IgAV children's humanized microbiota mice and the healthy children's humanized microbiota mice differed at the phylum, family, and genus levels (see Tables 9, 10, and 11). At the phylum level, the abundance of Firmicutes in the intestine of the IgAV children's humanized microbiota mice group was higher than that of the healthy children's humanized microbiota mice, and there was no significant difference between the groups. The abundance of Bacteroidetes, Proteobacteria, and Actinobacteria in the intestine of the IgAV children's humanized microbiota mice was lower than that of the healthy children's humanized microbiota mice, and the difference in the abundance of Actinobacteria was statistically significant (P<0.05). At the family level, the abundance of Lactobacillaceae in the intestine of the IgAV children's humanized microbiota mice was higher than that of the healthy children's humanized microbiota mice, and there was no significant difference between the groups. The abundance of S24-7, Helicobacteraceae, Lachnospiraceae, Rikenellaceae, and Rubracisaceae in the intestines of mice with human flora from gAV children were lower than those of mice with human flora from healthy children, and the difference in the abundance of Rubracisaceae was statistically significant (P<0.05). At the genus level, the abundance of Lactobacillus in the intestines of mice with human flora from IgAV children was higher than that of mice with human flora from healthy children, but there was no statistically significant difference between the groups. The abundance of [Prevotella], Allobaculum, Desulfovibrio, and Adlercreutzia in mice with human flora from IgAV children were lower than those of mice with human flora from healthy children, and the difference in the abundance of Adlercreutzia was statistically significant (P<0.05).

[0181] Table 9 Comparison of intestinal microbial abundance at the phylum level between the two groups of human bacterial flora mice

[0182]

[0183]

[0184] Note: a means the statistical method used was the independent sample t-test, b means the statistical method used was the Wilcoxon signed rank test, and P < 0.05 indicates statistical significance.

[0185] Table 10 Comparison of intestinal microbial abundance at the mouse family level between the two groups of human-derived flora

[0186]

[0187]

[0188] Note: a means the statistical method used was the independent sample t-test, b means the statistical method used was the Wilcoxon signed rank test, and P < 0.05 indicates statistical significance.

[0189] Table 11 Comparison of intestinal microbial abundance at the genus level in two groups of human-derived microbiota

[0190]

[0191] Note: a means the statistical method used was the independent sample t-test, b means the statistical method used was the Wilcoxon signed rank test, and P < 0.05 indicates statistical significance.

[0192] 9.3 Random Forest Analysis

[0193] In order to clarify the marker species that cause differences in the intestinal microbiome between IgAV children's human flora mice and healthy children's human flora mice, random forest analysis was used to analyze the differences in intestinal microbial composition between IgAV children's human flora mice and healthy children's human flora mice, so as to find the marker intestinal microorganisms that cause differences between the groups and arrange them according to importance (importance value>0.001 can distinguish the differences between the groups). Heat maps were used to display the abundance distribution of related intestinal microorganisms, and box plots were used to display the abundance of marker intestinal microorganisms in the intestines of IgAV children's human flora mice and healthy children's human flora mice.

[0194] The results showed that at the family level, the abundance of Alcaligenaceae and Coriobacteriaceae in the intestines of mice with human microbiota from children with IgAV was significantly lower than that of mice with human microbiota from healthy children, and the difference was statistically significant (P<0.05). The importance value of Alcaligenaceae was 0.180, and the importance value of Coriobacteriaceae was 0.146 (see Figure 5 ); at the genus level, the abundance of Adlercreutzia and Sutterella in the intestines of mice with human microbiota from children with IgAV was significantly lower than that of mice with human microbiota from healthy children, and the difference was statistically significant (P<0.05). The importance value of Adlercreutzia was 0.129 and that of Sutterella was 0.073 (see Figure 6 ).

[0195] 9.4 Abundance of mouse-differentiated gut microbes in the human gut

[0196] In order to clarify the abundance of characteristic intestinal microorganisms in the human intestine that can be used to distinguish between mice with human flora in IgAV children and mice with human flora in healthy children, the fecal microbiota sequencing results of 16 children with recurrent IgAV (IgAV group) and 23 healthy children (HC group) were selected for further analysis. There were no significant differences in gender and age between the two groups. The relative abundance of differential intestinal microorganisms used to distinguish between IgAV children's human flora mice and healthy children's human flora mice in the IgAV group and the HC group was compared at the family level. The results showed that compared with healthy children, the abundance of Burkholderiaceae in the feces of IgAV children was significantly reduced at the family level (P < 0.05) [due to the difference in species annotation systems, 16srDNA sequencing of human fecal specimens classified members of the Alcaligenes family (Sutterella and Parasutterella) into Burkholderiaceae, so Alcaligenes family is not shown here], and the abundance of Erysipelotrichaceae, Erysipelotrichaceae, and Enterobacteriaceae increased. The abundance differences of Veillonellaceae and Enterobacteriaceae between the two groups were statistically significant (P < 0.05) (see Figure 7 ); at the genus level, the abundance of Coprobacillus in the feces of children with IgAV was higher than that of healthy children (P<0.05), and the abundance of Sutterella was lower than that of healthy children (P>0.05). There was no significant difference in the other bacterial genera between the two groups.

[0197] 9.5 ROC Curve Analysis

[0198] To identify and quantify the potential diagnostic ability of Alcaligenesaceae for IgAV that are significantly differentially expressed in both mouse and human intestines, we plotted ROC curves based on their relative abundance in the human intestine (see Figure 8 ), the results suggested that Burkholderiaceae could well identify IgAV (specificity and sensitivity were 91.3% and 62.5%, respectively, and AUC was 0.766).

[0199] 9.6 Analysis of Burkholderiaceae composition in human feces

[0200] To further evaluate the composition of Burkholderiaceae in human fecal samples, pie charts were drawn to show their distribution (see Fig. 9 , Fig.10The results showed that Sutterella and Parasutterella (both belong to the Alcaligenes family) accounted for 99.7% and 99.9% of the Burkholderiaceae in the feces of children with IgAV and healthy children, respectively. Comparison between the two groups revealed that the abundance of Sutterella and Parasutterella in the feces of children with IgAV was lower than that of healthy children (see Fig.11 ).

[0201] 9.7 Mouse serum IgA and cytokine detection

[0202] ELISA test results are shown in Fig.12 The results showed that compared with mice with humanized flora from healthy children, the serum IgA, BAFF, IL-17A, and IL-21 levels of mice with humanized flora from children with IgAV were increased, and the serum TNF-α, IL-1β, and IL-6 were decreased. Among them, the differences in IL-17A and IL-21 between the groups were statistically significant (P<0.05).

[0203] According to the ROC curve ( Fig.13 , Fig.14 ), IL-17A and IL-21 can better identify IgAV children with human flora mice, and the optimal critical points of IL-17A and IL-21 are 25.10pg / ml and 89.95pg / ml, respectively. When IL-17A ≥ 25.10pg / ml and IL-21 ≥ 89.95pg / ml were classified as IgAV children with human flora mice, the prediction effect was the best.

[0204] 10 Conclusion

[0205] Alcaligenes family is a marker intestinal microorganism that causes differences between the humanized microbiota mice of IgAV children and the humanized microbiota mice of healthy children, and is also a characteristic intestinal microorganism of IgAV children and IgAV children humanized microbiota mice. By transplanting the characteristic intestinal microorganisms of IgAV children into the intestines of C57BL / 6J (SPF grade) mice, IgAV humanized microbiota mice can be constructed, and the serum IL-17A and IL-21 levels of the humanized microbiota mice are significantly increased. The combined application of Burkholderiaceae, IL-17A and IL-21 indicators to the judgment of the IgAV humanized microbiota mouse model can improve the accuracy of the judgment, and the AUC of the ROC curve can reach more than 0.95. The established mouse model lays the foundation for subsequent studies on the mechanism of intestinal microorganisms involved in the occurrence and development of IgAV, and also provides a screening model for IgAV therapeutic drugs.

[0206] Example 3

[0207] 1. Preparation of fecal bacteria capsules

[0208] (1) Collect 100 g of feces from children with recurrent IgAV, add 200 ml of normal saline, and filter to produce 180 ml of fecal bacterial suspension.

[0209] The structure of the collected fecal flora was as follows: at the family level, the abundance of Burkholderiaceae was 1.37%, the abundance of Rhodotorulaceae, Veillonellaceae and Enterobacteriaceae were 0.16%, 0.28% and 14.38% respectively; at the genus level, the abundance of Faecalibacterium was 0.12‰ and the abundance of Sutterella was 8.22‰.

[0210] (2) Every 200 ml of fecal bacteria suspension was mixed evenly with 40 ml of 100% glycerol, centrifuged at 400 g / min for 20 min, and the supernatant was discarded.

[0211] (3) The precipitate was centrifuged again at 10,000 g / min for 30 min at 2°C-8°C and the supernatant was discarded.

[0212] (4) The final precipitate is mixed, placed into an acid-resistant No. 2 capsule (volume about 0.37 ml) and sealed.

[0213] (5) The prepared capsules were quickly frozen on dry ice at -55°C and stored at -70°C.

[0214] Establishment of 2IgAV mouse model

[0215] Model group: The capsules were restored to room temperature, and SPF-grade C57BL / 6J mice were gavaged at a dose of 200 μl of capsule solution per mouse, once a day for 3 consecutive days.

[0216] Control group: SPF-grade C57BL / 6J mice were gavaged with 200 μl of normal saline per mouse, once a day for 3 consecutive days.

[0217] After the first oral administration, mice in both the model group and the control group were fed continuously for 7 days.

[0218] 3 Model Detection

[0219] Feces and serum of mice in the model group and control group were collected. Compared with the control group, Alcaligenes bacteria could be detected in the feces of mice in the model group. Serum IL-17A>25.10pg / ml and IL-21>89.95pg / ml of mice in the model group were higher than those in the control group.

[0220] Example 4

[0221] 1. Preparation of fecal bacteria capsules

[0222] (1) Collect 80 g of feces from children with recurrent IgAV, add 200 ml of normal saline, and filter to produce 180 ml of fecal bacterial suspension.

[0223] (2) Every 200 ml of fecal bacteria suspension was mixed evenly with 40 ml of 100% glycerol, centrifuged at 400 g / min for 20 min, and the supernatant was discarded.

[0224] (3) The precipitate was centrifuged again at 10,000 g / min for 30 min at 2°C-8°C and the supernatant was discarded.

[0225] (4) The final precipitate was mixed and placed into a No. 1 gelatin capsule (volume about 0.50 ml), and then resealed twice using a No. 0 capsule (volume about 0.68 ml) and a No. 00 capsule (volume about 0.93 ml), respectively.

[0226] (5) The prepared capsules were quickly frozen on dry ice at -55°C and stored at -70°C.

[0227] Establishment of 2IgAV mouse model

[0228] The capsules were returned to room temperature and administered to SPF C57BL / 6J mice by gavage at a dose of 250 μl of capsule solution per mouse, once a day for 3 consecutive days. After the first gavage, the mice were fed for 10 consecutive days.

[0229] Example 5

[0230] 1. Preparation of fecal bacteria capsules

[0231] (1) 90 g of feces was collected from children with recurrent IgAV, added to 200 ml of normal saline, and filtered to produce 180 ml of fecal bacterial suspension.

[0232] (2) Every 200 ml of fecal bacteria suspension was mixed evenly with 40 ml of 100% glycerol, centrifuged at 400 g / min for 20 min, and the supernatant was discarded.

[0233] (3) The precipitate was centrifuged again at 10,000 g / min for 30 min at 2°C-8°C and the supernatant was discarded.

[0234] (4) The final precipitate is mixed, placed in a No. 0 gelatin capsule (volume of about 0.68 ml) and sealed, and then sealed again with a No. 00 acid-resistant hydroxypropyl methylcellulose (HPMC) capsule (volume of about 0.93 ml). HPMC capsules can help protect dietary supplement ingredients from gastric acid without the cost and complexity of adding acid resistance properties during the manufacturing process, and can also help mask taste and odor.

[0235] (5) The prepared capsules were quickly frozen on dry ice at -55°C and stored at -70°C.

[0236] Establishment of 2IgAV mouse model

[0237] The capsules were returned to room temperature and gavage was performed on SPF-grade C57BL / 6J mice at a dose of 230 μl of capsule solution per mouse, once a day for 3 consecutive days. After the first gavage, the mice were fed for 9 consecutive days.

[0238] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. A method for constructing a human-derived bacterial flora mouse model of IgA vasculitis, including the construction of an initial IgA vasculitis model and a recurrent IgA vasculitis model, characterized in that: The following steps are involved: Preparation of fecal suspension: Collect 80-100 g of feces from patients with primary or recurrent IgA vasculitis, add 180-220 ml of normal saline, and filter to obtain fecal suspension; mix the fecal suspension with glycerol at a volume ratio of (4.5-5.5):1, centrifuge, and discard the supernatant; centrifuge the precipitate again at high speed and discard the supernatant; Preparation of fecal bacteria capsules: Place the precipitate in an acid-resistant No. 2 capsule and seal it; or place the precipitate in a No. 1 gelatin capsule, and then reseal it twice with No. 0 and No. 00 capsules respectively; or place the precipitate in a No. 0 gelatin capsule and seal it, and then use a No. 00 acid-resistant hydroxypropyl methylcellulose capsule for a second seal; Fecal microbiota transplantation: The capsules prepared in the previous step were gavaged into the stomach of mice for 3-4 consecutive days; the mice were selected from SPF grade C57BL / 6J mice; the gavage volume of the mice was 200-300 μL / time, and the gavage frequency was at least once a day; Model establishment: After the first oral administration, mice were housed for 21 consecutive days with a light / dark cycle alternating between day and night, and free access to food and water; Judgment method: Detection of characteristic bacteria Prevotella, Paraprevotella , Bifidobacterium, Staphylococcus, Enterobacteriaceae, Burkholderia, Sutterella, Collinsella, Catenibacterium or one or more bacteria from the genus Fusobacterium. Characteristic bacteria from the genus Alcaligenes, Clostridium, Staphylococcus, Exiguobacterium, Paenisporosarcina , Akkermansia , Acinetobacter, Brevibacter, F16 family or Fusobacterium.

2. An application of the method for constructing a human-derived bacterial flora mouse model of IgA vasculitis as claimed in claim 1 in preparing an IgA vasculitis detection kit, characterized in that: Initial IgA vasculitis test kit includes Prevotella spp., Paraprevotella , Bifidobacterium, Clostridium and Staphylococcus content detection reagents; the recurrent IgA vasculitis detection kit includes detection reagents for the abundance of Alcaligenesaceae bacteria, detection reagents for IL-17A and detection reagents for IL-21.

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