CN110698547B - Recombinant expression Ackermanella membrane protein Amuc _1100 and application thereof - Google Patents
Recombinant expression Ackermanella membrane protein Amuc _1100 and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant expression Ackermanella membrane protein Amuc _1100 and application thereof. According to the invention, the Ackermanella membrane protein Amuc _1100 is expressed in a recombinant mode, and through animal experiments, the inventor finds that the oral administration of the Ackermanella membrane protein Amuc _1100 can obviously improve the weight of a mouse, increase the length of a colon and the depth of a crypt, and activate the proliferation of intestinal stem cells so as to protect the mouse from being damaged by colitis. The colon lamina propria lymphocytes are separated to discover that the proportion of Treg cells is obviously increased and the proportion of Th17 cells is obviously reduced, which proves that the Amuc _1100 can restore the condition of damaged colon by adjusting the proportion of the immune cells Treg/Th 17. Therefore, the protein is expected to be developed into a protein product for preventing or treating colitis.
Description
Technical Field
The invention relates to a biological prevention and control technology, in particular to recombinant expression akkermansia membrane protein Amuc _1100 and application thereof, wherein the animal oral administration recombinant expression akkermansia membrane protein Amuc _1100 can promote the proliferation of intestinal stem cells so as to effectively relieve intestinal inflammation; meanwhile, the colon fixed layer lymphocytes are separated to detect the up-regulation of the proportion of Treg cells and the reduction of the proportion of Th17 cells, and the effect of regulating immunity in the process of repairing inflammation is proved.
Background
Ackermanella (Akkermansia muciniphila) is classified as a gram-negative bacterium, strictly anaerobic, non-spore-forming bacterium belonging to the phylum Verrucomicrobia, class Verrucomicrobiae, and family Akkermanaceae. Ackermansia can use mucin as its sole carbon and nitrogen source, can be cultured under anaerobic conditions on media containing gastric mucin, and can colonize the gastrointestinal tract of many animal species. Currently, extensive research is being conducted to understand the relationship of akkermansia to obesity, diabetes and cancer. Research shows that the abundance of Ackermanella in intestinal tracts of patients with enteritis is reduced; meanwhile, ackermanomyces can also be used for resisting obesity and type 2 diabetes and reducing fat burden of the body. Besides, the Ackermanomyces can also regulate intestinal immune response under the condition of steady state of the body.
Genomic and proteomic analysis of akkermansia identified proteins encoded by specific type IV pilus gene clusters that are rich in outer membrane proteins. Wherein the Amuc _1100 protein has the highest content of 32kDa. In addition, its presence on the gene cluster associated with pilus formation indicates that it may be involved in host interactions. Studies have demonstrated that Amuc _1100 can signal TLR 2-expressing cells in a similar manner to akkermansia, and furthermore, amuc _1100 exhibits relative thermostability, and a role in signal transduction can likewise be observed. Also, recent studies have found that Amuc _1100 may be involved in regulating metabolism and relieving type ii diabetes in the case of obesity.
At present, no report exists about the effect of recombinant expression of the membrane protein on colitis, and no report exists about the immunological effect and the effect on intestinal mucosa of the Acermann bacteria membrane protein Amuc _1100 which is used for researching the effect.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a recombinant expressed Ackermanella membrane protein Amuc _1100. Experimental research proves that the membrane protein Amuc _1100 taken by animals can obviously improve enteritis symptoms by stimulating the proliferation of intestinal stem cells; separating the colon lamina propria lymphocytes to detect the proportion of Treg/Th17 cells, secreting corresponding cell factors and regulating the immune response during enteritis.
The invention also solves the technical problem of providing the probiotics containing the recombinant expression Acermann mycoderm protein Amuc _1100.
The invention also aims to solve the technical problem of providing the application of the Ackermanella membrane protein Amuc _1100 or the probiotics in the preparation of medicaments for preventing or treating colitis.
The technical scheme is as follows: in order to solve the technical problem, the invention provides a recombinant expression akkermansia membrane protein Amuc _1100, wherein the recombinant expression akkermansia membrane protein Amuc _1100 is obtained by performing induced expression and purification on a recombinant bacterium BL21-pET-32a (+) -Amuc _1100, the recombinant bacterium BL21-pET-32a (+) -Amuc _1100 is obtained by converting a recombinant plasmid pET-32a (+) -Amuc _1100 into BL21, and the recombinant plasmid pET-32a (+) -Amuc _1100 is obtained by introducing a target gene Amuc _1100 into a pET-32a (+) plasmid.
Wherein the purity of the protein is 85.00% -91.00%.
The invention also comprises probiotics containing the recombinant expression Ackermanomyces avermitilis membrane protein Amuc _1100.
The invention also comprises the application of the Acermann mycoderm protein Amuc _1100 or the probiotics in the preparation of the medicine for preventing or treating colitis.
The invention also comprises the application of the Ackermanomyces avermitilis membrane protein Amuc _1100 or the probiotics in improving the length of a colon and increasing the depth of a crypt.
The invention also comprises the application of the Ackermanella membrane protein Amuc _1100 or the probiotics in promoting the proliferation of intestinal stem cells.
The invention also comprises the application of the Ackermanella membrane protein Amuc _1100 or the probiotics in regulating the proportion of Treg/Th17 cells of colon fixed layer lymphocytes.
Has the advantages that: compared with the prior art, the invention has the following characteristics and advantages: the invention successfully recombines and expresses the membrane protein Amuc _1100 of the Acermanium; the recombinant protein is heat-resistant, participates in regulation of metabolism, resists obesity, relieves type II diabetes, and can improve intestinal inflammation and regulate immune response of organisms; the oral administration of the membrane protein can improve the length of colon and the depth of colon crypt, stimulate the proliferation of intestinal stem cells so as to repair intestinal inflammation, and enhance the intestinal immunity level by regulating the proportion of Treg and Th17 cells, and the protein is expected to be developed into a probiotic protein for preventing or treating colitis.
Drawings
FIG. 1: a verification diagram of the recombinant expression Ackermanella membrane protein Amuc _1100, wherein the expression level of Amuc _1100 in the induced expression supernatant (a first lane), the expression level of Amuc _1100 in the induced expression precipitate (a second lane), the expression level of Amuc _1100 in the non-induced expression supernatant (a third lane), the expression level of Amuc _1100 in the non-induced expression precipitate (a fourth lane) and the protein Marker (a fifth lane) are respectively 15, 20, 25, 35, 50, 70, 100 and 130KDa from top to bottom;
FIG. 2: from left to right, respectively, an electrophoresis chart of Amuc _1100 in the recombinant expression Acermanium membrane protein passes through a Ni column, an electrophoresis chart of Amuc _1100 in the induction expression precipitation passes through the Ni column (a first lane), an electrophoresis chart of Amuc _1100 in the induction expression supernatant passes through the Ni column (a second lane), an electrophoresis chart of Amuc _1100 leaked through the Amuc _1100 (a third lane), a electrophoresis chart of a pre-washing buffer solution passes through the Ni column (a fourth lane), an elution buffer solution 1 (20 mM Imi) passes through the Ni column (a fifth lane), an elution buffer solution 2 (50 mM Imi) passes through the Ni column (a sixth lane and a seventh lane), an elution buffer solution 3 (260 mM Imi) passes through the Ni column (an eighth lane and a ninth lane) and a protein Marker (a tenth lane) are respectively 10, 15, 25, 35, 45, 60, 75 and 100KDa from top to bottom;
FIG. 3: FIG. 3A Experimental flow: mice orally take the akkermansia membrane protein Amuc _1100 through intragastric gavage recombinant expression every day, and Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% is added into drinking water on the 7 th day of feeding, and the weight change of the mice of the akkermansia membrane protein Amuc _1100 through oral recombinant expression in figure 3B (the abscissa is the feeding days, the ordinate is the weight, and the unit is g); control: negative control, 100ul of PBS was gavaged daily; DSS: adding Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% into drinking water on the 7 th day of feeding; DSS + Amuc _1100: each mouse is perfused with 3ug Ackermansin Amuc _1100 per day and fed continuously for 14 days, and Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% is added into drinking water on the 5 th day;
FIG. 4 is a schematic view of: figure 4 colon appearance of group a 3 mice: after the mice take the recombinant expression Ackermanella membrane protein Amuc _1100 orally, the colon length is increased, and the bleeding is reduced; 4B, after the mice take the recombinant Ackermanella membrane protein Amuc _1100 orally, the inflammatory state of the intestinal tract is reduced, and the LPS index in the serum is reduced; grouping is as in FIG. 3;
FIG. 5:3, colon HE staining pattern of mice of 3 groups, wherein histopathological symptoms of the mice are relieved after the mice orally take the recombinant expressed Ackermansin Amuc _1100, and the depth of colon crypts is increased; mice fed phosphate buffered saline (PBS, pH = 7.4) induced intestinal inflammation with DSS, showing marked histopathological changes; epithelial cell detachment, bleeding under the mucosa and in the intestinal cavity, inflammatory cell infiltration under the mucosa and in the lamina propria, hyperplasia and thickening of the intestinal wall; the histopathological change of enteritis is obviously reduced after the recombinant expression Acermann membrane protein Amuc _1100 is fed; no obvious bleeding point and inflammatory cell infiltration can be seen, epithelial cells are relatively complete, and no obvious hyperplasia exists in the intestinal wall; grouping is as in FIG. 3;
FIG. 6: FIG. 6A 3 Colon immunofluorescence staining PCNA Panel of mice: after the mice take the recombinant expression Ackermanella membrane protein Amuc _1100 orally, the proliferation of stem cells can be stimulated, and the proliferation is shown as the increase of the number of PCNA positive cells detected by immunofluorescence (the abscissa is the group, and the ordinate is the percentage of the PCNA positive cells); FIG. 6B shows that Wnt signal pathway related genes Wnt3a and cyc are significantly up-regulated after oral administration of recombinant expressed Ackermanella membrane protein Amuc _1100 through fluorescent quantitative PCR detection; the 6C fluorescent quantitative PCR detection of intestinal stem cell marker genes Lgr5 and Bmi1 is also obviously up-regulated after the oral administration of the recombinant expressed Ackermanella film protein Amuc _1100; grouping is as in FIG. 3;
FIG. 7 is a schematic view of: FIG. 7A Experimental flow: mice orally take the recombinant Ackermanella membrane protein Amuc _1100 by intragastric administration every day and add Dextran Sulfate Sodium (DSS) with the mass percentage of 3.5% in drinking water on the 7 th day of feeding; figure 7B flow staining detects the proportion of immune cell tregs; FIG. 7C flow staining to detect the proportion of Th17 immune cells; after the cell mice take the recombinant Acermann membrane protein Amuc _1100 orally, the cell mice induce the up-regulation of Treg cells and the reduction of Th17 cells, regulate the proportion of the Treg/Th17 cells and regulate the immune state after being damaged; control: negative control, 100ul of PBS was gavaged daily; DSS: adding Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% into drinking water on the 7 th day of feeding; amuc _1100: each mouse was gavaged with 3ug of akkermansia mycoderm protein Amuc _1100 every day, and fed continuously for 14 days; DSS + Amuc _1100: each mouse was gavaged with 3ug akkermansia mycoprotein Amuc _1100 every day, fed continuously for 14 days, and added with Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% in drinking water on day 5;
FIG. 8: after the mice of FIGS. 8A and 8B take the recombinant expression Ackermanella membrane protein Amuc _1100 orally, the cytokines TGF-beta, IL-2 and IL-10 which are secreted by the colon and are related to Treg cells are all obviously up-regulated (the ordinate is the concentration of the cytokines, and the units are ng/g, pg/mg and ng/g respectively); FIG. 8C shows that the Th 17-associated cytokines IL-6 and IL-17 secreted from the colon of mice orally administered with the recombinant Aceman fungal membrane protein Amuc-1100 are significantly reduced after treatment (the ordinate is the concentration of the cytokine, and the unit is pg/mg and pg/mg, respectively); the grouping is the same as in FIG. 7.
Detailed Description
The invention is further illustrated by the following specific examples and figures. The methods used in the following examples are conventional reagents and conventional methods unless otherwise specified.
Example 1 recombinant expression of the Ackermanella membrane protein Amuc _1100
1.1 PCR amplification of the Amuc _1100 Gene in Ackermanella
The genome of Ackermanella (awarded by Liu-Li-Shi, university of Nanjing agriculture technology school) was extracted using a bacterial genomic DNA extraction kit (Tiangen, china), and the extracted bacterial genome was PCR-amplified by autonomously designing primers P1 and P2 for homologous recombination with reference to the sequence in AMUC _ RS05900 recombinant protein [ Akkermansia muciniphila ATCC BAA-835] (Gene ID: 34174504) on NCBI, P1:5 '-CATGGCTGATATCGGATCCATGAGCAATTGGATTACAGACAAGC-3' P2: 5's of CGAGTGCGGCCGCAAGCTTTTAATCTTCAGAGCGGTTCCTGAGCC-3
The above primers were synthesized by Nanjing Kinseri company using 25. Mu.l of a system (12.5. Mu.l BU-Taq 2 × Master PCR mix (Takara, china), 2. Mu.l of template DNA (bacterial genome), 1. Mu.l of each of upstream and downstream primers, 9.5. Mu.l of ddH 2 O) was performed on a Biometra PCR instrument (Bio-Rad, USA) and the reaction program was: pre-denaturation at 95 ℃ for 3min, 30s at 94 ℃ for 1min, and 72 ℃ for 1min, 30 cycles in total, and final extension at 72 ℃ for 15min.
And (3) carrying out electrophoresis on the amplified product through 1% agarose gel, quickly cutting gel containing the target fragment under an ultraviolet lamp, and carrying out operation recovery according to the instruction of a DNA gel recovery kit (Biyuntian), so as to obtain the target gene.
1.2 construction of recombinant expression plasmids
After the target gene and a vector pET-32a (+) are subjected to enzyme digestion and recovery respectively, the molar ratio of the target gene to the vector pET-32a (+) is 6:1, mixing, connecting by T4 DNA ligase for 1h at 22.5 ℃, and transforming competent cell DH5 alpha. The transformant DH 5. Alpha. Was inoculated into LB solid medium containing 50. Mu.g/mL ampicillin and cultured at 37 ℃ for 12 hours. Individual colonies were picked, inoculated into LB liquid medium containing 100. Mu.g/mL ampicillin, and cultured at 37 ℃ for 12 hours. Plasmid DNA was extracted with a small plasmid extraction kit (Biyuntian), and subjected to BamHI and HindIII double digestion analysis and PCR identification.
1.3 inducible expression and purification of Amuc _1100gene
Extracting pET-32a (+) -Amuc _1100 plasmid which is subjected to double enzyme digestion and PCR verification to transform BL21 (DE 3) strain. Transformed BL21 (DE 3) E.coli was inoculated into 5mLLB liquid medium and cultured with shaking at 37 ℃ for 12 hours with pET-32a (+) as a negative control. The next day, the bacterial solution was inoculated into LB liquid medium at a ratio of 1: 100, cultured at 37 ℃ until OD600 value became 0.7. + -. 0.1, IPTG was added to a final concentration of 1mmol/L, shaking culture was continued at 37 ℃ for 4 hours, an appropriate amount of the induced expression bacterial solution and the control bacterial solution (i.e., non-induced bacterial solution) were taken, analyzed by 10% SDS-PAGE, stained with Coomassie Brilliant blue R250 and decolored, and the results were observed. As can be seen from FIG. 1, the induction expression supernatant has a large amount of Amuc _1100 expression, a small amount of Amuc _1100 expression is expressed in the precipitate, which proves successful induction expression, and then purification is carried out, specifically as follows:
four buffers were prepared in advance, respectively: and (3) an equilibrium buffer: 20mM Tris-HCl,300mM NaCl, pH8.0; prewashing buffer solution: 20mM Tris-HCl,300mM NaCl, pH8.0; elution buffer 1:20mM Tris-HCl,300mM NaCl,20mM Imi (imidazole), pH8.0; elution buffer 2:20mM Tris-HCl,300mM NaCl,50mM Imi (imidazole), pH8.0; elution buffer 3:20mM Tris-HCl,300mM NaCl,260mM Imi (imidazole), pH8.0; dialysis buffer: 20mM Tris-HCl, pH8.0.
Protein purification process: ultrasonically crushing the transformed BL21 (DE 3) escherichia coli thallus, and ultrasonically crushing (400 w/min) for 10min at the temperature of 4 ℃, and stopping for 6s every 4 s. Centrifuging the crushed bacterial liquid for 15min at the temperature of 4 ℃ (12000 rpm/min), and purifying the supernatant; the column chromatography purification method was used, GE Ni Sepharose 6Fast Flow (Code No. 10257810) (Sigma, USA) chromatography packing, volume 3.0ml; 30ml of deionized water with 10 times of column volume and 30ml of equilibrium buffer with 10 times of column volume are used for equilibrium packing, and the flow rate is 0.5ml/min; sample loading is carried out after balance, and the flow rate is 0.5ml/min; after protein adsorption, using 30ml of prewashing buffer solution with 10 times of column volume to wash the filler, wherein the flow rate is 0.5ml/min; sequentially eluting the Ni filler step by step according to the sequence of the elution buffer solutions 1, 2 and 3, collecting eluent of each part, tracking the protein elution condition by using a Coomassie brilliant blue method in the elution process, and stopping collecting when the color is changed to negative control; 20ul of each component is taken, 5ul of 5 xSDS Loading Buffer is added, the mixture is heated at 100 ℃ for 10min, and SDS-PAGE analysis is carried out, as shown in figure 2, and the content of the target protein in the target protein supernatant is high, but the His label is not fully exposed, so that fewer hanging columns are formed, and most of the target protein flows through. The purity of the target protein in the eluate in lane 50-2 of FIG. 2 reaches 90.93%, and it is directly dialyzed, while the purity of the target protein in the other lanes from left to right (i.e. lane 20, lane 50-1, lane 260) is 33.04%, 65.17%, 80.79%, and 84.55%, respectively, and the purity of the target protein is insufficient and is not used. The proteins in lane 50-2 were then dialyzed to remove salt ions and then quantitated (0.9 mg/ml, quantitation by Coomassie Brilliant blue) and frozen.
Example 2 testing of the Effect of oral recombinant expression of Acermann membrane protein Amuc _1100 on colitis in mice
Mice (strain C57BL/6, purchased from the university of promiscuous comparative animal medicine center) were gavaged daily with 3ug of the membrane protein Amuc — 1100 purified according to example 1 (protein purity 91%), and colitis was induced by addition of 3.5% by mass Dextran Sodium Sulfate (DSS) to the drinking water after 7 days of continuous feeding, during which time changes in body weight of the mice were recorded. Mice sacrificed by cervical dislocation after a total of 14 days to observe changes in colon length and perform HE staining of colon for crypt depth and histopathological changes.
The oral administration of the membrane protein Amuc _1100 in mice is found to increase the weight, the length of colon and the intestinal inflammation caused by DSS. Control: negative control, 100ul of PBS was gavaged daily; DSS: adding Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% into drinking water on the 7 th day of feeding; DSS + Amuc _1100: each mouse is perfused with 3ug Ackermansin Amuc _1100 per day and fed continuously for 14 days, and Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% is added into drinking water on the 5 th day; as shown in fig. 3 and 4, compared with the DSS injured group, the membrane protein Amuc _1100 repair group showed a slight increase in body weight, an increase in colon length of about 33.3%, a decrease in inflammation index LPS of about 25%, and a reduction in colitis symptoms; as shown in figure 5, the histopathological symptoms of the colitis of the mice are relieved after the oral administration of the membrane protein Amuc _1100, and the depth of the crypt is increased by about 50 percent. After the mice in the injured group are fed with DSS, obvious enteritis histopathological changes are shown: epithelial cell detachment, bleeding under the mucosa and in the intestinal lumen, infiltration of inflammatory cells under the mucosa and in the lamina propria, thickening of the intestinal wall hyperplasia. The pathological change of enteritis tissue is obviously reduced after the membrane protein Amuc _1100 is fed to a membrane protein Amuc _1100 repairing group: there are no bleeding points and inflammatory cell infiltration, the epithelial cells are relatively intact, and the intestinal wall has no obvious hyperplasia.
Example 3 examination of the Effect of oral administration of the recombinant Acermanium membrane protein Amuc _1100 on mouse intestinal Stem cells
Mice (strain C57BL/6, purchased from the university of promiscuous comparative animal medicine center) were gavaged daily for 3ug of the purified membrane protein Amuc _1100 of example 1 (protein purity 91%), fed continuously for 7 days and then treated with 3.5% by mass Dextran Sodium Sulfate (DSS) added to the drinking water to induce colitis. After 14 days, the mice are killed by dislocation of cervical vertebrae to extract colon RNA, and the colon is subjected to immunofluorescence staining to detect the expression of genes related to proliferation.
As shown in fig. 6, the Wnt3a and cyc genes were detected by fluorescent quantitative PCR, and the gene expression level of the mice was significantly increased after oral administration of akkermansia membrane protein Amuc _1100 for repair, indicating that the Wnt signaling pathway was activated, and the active stem cell marker Lgr5 and the quiescent stem cell marker Bmi1 genes were significantly upregulated, indicating that the intestinal stem cells of the mice were significantly proliferated. Meanwhile, intestinal PCNA positive cells are counted through immunofluorescence, the number of proliferating cells of a mouse at a crypt is remarkably increased (9.4% +/-1.077) after the oral administration of the membrane protein Amuc _1100, and the fact that the oral administration of the recombinant expression Acermann mycodermic protein Amuc _1100 can regulate and control the proliferation of intestinal stem cells through activating a Wnt signal path is further proved.
Example 4 detection of changes in immune cells and their associated cytokines in lymphocytes in the colon lamina propria
4.1 isolation of lamina propria lymphocytes
4.1.1 in the absence of Ca 2+ 、Mg 2+ To 100ml of Hanks equilibrium solution (Yuansei, china), 5g of bovine serum albumin (Sigma, USA), 58mg of EDTA, and 15.4mg of dithiothreitol (Sigma, USA) were added to prepare a separation solution. To 100ml of PBS were added 5g of bovine serum albumin, 0.15g of collagenase type VIII (Sigma, USA) and 100U of DNase I (Sigma, USA), and the mixture was incubated at 37 ℃ for 5min to prepare a digestion solution. Percoll cell separation medium (Unico, china) and 10 XPBS according to the volume ratio of 9:1, mixing and preparing 100% isotonic Percoll mother liquor. The mixture is prepared by Percoll mother liquor, DMEM high-sugar solution and FCS according to the volume ratio of 8:1:1 and 4:5:1 and preparing 80% isotonic Percoll solution and 40% isotonic Percoll solution.
4.1.2 cervical dislocation method to sacrifice mice, immediately taking out about 7-8cm of colon tissue, placing in precooled calcium and magnesium-free PBS (PBS) -/- ) Removing fat, mesenteric connective tissue and collecting lymph nodes of small intestine; the intestine was longitudinally sectioned along one mesenteric side, gently rinsed in calcium-and magnesium-free, precooled PBS until the feces were completely rinsed, and transversely sectioned into intestinal tissue segments of about 0.5-1.0 cm.
4.1.3 transferring the intestinal tissue segment into a 50ml centrifuge tube, adding 5ml of separation solution, placing in a constant temperature shaking box, and shaking (250 r/min) at 37 ℃ for 15min; placing on a vortex mixer, vortexing for 30s, and filtering the vortexed intestinal tissue segment through a 100 μm nylon filter screen, wherein the filtrate is intestinal intraepithelial lymphocyte and intestinal epithelial cell; the filtered intestinal tissue fragments were transferred into 50ml centrifuge tubes again, 5ml of separation medium was added and the above shaking, vortexing and filtering steps were repeated.
4.1.4 transferring the intestinal tissue fragments after filtration treatment into a new 50ml centrifuge tube, adding 5ml of digestive juice, placing in a constant temperature shaking box, and shaking (250 r/min) at 37 ℃ for 45min; the mixture was vortexed for 30 seconds in a vortex mixer, the vortexed intestinal tissue fragments were filtered through a 100 μm nylon filter, the filtrate was collected in a 15ml centrifuge tube, centrifuged (400 g) at 4 ℃ for 10min, and the supernatant was discarded, whereupon pellets containing Lamina Propria Lymphocytes (LPLs).
4.1.5 spreading 4ml of 80% isotonic Percoll solution on the bottom of a new 15ml centrifuge tube; resuspending the precipitate with 8ml of 40% isotonic Percoll solution, blowing thoroughly and uniformly, spreading on 80% isotonic Percoll solution, inclining the centrifugal tube by 180 degrees to slowly add the liquid along the tube wall, and forming a clear interface between the two layers of liquid; density gradient centrifugation (500 Xg) for 20min; discarding the supernatant until the residual volume is 7ml, sucking out the opaque cell layer between the two layers, transferring into a new 15ml centrifuge tube, adding PBS -/- The volume is 15ml, after fully mixing, centrifuging (400 Xg) for 8min at 4 ℃, discarding the supernatant, resuspending the sediment by 5ml of RPMI 1640 culture solution containing 10% by mass of fetal calf serum, fully and uniformly blowing and beating to prepare cell suspension, and then obtaining the lamina propria lymphocytes.
4.2 flow cytostaining assay
4.2.1 resuspension of the collected colon lamina propria lymphocytes with 100. Mu.l of 0.01MPBS after centrifugation;
4.2.2 surface molecules are usually first labeled and approximately 1. Mu.l FITC-CD4 antibody (BioLegend, USA) is added to 100. Mu.l PBS buffer from the previous step. Incubating at 4 deg.C in dark for 30min;
4.2.3 washing with PBS buffer solution, centrifuging and discarding 1500rmp of washing liquid, centrifuging for 5 minutes;
4.2.4 preparing a membrane breaking solution, and diluting 1 part of fixed membrane breaking concentrated solution (Invitrogen, USA) into 3 parts of fixed membrane breaking diluent (Invitrogen, USA) to a desired volume of working solution; the membrane-breaking buffer (Invitrogen, usa) is 10-fold concentrated and should be diluted with distilled water to a 1-fold solution before use;
4.2.5 suspending the cell pellet, adding 300. Mu.l of prepared fresh rupture of membranes working solution to each sample, and suspending the solution again;
4.2.6 incubation in the dark at 4 ℃ for 30min;
4.2.7 washing the cells with 1ml of membrane-breaking buffer (Invitrogen, USA), centrifuging and discarding the supernatant; 2500rmp centrifugation for 5min
4.2.8 then add 1. Mu.l of mouse PE-FOXP3 antibody (BioLegend, USA) or PE-IL-17A antibody (BioLegend, USA) to 100. Mu.l of buffer solution, divide into two different groups of stained cells, incubate for at least 30min at 4 ℃ in the dark;
4.2.9 washing the cells with 1ml of membrane breaking buffer, centrifuging and discarding the supernatant; 3500rmp centrifugation, 5min
4.2.10 suspend appropriate flow cytometric staining buffer volumes, detected and recorded on the up-flow cytometer;
4.2.11 statistical analysis was performed using FlowJo software.
Control: negative control, 100ul of PBS was gavaged daily; DSS: adding Dextran Sodium Sulfate (DSS) with the mass percentage of 3.5% into drinking water on the 7 th day of feeding; amuc _1100: each mouse was gavaged daily for 3ug of akkermansia membrane protein Amuc _1100 (protein purity 91%) prepared in example 1, and fed continuously for 14 days; DSS + Amuc _1100: example of gavage each mouse daily 1.3 ug of akkermansia membrane protein Amuc _1100 purified according to the present patent was fed continuously for 14 days, and Dextran Sulfate Sodium (DSS) was added to the drinking water at 3.5% by mass on day 5; the statistical result shows that, as shown in fig. 7, the flow detection result shows that the mouse oral membrane protein Amuc _1100 can significantly improve the CD4 in the colon fixed layer lymphocytes + FOXP3 + Proportion of Treg cells (around 50%) and downregulation of CD4 + IL-17A + The proportion (about 50%) of Th17 cells proves that the oral recombinant Acermann mycoderm protein Amuc _1100 can regulate the proportion of immune cells Treg/Th17 in intestinal tracts so as to relieve intestinal inflammation.
4.3 expression assay for Treg and Th17 associated cytokines
The protein sample at the colon is extracted by PMSF through PBS +1 percent, the change of the cell factors at the colon is detected by enzyme-linked immunosorbent assay (ELISA), as shown in figure 8, TGF-beta, IL-2 and IL-10 are all relevant cell factors secreted by Treg cells, and are remarkably increased after the Acermann mycodermic protein Amuc-1100 is treated, and the cell factors IL-6 and IL-17 secreted by Th17 cells are remarkably reduced after the Acermann mycodermic protein Amuc-1100 is treated, which indicates that the cell factors secreted by the cell are regulated by the Amuc-1100 so as to relieve the intestinal inflammation caused by DSS.
Sequence listing
<110> Nanjing university of agriculture
<120> recombinant expression Ackermanella membrane protein Amuc _1100 and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 47
<212> DNA
<213> upstream primer P1 (Artificial Sequence)
<400> 1
catggctgat atcggatcca tgagcaattg gattacagac aacaagc 47
<210> 2
<211> 44
<212> DNA
<213> downstream primer P2 (Artificial Sequence)
<400> 2
cgagtgcggc cgcaagcttt taatcttcag acggttcctg agcc 44
<210> 3
<211> 954
<212> DNA
<213> Amuc_1100
<400> 3
atgagcaatt ggattacaga caacaagccc gccgccatgg tcgcgggcgt gggacttctc 60
ttattcctgg ggttatccgc gacagggtac atcgtcaatt ccaaacgcag tgaactggac 120
aaaaaaatca gcatcgccgc caaggaaatc aagtccgcca atgctgcgga aatcactccg 180
agccgatcat ccaacgaaga gctggaaaaa gaactgaacc gctatgccaa ggccgtgggc 240
agcctggaaa cggcctacaa gcccttcctt gcctcctccg cgctggtccc caccacgccc 300
acggcattcc agaatgaact gaaaacattc agggattccc tgatctcctc ctgcaagaaa 360
aagaacattc tcataacgga cacatcctcc tggctcggtt tccaggttta cagcacccag 420
gctccctctg ttcaggcggc ctccacgctg ggttttgaat tgaaagccat caacagcctg 480
gtcaacaaac tggcggaatg cggcctgtcc aaattcatca aggtgtaccg cccccagctc 540
cccattgaaa ccccggcgaa caatccggaa gaatcggacg aagccgacca ggccccatgg 600
actcccatgc ctctggaaat agccttccag ggcgaccggg aaagtgtatt gaaagccatg 660
aacgccataa ccggcatgca ggactatctg ttcacggtca actccatccg tatccgcaac 720
gaacggatga tgccccctcc catcgccaat ccggcagccg ccaaacctgc cgcggcccaa 780
cccgccacgg gtgcggcttc cctgactccg gcggatgagg cggctgcacc tgcagccccg 840
gccatccagc aagtcatcaa gccttacatg ggcaaggagc aggtctttgt ccaggtctcc 900
ctgaatctgg tccacttcaa ccagcccaag gctcaggaac cgtctgaaga ttaa 954
Claims (1)
1. The application of the recombinant expression akkermansia membrane protein Amuc _1100 or probiotics containing the recombinant expression akkermansia membrane protein Amuc _1100 in preparing a medicine for treating colitis is characterized in that the recombinant expression akkermansia membrane protein Amuc _1100 is obtained by inducing, expressing and purifying a recombinant bacterium BL21-pET-32a (+) -Amuc _1100, the recombinant bacterium BL21-pET-32a (+) -Amuc _1100 is obtained by converting a recombinant plasmid pET-32a (+) -Amuc _1100 into BL21, the recombinant plasmid pET-32a (+) -Amuc _1100 is obtained by introducing a target gene Amuc _1100 into a pET-32a (+) plasmid, the purity of the protein is 85.00% -91.00%, and the coding nucleotide sequence of the recombinant expression akmansia membrane protein Amuc _1100 is shown as SEQ ID NO:3, respectively.
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| CN111450233B (en) * | 2020-03-19 | 2021-04-13 | 中国农业大学 | Application of microbial metabolite in relieving glycolipid metabolic disorder |
| CN111690044B (en) * | 2020-05-06 | 2022-03-01 | 中南大学湘雅二医院 | Application of Amuc1100 protein |
| CN113278643B (en) * | 2021-05-19 | 2022-08-23 | 中国农业科学院饲料研究所 | Method for constructing genetic engineering bacteria with functions of reducing liver fat accumulation and protecting intestinal barrier and application |
| WO2023093883A1 (en) * | 2021-11-26 | 2023-06-01 | 和度生物技术(上海)有限公司 | Genetically modified microorganism and use thereof |
| CN114369146B (en) * | 2022-01-14 | 2023-05-23 | 上海交通大学医学院附属仁济医院 | Acremonium Amuc-2172 protein and preparation method and application thereof |
| CN117065002B (en) * | 2023-02-02 | 2024-03-22 | 浙江大学 | Application of Acremonium membrane protein Amuc_1100 in preparation of product for preventing and/or treating age-related cognitive decline |
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Non-Patent Citations (2)
| Title |
|---|
| Akkermansia muciniphila ATCC BAA-835, complete genome;GenBank;《GenBank》;20131211;Sequence ID:CP001071.1 * |
| hypothetical protein Amuc_1100 [Akkermansia muciniphila ATCC BAA-835];GenBank;《GenBank》;20131211;GenBank: ACD04926.1 * |
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