CN108339111B - Medical application of MG53 protein - Google Patents
Medical application of MG53 protein Download PDFInfo
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- CN108339111B CN108339111B CN201810271994.9A CN201810271994A CN108339111B CN 108339111 B CN108339111 B CN 108339111B CN 201810271994 A CN201810271994 A CN 201810271994A CN 108339111 B CN108339111 B CN 108339111B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a medical application of MG53 protein, which comprises the application of recombinant human secreted MG53 lactococcus lactis in preparing medicaments, pharmaceutical compositions, foods, health-care products, food additives and the like for preventing and/or treating inflammatory bowel diseases. The in vivo and in vitro related experiments prove that the recombinant human MG53 protein has excellent prevention and treatment effects on inflammatory bowel diseases including ulcerative colitis or Crohn's disease, has no toxic or side effect, can be durably and effectively applied to preparation of medicines, pharmaceutical compositions, foods, health-care products or food additives for preventing and/or treating the inflammatory bowel diseases, can be used for preventing and treating the inflammatory bowel diseases, and has great application value.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to an application of recombinant human MG53 protein in preventing and/or treating inflammatory bowel diseases.
Background
Inflammatory Bowel Disease (IBD) is a chronic, recurrent autoimmune Disease, and is characterized by alternating periods of activity and remission, with two types, Ulcerative Colitis (ulcerous Colitis) and Crohn's Disease. Ulcerative colitis is a chronic nonspecific inflammation of the colon with diarrhea, abdominal pain and mucopurulent bloody stool as the main clinical manifestations. Crohn's disease is a chronic granulomatous inflammation with abdominal pain, diarrhea, fistulas, anal lesions and various systemic symptoms as the main clinical manifestations. In recent years, the prevalence rate of IBD is gradually increasing in China with increasingly western lifestyle. The pathogenesis of IBD is not completely understood, resulting in a low level of IBD control, causing the duration of IBD to be prolonged and repeated, affecting the quality of life of patients.
The main pathological changes in inflammatory bowel disease are extensive acute and chronic inflammation of the intestinal mucosa with ulceration, erosion and necrosis, and this pathological change in the intestinal mucosa leads to an impairment of the intestinal epithelial barrier function. The intestinal epithelial cells, which are the basis for the intestinal epithelial barrier, together with the tight and adhesive junctions between cells. Under pathological conditions, necrosis of intestinal epithelial cells can cause increase of cell permeability, so that intestinal pathogenic bacteria, endotoxin and macromolecular substances enter other tissues and organs or a circulatory system through an intestinal barrier to induce immune inflammatory reaction, and meanwhile, after inflammation occurs in the intestinal tract, further necrosis of the intestinal epithelial cells can be caused, so that the inflammatory reaction is amplified, malignant circulation is formed, and IBD is finally delayed and repeated. The repair of necrosis of intestinal epithelial cells is a major direction of research in the prevention and treatment of IBD.
Intestinal epithelial cell membrane disruption is the primary pathological basis for intestinal epithelial cell necrosis and intestinal epithelial barrier disruption. The cell membrane repair protein MG53 is one of family members of tripartite motif consisting of 477 amino acids, and the structure of the protein comprises three structural domains, namely a zinc finger structural domain, a B-box and a coiled coil structure from the N end to the C end, and the protein is specifically expressed in cardiac muscle and skeletal muscle cells. The previous research shows that after the cell membrane is damaged, MG53 can be transferred from cytoplasm to cell membrane, gather at damaged part of cell membrane, close the gap of cell membrane, and repair cell membrane. MG53 administered in the model of heart brain kidney lung injury and in the mouse model of muscular atrophy can promote cell survival through membrane repair function to achieve the effect of treating diseases, but is not reported in the repair of intestinal epithelial cells. On the other hand, the E3 ubiquitin ligase Ring finger domain in the structure of MG53 protein can play a role of E3 ubiquitin ligase, induce ubiquitination of insulin receptor 1 (IRS 1), and accelerate attenuation of IRS1, so MG53 protein can also be an important regulator in protein attenuation.
However, due to the specificity of the protein, the administration mode is limited, so that it is necessary to make a method for locally expressing the activity in the intestinal tract, having no antigenicity, being safe to use, convenient to take, facilitating the mass production of the MG53 protein, and possibly exerting a better therapeutic effect on the colon injury.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of MG53 as a marker for diagnosing or treating ulcerative colitis; the invention also aims to provide the application of the recombinant human MG53 protein in the preparation of medicaments or health-care products for preventing and/or treating inflammatory bowel diseases; the invention also aims to provide the application of recombinant lactobacillus secreting human MG53 protein in the preparation of medicaments or health-care products for preventing and treating inflammatory bowel diseases; the fourth purpose of the invention is to provide the application of the recombinant human MG53 protein in the preparation of drugs or health products for repairing the intestinal epithelial cell injury.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the invention can detect that the content of MG53 in circulating blood in an ulcerative colitis model is lower than that of a normal mouse, and the content of MG53 in colon is higher than that of the normal mouse, and the result proves that MG53 can be used as a diagnostic biomarker of the ulcerative colitis; targeted serum MG53 may be a potential therapeutic for the treatment of colon injury in ulcerative colitis. Therefore, MG53 can be used as a marker for diagnosing or treating ulcerative colitis.
2. The MG53 knock-out mouse can obviously promote colitis of mice induced by dextran sodium sulfate, aggravate the phenomena of weight reduction, diarrhea and occult blood of the mice, promote the length of colon to be shortened and reduce the survival rate of the mice with colitis by utilizing an MG53 knock-out mouse ulcerative colitis model. The recombinant rhMG53 has obvious effect on treating inflammatory bowel diseases, particularly ulcerative colitis and Crohn's disease. Therefore, the recombinant human MG53 protein can be applied to the preparation of medicaments, compositions, health products, foods or additives for preventing and/or treating inflammatory bowel diseases.
Preferably, the inflammatory bowel disease is ulcerative colitis.
Preferably, the inflammatory bowel disease is crohn's disease.
3. The recombinant rhMG53 protein and the secretory active rhMG53 streptococcus lactis are respectively injected intramuscularly or orally, can obviously inhibit mouse colitis (a recognized ulcerative colitis experimental animal model) induced by dextran sodium sulfate, improve the phenomena of weight reduction, diarrhea and occult blood of the mouse, inhibit the colon length from being shortened, and improve the survival rate of the mouse with colitis. The results show that the recombinant rhMG53 has good improvement effect on the mouse ulcerative colitis and can be used for preventing and treating the ulcerative colitis. In addition, the recombinant rhMG53 protein and the secretion-active rhMG53 streptococcus lactis can reduce 2,4, 6-trinitrobenzenesulfonic acid-induced colitis of mice (a recognized experimental animal model of Crohn's disease), relieve the phenomenon of weight loss, improve the survival rate and inhibit the shortening of colon length. The results show that the recombinant rhMG53 protein and the secretion-active rhMG53 streptococcus lactis have good improvement effect on the mouse Crohn's disease. Therefore, the recombinant lactobacillus secreting human MG53 protein is applied to the preparation of medicines, compositions, health products, foods or additives for preventing and treating inflammatory bowel diseases.
Preferably, the inflammatory bowel disease is ulcerative colitis.
Preferably, the inflammatory bowel disease is crohn's disease.
More preferably, the recombinant lactobacillus secreting human MG53 protein contains an expression vector for expressing MG53 gene.
More preferably, the expression vector is ligated into the pNZ8148 vector at the cloning site by the MG53 gene.
1. The recombinant human MG53 protein is applied to the preparation of drugs or health products for repairing intestinal epithelial cell injury.
The recombinant rhMG53 protein can be mixed with a pharmaceutically acceptable carrier to prepare various preparations of medicines for preventing and treating ulcerative colitis and Crohn's disease. Can be administered orally, sublingually, transdermally, by injection, instillation, mucosally, by spray, by infusion, rectally, or parenterally.
The invention has the beneficial effects that: the invention discloses MG53 and medical application of recombinant MG53 thereof, provides a new way and a new medicine for diagnosing and treating inflammatory bowel diseases, and has important significance for monitoring and treating clinical inflammatory bowel diseases.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the effect of MG53 content and location in blood and colon of ulcerative colitis model mouse (A: the effect of MG53 in serum of ulcerative colitis mouse; B: the tissue location of MG53 in colon of ulcerative colitis mouse; C: the expression level of MG53 in serum of ulcerative colitis mouse).
FIG. 2 shows the effect of MG53 knockout mouse model on body weight, activity index and survival (A: body weight; B: disease activity index; C: survival).
FIG. 3 shows the effect of recombinant rhMG53 protein on the disease activity index and survival rate of ulcerative colitis (A: colitis model; B: disease activity index; C: survival rate).
FIG. 4 shows the effect of recombinant rhMG53 protein on the colon length and pathological changes in the mouse model of ulcerative colitis (A: colon length in mouse; B: pathological changes).
FIG. 5 Effect of recombinant rhMG53 protein on body weight and survival in a model of 2,4, 6-trinitrobenzenesulfonic acid-induced colitis in mice (A: body weight; B: survival).
FIG. 6 preparation and identification of secretion-active rhMG53 Streptococcus lactis (A: vector construction; B: PCR detection; C: Westernblot detection).
FIG. 7 is a graph of the effect of secretable active rhMG53 Streptococcus lactis on survival in a model of ulcerative colitis.
FIG. 8 is a graph of the effect of secretionally active rhMG53 Streptococcus lactis on survival in a Crohn's disease model.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Example 1 preparation of ulcerative colitis model
All animals are tested by male C57BL/6 mice provided by the experimental animal center of the university of army and military medical university, the weight is 22-25 g, and the operation in the experimental process conforms to the regulations of the experimental animals of the university of army and military medical. The mice were randomly divided into a normal group and a model group, the control group was drunk with ordinary sterilized water, the model group was freely drunk with 4% Dextran Sodium Sulfate (DSS) for 7 days, and the change of the signs of the mice was observed. And (4) sampling blood for eye sockets of each group of mice on the 8 th day of model building, centrifuging for 10 minutes at the room temperature of 5000 r, and collecting supernatant serum. Reserving colon extracted tissue sample protein for preservation at a temperature of-80 ℃, fixing by 4% paraformaldehyde, conventionally extracting tissue sample protein for quantification, embedding in paraffin, slicing, and detecting the content and location of MG53 in the tissue sample, wherein the detection method of the content of serum MG53 is disclosed in Chinese patent with publication number CN 103430023A, and the result is shown in figure 1. As shown in FIG. 1, in the mice in the ulcerative colitis model group, the content of MG53 in the circulating blood was lower than that in normal mice, the content of MG53 in colon tissues was higher than that in normal mice, and MG53 was enriched in intestinal epithelial cells.
Example 2 preparation of MG53 knock-out mouse model for ulcerative colitis
MG53 raised in SPF level in the center of experimental animals in the university of army and military medical university large-area hospital is taken and knocked out of a mouse (the source of the mouse is a donation provided by professor Majianji) with the weight of 22-25 g, and the operation in the experimental process conforms to the regulations of the experimental animals in the university of army and military medical university. The mice were randomly divided into a normal group, a model group (4% DSS), and a MG53 knock-out mouse model group (4% DSS). The remaining 2 groups, except the normal control group, were free to drink 4% Dextran Sodium Sulfate (DSS) for 7 days, followed by changing to single distilled water for continued free drinking for 7 days. The effects of mouse body weight, disease activity index, survival rate were continuously observed.
Disease activity index of ulcerative colitis model mice Disease Activity Index (DAI) was used to accurately quantify inflammatory bowel disease severity, with scoring criteria: body mass index (body weight does not become 0, 1 point for 1% reduction 1% -5%, 2 point for 2% reduction 5% -10%, 3 point for 10% -15%, 4 point for more than 15%), stool character score (normal 0, loose 2 points, diarrhea 4 points), and stool bleeding score (normal 0 points, recessive bleeding 2 points, dominant bleeding 4 points).
DAI score = (body mass index + stool trait score + stool bleeding score)
The results of MG53 knock-out mouse ulcerative colitis model mouse body weight, disease activity index, survival rate are shown in fig. 2. As can be seen from fig. 2, the disease activity index of the model group mice was significantly increased from day 3, and there were weight loss, diarrhea, and as the test continued to be gradually worsened, fecal occult blood and stool blood appeared, and death began on day 9, and the mortality rate was 50% at day 14. MG53 knocks out mouse ulcerative colitis model mouse and starts disease activity index to rise remarkably from day 2, and disease activity index, weight, diarrhea, fecal occult blood and hematochezia are all aggravated compared with model group; and the survival rate started to die on day 6, with a 90% mortality rate in day 10.
Example 3 protection of DSS-induced ulcerative colitis model by recombinant rhMG53 protein
The weight of a C57 mouse bred at SPF level in the center of experimental animals in a large-lawn hospital of army-military-medical university is 22-25 g, and the operation in the experimental process accords with the regulations of the experimental animals of the army-military-medical university. The treatment groups were randomly divided into a normal group, a model group (2.5% DSS), and an rhMG53 treatment group. The remaining 2 groups were free to drink 4% Dextran Sodium Sulfate (DSS) for 7 days, except for the normal control group, and the rhMG53 treated group was given 1mg/kg protein (a gift from professor makejie) intramuscularly per day, followed by changing to single distilled water for continued free drinking for 7 days. The influence of the weight, disease activity index and survival rate of the mice was continuously observed.
Pathological scoring of ulcerative colitis model mouse colitis mouse colon tissues (day 7 after time point DSS treatment) are subjected to paraffin embedding, sectioning and HE staining, pathological damage degree is observed and scored, and scoring is marked as: ulcer (no is 0 point, less than or equal to 3mm is 1 point, more than 3mm is 2 points), inflammation (normal is 0 point, mild is 1 point, severe is 2 points), granulation tissue (no is 0 point, there is 1 point), depth of lesion (normal is 0 point, submucosa is 1 point, muscle layer is 2 points, serosal layer is 3 points), and fibrosis (normal 0 point, mild is 1 point, severe is 2 points).
The results of the recombinant rhMG53 protein protecting DSS-induced ulcerative colitis model mouse are shown in fig. 3. The mice containing 4% DSS-induced C57BL/6 strain are drunk to construct a mouse acute IBD model, and the modeling condition of the animal model is comprehensively evaluated through the disease activity index, colitis pathological scoring standard, survival rate and the like of the mice. Mice in the acute IBD model had reduced body weight and increased disease activity index compared to the negative control group (fig. 3, a and B). The rhMG53(1mg/kg) was administered in combination, and it was found that the mice in the rhMG 53-treated group did not undergo significant weight loss, stools formed, and no significant macroscopic bloody stools, as compared to the model control group. FIG. 3 shows that rhMG53 can effectively reduce the body weight disease activity index and survival rate of mice in DSS-induced acute IBD model. The MG53 is shown to have good protective effect on the ulcerative colitis model mouse.
Colon Length in mice model for ulcerative colitis Colon tissue from the ileocecal region to the anus was taken and the length of the colon was measured and the results are shown in FIG. 4. The results show that colon is shortened (figure 4, A), normal structure of pathological mucosa of colon tissue is destroyed, crypt structure is disordered (figure 4, B), and colitis pathological score of a model group is obviously higher than that of a control group (figure 4, B); the survival rate of the 4% DSS-induced ulcerative colitis model group was lower than that of the control group. The results show that the mouse acute colitis model is successfully induced. Compared with a blank control group, the DAI of the mice of the colitis modeling group is obviously increased on the 3 rd day, and the mice show weight reduction, watery stool and macroscopic bloody stool and acute moderate and severe colitis. The above results indicate that the rhMG 53-treated group can significantly inhibit colon length shortening in model mice. It can also be seen from fig. 4 that colon tissue lesions mainly affect the mucosal layer and submucosa in the DSS model group of mice, and inflammatory cell types are mainly mononuclear macrophages and neutrophils. The local mucosa in the severe inflammation area is necrosed in a whole layer to form ulcer. rhMG53(1mg/kg) significantly reduced the infiltration of mononuclear macrophages and neutrophils in colon tissue of model mice, and reduced mucosal layer necrosis and crypt disappearance.
Example 4 anti-Crohn's disease Effect
BALB/c mice, male, 6-8 weeks old, weighing 22-25 g, were randomly divided into normal group, model group, rhMG53 (1mg/kg) group. Except for the normal control group, mice in other groups were anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital, the flexible tubules were carefully inserted into the colon (3.5 cm proximal anus) and 100. mu.L of 2% 2,4, 6-trinitrobenzenesulfonic acid was injected and the mice were vertically inverted for 1 minute. From the first day, rhMG53 (1mg/kg) was administered intramuscularly, and the normal group and the 2,4, 6-trinitrobenzenesulfonic acid model group were administered with an equivalent amount of vehicle 0.5% CMC-Na by injection for 7 consecutive days. Mice were weighed daily and observed for diarrhea and hematochezia. The results are shown in FIG. 5.
As shown in fig. 5, the body weight of the 2,4, 6-trinitrobenzenesulfonic acid model group mice decreased significantly from day 2, with diarrhea, fecal occult blood, and hematochezia, and gradually increased as the experiment continued. The rhMG53 (1mg/kg) can effectively relieve the weight loss of mice, which indicates that the rhMG53 has good protective effect on the Crohn disease model mice. The effect of value 2 on survival in crohn's disease model mice.
Fig. 5 also shows that after 7 days of molding, the survival rate of 2,4, 6-trinitrobenzenesulfonic acid model group mice is only 50%, while rhMG53 (1mg/kg) significantly improved the survival rate of colitis mice.
Example 5 construction of lactococcus lactis engineering bacteria secreting human MG53
Experimental strains and plasmids: food grade lactococcus lactis NZ9000 and secretory expression vector pNZ8148 were purchased from Biovector plasmid vector cell Gene Collection. The human TRIM72 gene (MG 53 protein coding gene), the signal peptide and the lactobacillus acidophilus full sequence are searched in an NCBI gene bank, primers and the human TRIM72 gene are synthesized and provided by Shanghai chemical company, the SPusp45 signal peptide sequence is added at the upstream of the TRIM72 gene, and the His gene sequence added at the downstream is shown as SEQ ID No. 1. The plasmid minipill kit is a product of Tiangen company; tricine and Nisin are products of Sigma company; the Coomassie brilliant blue kit is a product of Biyuntian company; MG53 antibody was given by professor Majianji.
Connection of the secretory expression vector pNZ8148 and the MG53 gene: the attachment site is shown as a in fig. 6.
1) Recombination ligation
Treatment of pNZ8148 plasmid linearized vector with NcoI-KpnI:
a recombination reaction system:
20 ng/. mu.l 3. mu.l of product containing TRIM72 gene
pNZ8148 (linearized vector) 20 ng/. mu.l 2.5. mu.l
10×buffer 0.5μl
ddH2O 3μl
The total volume is 10ul, and the mixture is gently mixed and bathed for 25min at 50 ℃.
2) Transformation of
Mu.l of the ligation product was added to 50. mu.l of competent cells (Tiangen), ice-cooled for 30min, at 42 ℃ for 90sec, and immediately ice-cooled for 2 min. Sterile LB medium (150. mu.l) was added thereto and the mixture was cultured with shaking at 37 ℃ for 1 hour. The cells were plated on LB plates containing chloramphenicol and cultured.
3) Colony screening assay
a. Single colonies were picked from overnight plates
b. Colony PCR with TRIM72 primer
c. Identification of Positive clones by electrophoresis
d. Randomly selecting 4 positive bacteria, and shake culturing overnight at 37 deg.C in 4ml single tube
TRIM72-seqF：5’-caccgttctctgcccctg-3’（SEQ ID NO.2）
TRIM72-seqR：5’-ctgtgtcttgaggcgtgc-3’（ SEQ ID NO.3）
4) Extraction of plasmid
The overnight culture solution in 3.2.5 was used to extract plasmids (plasmid extraction kit from Tiangen corporation), and sequencing was performed, the result is shown in SEQ ID NO. 4. The correct plasmid was obtained and named Pnz8148-SPUsp45-TRIM72, which was stored at-80 ℃ until use.
Preparation and electrotransformation of lactic acid bacteria NZ9000 competence:
1) 4ml of G/L-SGM17B culture medium is inoculated with 4 mul of lactococcus lactis NZ9000 according to the inoculation amount of L percent, and the mixture is subjected to static culture at the temperature of 30 ℃ for 12 hours;
2) adding all the bacterial liquid obtained in the step 1 into 80ml of fresh G/L-SGM17B culture medium, and culturing until OD600= 0.3-0.35;
3) centrifuging at 4 deg.C and 5000g for 20min, and removing supernatant;
4) resuspending the precipitate with 80ml of 0.5M sucrose/10% glycerol, centrifuging at 4 ℃ and 6000g for 20min, and discarding the supernatant;
5) resuspend pellet with 40ml of 0.5M sucrose/10% glycerol/0.5 mM EDTA, centrifuge at 6000g for 20min at 4 ℃ after 15min ice pre-harvest, discard supernatant:
6) resuspending the precipitate with 20ml of 0.5M sucrose/10% glycerol, centrifuging at 4 ℃ and 6000g for 20min, and discarding the supernatant;
7) the pellet was resuspended in 80ml of 0.5M sucrose/l 0% glycerol and dispensed into 40. mu.l/tube and stored at-80 ℃.
Recombinant electrotransformation:
1) the electric rotor is placed on ice for precooling, and ultraviolet sterilization is carried out in a superclean workbench.
2) Tubes (40. mu.l/tube) were removed from the-80 ℃ freezer and thawed on ice. Taking out the granules from a refrigerator at-20 deg.C
Placing on ice for thawing.
3) Pnz8148 linked MG53 product was added to 40ul of competence and after tapping, the mixture was placed on ice for 10 min.
4) Transferring the mixture of step 3 to a pre-cooled electric rotor to ensure that the mixture is at the bottom of the electric rotor and no bubbles are present.
5) And removing the condensed water on the outer wall of the electric revolving cup, and putting the electric revolving cup into an electric shock instrument.
6) The voltage of the shocker was set to 2000V and the shock time was set to 4 ms.
7) And 4, electric shock is carried out once.
And (3) recovering recombinants and identifying PCR:
1) and after the electric shock is finished, the electric rotating cup is quickly taken out of the electric shock groove.
2) GM17MC in an lml ice bath was added to regenerate the liquid medium.
3) Mixing gently with pipette, ice-cooling for 5min, transferring to 1.5ml EP tube, and standing at 30 deg.C for 1-1.5 h.
4) Mu.l of the suspension was applied to a BCP plate at 100. mu.l, 900. mu.l, and allowed to stand anaerobically at 30 ℃ for l-2 d.
5) Picking a single yellow colony from a BCP plate, placing the single yellow colony in 3ml of GM17 liquid culture medium, and standing and culturing at 30 ℃ overnight;
6) preparing bacterial liquid template DNA, namely taking 200 mu l of bacterial liquid in a 1.5ml sterile EP centrifuge tube, centrifuging at 12,000 revolutions, discarding a culture medium, and adding 100 mu l of sterile ultrapure water to resuspend the bacterial. Then placing the EP tube at-80 deg.C for 15min, heating in boiling water bath for 15min to fully crack thallus, centrifuging at 12000 r for 3min, and collecting supernatant as template DNA;
7) Taking 1 mul of bacterial liquid DNA as a template, taking MG53 as an upstream primer and a downstream primer, respectively taking 0.3 mul of the primers as the upstream primer: 5'-caccgttctctgcccctg-3' (SEQ ID NO. 2); a downstream primer: 5'-ctgtgtcttgaggcgtgc-3' (SEQ ID NO. 3), molecular weight: 250 bp. 2 mul of 10 XPCR buffer solution, 2 mul of dNTP solution and 0.2 mul of TaqDNA polymerase are denatured for 5min and then enter into circulation, the circulation temperature and the circulation time are 94 ℃, and water is added for 30s until the total volume is 20 mul; performing 25 cycles at 94 deg.C and 55 deg.C, and then performing extension at 72 deg.C for 10 min; the product was detected by 1.0% agarose gel electrophoresis, and the result is shown as B in FIG. 6.
Expression of the protein of interest:
1) selecting recombinants identified as positive by PCR in 7, inoculating 150 mu l of glycerol strain into 3ml of GM17 liquid culture medium according to the inoculation amount of 5%, and standing and culturing for 48h at 30 ℃;
2) taking 2ml of bacterial liquid at 12000rpm, centrifuging at 4 ℃ for 5 min;
3) taking 2ml of centrifugal supernatant, adding 200 mul of 150% (w/v) trichloroacetic acid (TCA), standing at 4 ℃ in the dark for 12h, then centrifuging at 12000rpm and 4 ℃ for 10min, and re-suspending the precipitate with lml precooled acetone; centrifuging at 12000rpm and 4 deg.C for 10min, and resuspending the precipitate with 1ml of precooled acetone; centrifuging at 12000rpm and 4 deg.C for 10min, carefully sucking the supernatant with 1ml pipette gun, retaining the precipitate, air drying, and adding 100 μ l ddH 2O and 20 mul loading buffer (6 x), boiling in boiling water for 10min, 12000rpm, room temperature, centrifuging for 10min for later use;
4) 500. mu.l ddH for bacteria2Resuspending O, 12000rpm, at room temperature, centrifuging for 5min, discarding the supernatant, resuspending the mycelia with 200. mu. lpH (8.0) Phosphate Buffered Saline (PBS), adding 5. mu.l lysozyme (50mg/ml), water bathing at 37 ℃ for 1.5h, adding 20. mu.l SDS solution (20%), boiling in boiling water for 10min, adding 50. mu.l loading buffer (6X), boiling in boiling water for 10min, 12000rpm, centrifuging at room temperature for 10min, and keeping the mixture for use.
5) An equal amount of total protein was electrophoresed on a 10% SDS polyacrylamide gel and transferred to nitrocellulose. Non-specific binding sites were blocked with 5% skim milk powder at room temperature, and the membrane was incubated with MG53 antibody (antibody dilution 1: 800) overnight at 4 ℃. Washing the membrane for 3 times by TBST, adding goat anti-rabbit secondary antibody (dilution is 1: 10000), and incubating for 1h at room temperature. After washing the membrane for 3 times with TBST, scanning and analyzing by using an Odys-sey two-color infrared fluorescence imaging system of LI-COR company in the United states. As a result, it was found that MG53 was expressed in the bacterial suspension of NZ9000-TRIM72, whereas MG53 was not expressed in NZ9000-VC (FIG. 6, C).
Example 6 lactococcus lactis secreting human MG53 protection of the DSS-induced ulcerative colitis model and TNBS-induced effects against Crohn's disease
The weight of a C57 mouse bred at SPF level in the center of experimental animals in the university of army and military medical university large-lawn hospital is 22-25 g, and the operation in the experimental process conforms to the regulations of the experimental animals of the university of army and military medical. The treatment groups were randomly divided into a normal group, a model group (4% DSS), a lactococcus lactis group secreting human MG53, and a lactococcus lactis control group. Except for the normal control group and the lactococcus lactis control group, the other 2 groups were freely drunk with 4% Dextran Sodium Sulfate (DSS) for 7 days, and the lactococcus lactis treatment group secreting human MG53 was gavaged daily. Then changed to single distilled water for free drinking for 7 days. The effect of survival of mice was continuously observed and the results are shown in fig. 7. The results show that lactococcus lactis secreting human MG53 can improve survival in mice model for ulcerative colitis.
Example 7 lactococcus lactis secreting human MG53 protection of TNBS-induced effects against Crohn's disease
The weight of a C57 mouse bred at SPF level in the center of experimental animals in the university of army and military medical university large-lawn hospital is 22-25 g, and the operation in the experimental process conforms to the regulations of the experimental animals of the university of army and military medical. The group was randomly divided into a normal group, a model group (TNBS), a lactococcus lactis treatment group secreting human MG53, and a lactococcus lactis control group. As shown in fig. 8, 2,4, 6-trinitrobenzenesulfonic acid model group mice had significantly reduced body weight starting on day 2, with diarrhea, fecal occult blood, and hematochezia, and gradually increased as the experiment continued. The lactococcus lactis treatment group secreting human MG53 can effectively relieve the weight loss of mice and improve the influence of the survival rate of mice of a Crohn's disease model.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> third Hospital of civil liberation army and military medical university (field operations department of research)
Medical application of <120> MG53 protein
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cctaggatga aaaaaaagat tatctcagct attttaatgt ctacagtgat actttcggct 60
gcgcccggcc tcctgcacca ggagctgtcc tgcccgctgt gcctgcagct gttcgacgcg 120
cccgtgacag ccgagtgcgg ccacagtttc tgccgcgcct gcctaggccg cgtggccggg 180
gagccggcgg cggatggcac cgttctctgc ccctgctgcc aggcccccac gcggccgcag 240
gcactcagca ccaacctgca gctggcgcgc ctggtggagg ggctggccca ggtgccgcag 300
ggccactgcg aggagcacct ggacccgctg agcatctact gcgagcagga ccgcgcgctg 360
gtgtgcggag tgtgcgcctc actcggctcg caccgcggtc atcgcctcct gcctgccgcc 420
gaggcccacg cacgcctcaa gacacagctg ccacagcaga aactgcagct gcaggaggca 480
tgcatgcgca aggagaagag tgtggctgtg ctggagcatc agctggtgga ggtggaggag 540
acagtgcgtc agttccgggg ggccgtgggg gagcagctgg gcaagatgcg ggtgttcctg 600
gctgcactgg agggctcctt ggaccgcgag gcagagcgtg tacggggtga ggcaggggtc 660
gccttgcgcc gggagctggg gagcctgaac tcttacctgg agcagctgcg gcagatggag 720
aaggtcctgg aggaggtggc ggacaagccg cagactgagt tcctcatgaa atactgcctg 780
gtgaccagca ggctgcagaa gatcctggca gagtctcccc cacccgcccg tctggacatc 840
cagctgccaa ttatctcaga tgacttcaaa ttccaggtgt ggaggaagat gttccgggct 900
ctgatgccag cgctggagga gctgaccttt gacccgagct ctgcgcaccc gagcctggtg 960
gtgtcttcct ctggccgccg cgtggagtgc tcggagcaga aggcgccgcc ggccggggag 1020
gacccgcgcc agttcgacaa ggcggtggcg gtggtggcgc accagcagct ctccgagggc 1080
gagcactact gggaggtgga tgttggcgac aagccgcgct gggcgctggg cgtgatcgcg 1140
gccgaggccc cccgccgcgg gcgcctgcac gcggtgccct cgcagggcct gtggctgctg 1200
gggctgcgcg agggcaagat cctggaggca cacgtggagg ccaaggagcc gcgcgctctg 1260
cgcagccccg agaggcggcc cacgcgcatt ggcctttacc tgagcttcgg cgacggcgtc 1320
ctctccttct acgatgccag cgacgccgac gcgctcgtgc cgctttttgc cttccacgag 1380
cgcctgccca ggcccgtgta ccccttcttc gacgtgtgct ggcacgacaa gggcaagaat 1440
gcccagccgc tgctgctcgt gggtcccgaa ggcgccgagg cccaccacca ccatcaccac 1500
tgaggtacc 1509
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caccgttctc tgcccctg 18
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctgtgtcttg aggcgtgc 18
<210> 4
<211> 4649
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agatctagtc ttataactat actgacaata gaaacattaa caaatctaaa acagtcttaa 60
ttctatcttg agaaagtatt ggtaataata ttattgtcga taacgcgagc ataataaacg 120
gctctgatta aattctgaag tttgttagat acaatgattt cgttcgaagg aactacaaaa 180
taaattataa ggaggcactc acctaggatg aaaaaaaaga ttatctcagc tattttaatg 240
tctacagtga tactttcggc tgcgcccggc ctcctgcacc aggagctgtc ctgcccgctg 300
tgcctgcagc tgttcgacgc gcccgtgaca gccgagtgcg gccacagttt ctgccgcgcc 360
tgcctaggcc gcgtggccgg ggagccggcg gcggatggca ccgttctctg cccctgctgc 420
caggccccca cgcggccgca ggcactcagc accaacctgc agctggcgcg cctggtggag 480
gggctggccc aggtgccgca gggccactgc gaggagcacc tggacccgct gagcatctac 540
tgcgagcagg accgcgcgct ggtgtgcgga gtgtgcgcct cactcggctc gcaccgcggt 600
catcgcctcc tgcctgccgc cgaggcccac gcacgcctca agacacagct gccacagcag 660
aaactgcagc tgcaggaggc atgcatgcgc aaggagaaga gtgtggctgt gctggagcat 720
cagctggtgg aggtggagga gacagtgcgt cagttccggg gggccgtggg ggagcagctg 780
ggcaagatgc gggtgttcct ggctgcactg gagggctcct tggaccgcga ggcagagcgt 840
gtacggggtg aggcaggggt cgccttgcgc cgggagctgg ggagcctgaa ctcttacctg 900
gagcagctgc ggcagatgga gaaggtcctg gaggaggtgg cggacaagcc gcagactgag 960
ttcctcatga aatactgcct ggtgaccagc aggctgcaga agatcctggc agagtctccc 1020
ccacccgccc gtctggacat ccagctgcca attatctcag atgacttcaa attccaggtg 1080
tggaggaaga tgttccgggc tctgatgcca gcgctggagg agctgacctt tgacccgagc 1140
tctgcgcacc cgagcctggt ggtgtcttcc tctggccgcc gcgtggagtg ctcggagcag 1200
aaggcgccgc cggccgggga ggacccgcgc cagttcgaca aggcggtggc ggtggtggcg 1260
caccagcagc tctccgaggg cgagcactac tgggaggtgg atgttggcga caagccgcgc 1320
tgggcgctgg gcgtgatcgc ggccgaggcc ccccgccgcg ggcgcctgca cgcggtgccc 1380
tcgcagggcc tgtggctgct ggggctgcgc gagggcaaga tcctggaggc acacgtggag 1440
gccaaggagc cgcgcgctct gcgcagcccc gagaggcggc ccacgcgcat tggcctttac 1500
ctgagcttcg gcgacggcgt cctctccttc tacgatgcca gcgacgccga cgcgctcgtg 1560
ccgctttttg ccttccacga gcgcctgccc aggcccgtgt accccttctt cgacgtgtgc 1620
tggcacgaca agggcaagaa tgcccagccg ctgctgctcg tgggtcccga aggcgccgag 1680
gcccaccacc accatcacca ctgaggtacc actagttcta gagagctcaa gctttctttg 1740
aaccaaaatt agaaaaccaa ggcttgaaac gttcaattga aatggcaatt aaacaaatta 1800
cagcacgtgt tgctttgatt gatagccaaa aagcagcagt tgataaagca attactgata 1860
ttgctgaaaa attgtaattt ataaataaaa atcacctttt agaggtggtt tttttattta 1920
taaattattc gtttgatttc gctttcgata gaacaatcaa atcgtttctg agacgtttta 1980
gcgtttattt cgtttagtta tcggcataat cgttaaaaca ggcgttatcg tagcgtaaaa 2040
gcccttgagc gtagcgtggc tttgcagcga agatgttgtc tgttagatta tgaaagccga 2100
tgactgaatg aaataataag cgcagcgtcc ttctatttcg gttggaggag gctcaaggga 2160
gtttgaggga atgaaattcc ctcatgggtt tgattttaaa aattgcttgc aattttgccg 2220
agcggtagcg ctggaaaatt tttgaaaaaa atttggaatt tggaaaaaaa tggggggaaa 2280
ggaagcgaat tttgcttccg tactacgacc ccccattaag tgccgagtgc caatttttgt 2340
gccaaaaacg ctctatccca actggctcaa gggtttgagg ggtttttcaa tcgccaacga 2400
atcgccaacg ttttcgccaa cgttttttat aaatctatat ttaagtagct ttatttttgt 2460
ttttatgatt acaaagtgat acactaattt tataaaatta tttgattgga gttttttaaa 2520
tggtgatttc agaatcgaaa aaaagagtta tgatttctct gacaaaagag caagataaaa 2580
aattaacaga tatggcgaaa caaaaagatt tttcaaaatc tgcggttgcg gcgttagcta 2640
tagaagaata tgcaagaaag gaatcagaac aaaaaaaata agcgaaagct cgcgttttta 2700
gaaggatacg agttttcgct acttgttttt gataaggtaa ttatatcatg gctattaaaa 2760
atactaaagc tagaaatttt ggatttttat tatatcctga ctcaattcct aatgattgga 2820
aagaaaaatt agagagtttg ggcgtatcta tggctgtcag tcctttacac gatatggacg 2880
aaaaaaaaga taaagataca tggaatagta gtgatgttat acgaaatgga aagcactata 2940
aaaaaccaca ctatcacgtt atatatattg cacgaaatcc tgtaacaata gaaagcgtta 3000
ggaacaagat taagcgaaaa ttggggaata gttcagttgc tcatgttgag atacttgatt 3060
atatcaaagg ttcatatgaa tatttgactc atgaatcaaa ggacgctatt gctaagaata 3120
aacatatata cgacaaaaaa gatattttga acattaatga ttttgatatt gaccgctata 3180
taacacttga tgaaagccaa aaaagagaat tgaagaattt acttttagat atagtggatg 3240
actataattt ggtaaataca aaagatttaa tggcttttat tcgccttagg ggagcggagt 3300
ttggaatttt aaatacgaat gatgtaaaag atattgtttc aacaaactct agcgccttta 3360
gattatggtt tgagggcaat tatcagtgtg gatatagagc aagttatgca aaggttcttg 3420
atgctgaaac gggggaaata aaatgacaaa caaagaaaaa gagttatttg ctgaaaatga 3480
ggaattaaaa aaagaaatta aggacttaaa agagcgtatt gaaagataca gagaaatgga 3540
agttgaatta agtacaacaa tagatttatt gagaggaggg attattgaat aaataaaagc 3600
ccccctgacg aaagtcgacg gcaatagtta cccttattat caagataaga aagaaaagga 3660
tttttcgcta cgctcaaatc ctttaaaaaa acacaaaaga ccacattttt taatgtggtc 3720
ttttattctt caactaaagc acccattagt tcaacaaacg aaaattggat aaagtgggat 3780
atttttaaaa tatatattta tgttacagta atattgactt ttaaaaaagg attgattcta 3840
atgaagaaag cagacaagta agcctcctaa attcacttta gataaaaatt taggaggcat 3900
atcaaatgaa ctttaataaa attgatttag acaattggaa gagaaaagag atatttaatc 3960
attatttgaa ccaacaaacg acttttagta taaccacaga aattgatatt agtgttttat 4020
accgaaacat aaaacaagaa ggatataaat tttaccctgc atttattttc ttagtgacaa 4080
gggtgataaa ctcaaataca gcttttagaa ctggttacaa tagcgacgga gagttaggtt 4140
attgggataa gttagagcca ctttatacaa tttttgatgg tgtatctaaa acattctctg 4200
gtatttggac tcctgtaaag aatgacttca aagagtttta tgatttatac ctttctgatg 4260
tagagaaata taatggttcg gggaaattgt ttcccaaaac acctatacct gaaaatgctt 4320
tttctctttc tattattcct tggacttcat ttactgggtt taacttaaat atcaataata 4380
atagtaatta ccttctaccc attattacag caggaaaatt cattaataaa ggtaattcaa 4440
tatatttacc gctatcttta caggtacatc attctgtttg tgatggttat catgctggat 4500
tgtttatgaa ctctattcag gaattgtcag ataggcctaa tgactggctt ttataatatg 4560
agataatgcc gactgtactt tttacagtcg gttttctaat gtcactaacc tgccccgtta 4620
gttgaagaag gtttttatat tacagctcc 4649
Claims (1)
1. The application of the recombinant human MG53 protein in preparing a medicament for treating inflammatory bowel disease is characterized in that the inflammatory bowel disease is ulcerative colitis, the medicament is prepared by mixing the recombinant human MG53 protein with a pharmaceutically acceptable carrier, the medicament is in an injection form, and the recombinant human MG53 protein is generated by recombinant lactic acid bacteria secreting human MG53 protein; the recombinant lactobacillus secreting human MG53 protein contains an expression vector for expressing MG53 gene; the expression vector is connected to a pNZ8148 vector polyclonal enzyme cutting site through an MG53 gene, and the sequence of the MG53 gene connecting vector is SEQ ID NO. 1.
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| US9458465B2 (en) * | 2007-12-04 | 2016-10-04 | Rutgers, The State University Of New Jersey | Compositions and methods to modulate cell membrane resealing |
| JP5248494B2 (en) * | 2006-07-11 | 2013-07-31 | ユニバーシティ・オブ・メディシン・アンド・デンティストリー・オブ・ニュージャージー | Protein, nucleic acid encoding it and related methods of use |
| US9139630B2 (en) * | 2006-07-11 | 2015-09-22 | Rutgers, The State University Of New Jersey | Compositions and methods for preparing recombinant MG53 and methods for optimizing same |
| EP2635908B1 (en) * | 2010-11-05 | 2015-08-19 | University of Medicine and Dentistry of New Jersey | Serum mg53 as a diagnostic marker for tissue injury |
| US20120213737A1 (en) * | 2011-02-18 | 2012-08-23 | University Of Medicine And Dentistry Of New Jersey | Compositions and methods for therapeutic membrane repair |
| CN102329766A (en) * | 2011-09-16 | 2012-01-25 | 暨南大学 | Recombination food-grade lactic acid bacterium capable of being used for preventing or treating human ulcerative colitis as well as preparation method and application of recombination food-grade lactic acid bacterium |
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