CN116549617A - Use of RNASE4 in preparation of medicament for preventing or treating inflammatory bowel disease - Google Patents
Use of RNASE4 in preparation of medicament for preventing or treating inflammatory bowel disease Download PDFInfo
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- CN116549617A CN116549617A CN202310613079.4A CN202310613079A CN116549617A CN 116549617 A CN116549617 A CN 116549617A CN 202310613079 A CN202310613079 A CN 202310613079A CN 116549617 A CN116549617 A CN 116549617A
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- rnase4
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Abstract
The invention discloses application of RNASE4 in preparation of medicines for preventing or treating inflammatory bowel diseases. The invention discovers that ribonuclease 4 (RNASE 4) can effectively improve the symptoms of weight reduction, disease activity index increase, colon shortening, intestinal epithelial destruction, expression level increase of inflammatory related factors and the like caused by inflammatory bowel disease; in vitro experiments show that the RNASE4 with low concentration can effectively kill the helicobacter, and can effectively treat inflammatory bowel diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of RNASE4 in preparation of medicines for preventing or treating inflammatory bowel diseases.
Background
Inflammatory bowel disease (inflammatory bowel diseases, IBD) is a collective term for gastrointestinal diseases that exhibit chronic or recurrent immune responses and inflammatory symptoms. With the increasing changes in people's lifestyle and the increasing pressure of life, the global incidence of IBD is on a year-by-year trend, with our country suffering from disease in asia. Clinical manifestations of IBD patients mainly include: intestinal manifestations such as abdominal pain, diarrhea, mucous bloody stool, fistula formation, intestinal manifestations such as perianal abscess, bloody stool, abdominal cramps, fever, fatigue, severe cramps in the pelvic region, muscle cramps, loss of appetite, weight loss, and the like. At present, the lack of medicine varieties for treating IBD has no effective treatment means; although the conventional treatment can improve symptoms, the recurrence rate is high, the curative effect on severe patients is poor, and the overall clinical remission rate is not more than 50%. Innovative drug development for the treatment of IBD has become a clinical urgent need.
Currently, treatments for IBD mainly include drug therapy, biological therapy, surgical therapy, and nutritional support. Drug therapy includes the use of anti-inflammatory drugs (e.g., 5-aminosalicylic acid drugs, glucocorticoids, etc.), immunosuppressants (e.g., azathioprine, methotrexate, etc.), and antibiotics. Biological therapy is mainly carried out by using biological agents (such as anti-tumor necrosis factor alpha monoclonal antibodies) targeting immune mediators. Surgical treatment may be selected when drug and biological treatments are ineffective, but have some impact on the quality of life of the patient. Nutritional support including dietary modification and parenteral nutritional support can relieve symptoms and improve quality of life.
RNASE 4is a newly discovered antibacterial protein that acts against uropathogenic escherichia coli in the human kidney and bladder. We have found that RNASE 4is highly expressed in cells (Panzem cells and goblet cells) secreting antibacterial proteins in the intestinal tract, regulates the homeostasis of intestinal flora, and is involved in the occurrence and development of inflammatory bowel disease, thus being a potential biomarker and therapeutic target thereof. At present, the application of the patent RNASE4 as a drug target in a glioma inhibition drug (patent publication number: CN 105727311A) informs the application of the RNASE4 as a glioma treatment target; the patent "method for treatment and diagnosis of prostate cancer" (patent publication No. CN 111417855A) tells the use of RNASE4 as a diagnostic and therapeutic for prostate cancer; the patent "application of RNASE4 as a drug target for treating and/or preventing diabetes" (patent publication No. CN 114404589A) teaches the application of RNASE4 as a drug target for preventing and treating diabetes. Based on the application of RNASE4 as an inflammatory bowel disease diagnosis marker in the prior art, no report is available at present.
Disclosure of Invention
In view of the above problems in the prior art, it is an object of the present invention to provide the use of RNASE4 in the preparation of a diagnostic product for inflammatory bowel disease, for solving the problems in the prior art.
In the process of screening a large number of compounds for the influence of inflammatory bowel disease, the RNASE4 protein is found to improve symptoms caused by inflammatory bowel disease, such as weight loss, disease activity index increase, colon shortening, intestinal epithelial structure destruction, expression level increase of inflammatory related factors and the like. The present invention has been completed on the basis of this finding.
To achieve the above and other related objects, the present invention is achieved by the following technical means.
The use of an RNASE4 protein or an RNASE4 agonist in the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease in accordance with the first aspect of the present invention.
In the first aspect, the inflammatory bowel disease is selected from Ulcerative Colitis (UC) or Crohn's Disease (CD).
In the first aspect, the inflammatory bowel disease is at least one of the following items A1) to A6):
a1 Weight loss;
a2 A) an increase in disease activity index;
a3 Colorectal shortening;
a4 Intestinal epithelial structural disruption;
a5 Elevated inflammatory factor expression levels;
a6 Dysbacteriosis in the intestinal tract.
In the first aspect, the medicament for pre-preventing or treating inflammatory bowel disease medicament has the function of at least one of the following items B1) to B6):
b1 Increasing body weight;
b2 Reducing disease activity index;
b3 Colorectal lengthening;
b4 Slowing down the disruption of the intestinal epithelial structure;
b5 Reduced inflammatory factor expression levels;
b6 Maintaining intestinal flora balance.
Preferably, the inflammatory factor is selected from one or more of CCL2, CCL3, CXCL1, CXCL2, GCSF, IL-6, IL-1β, IL-17A, S A8 and TNF α.
In the first aspect described above, the RNASE4 agonist is a substance that increases the level of the RNASE4 gene or protein.
Preferably, the RNASE4 protein is delivered to a patient suffering from inflammatory bowel disease to directly increase the level of RNASE4 in the inflammatory bowel disease cells, or to increase RNASE4 activity or promote RNASE4 transcription or expression via an RNASE4 agonist, thereby increasing the level of RNASE4 in the inflammatory bowel disease cells. Promoting RNASE4 transcription or expression refers to: the RNASE 4is highly expressed or RNASE4 transcriptional activity is increased, and the RNASE4 transcription or expression can be controlled by one skilled in the art using conventional methods. Preferably, the RNASE4 activity is increased by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by 70%, most preferably by at least 90% compared to that before the increase.
In the first aspect, the therapeutic agent for inflammatory bowel disease necessarily includes RNASE4 protein or RNASE4 agonist, and uses RNASE4 protein or RNASE4 agonist as the active ingredient of the aforementioned functions, and in the agent for preventing and/or treating inflammatory bowel disease, the active ingredient that performs the aforementioned functions may be only RNASE4 protein or RNASE4 agonist, or may include other molecules that perform similar functions.
In the first aspect described above, the RNASE 4is used either alone or in combination with other drugs. The RNASE4 protein or RNASE4 agonist is the only active ingredient or one of the active ingredients of the inflammatory bowel disease therapeutic drug.
Preferably, the RNASE4 or a pharmaceutically acceptable auxiliary material thereof forms a pharmaceutical composition. By pharmaceutically acceptable is meant that when the drug is properly administered to an animal or human, they do not produce adverse, allergic or other untoward reactions. The pharmaceutically acceptable adjuvant should be compatible with, i.e. capable of being blended with, the RNASE4 or its effect without substantially reducing the effect of the RNASE4 or its effect in the usual case.
More preferably, the pharmaceutically acceptable auxiliary materials are selected from one or more of carriers, diluents, binders, lubricants and wetting agents. Specific examples of some substances that may be pharmaceutically acceptable carriers, diluents, binders, lubricants and wetting agents are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.
More preferably, the pharmaceutical composition is one or more of solution, injection, spray, nose drops, aerosol, powder fog, tablet, capsule and granule. The medicaments of the various formulations can be prepared according to the conventional method in the pharmaceutical field. Preferably a solution.
More preferably, the pharmaceutical composition is introduced into the body by injection, spraying, nasal drip, eye drip, permeation, absorption, physical or chemical mediated methods such as intramuscular, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped with other substances and introduced into the body. Preferably, administration is by lavage. The pharmaceutical compositions may also be used in combination with other therapeutic means including surgery, radiation therapy, chemotherapy, targeted therapies.
In the present invention, when the medicament is used for treating inflammatory bowel disease in a subject, an effective dose of the product is required to be administered to the subject. With this method, the inflammatory bowel disease causes weight loss, disease activity index increase, colon shortening, intestinal epithelial structure destruction or inflammatory-related factor expression level is inhibited, reduced or alleviated. The subject is an organism, including a mammal, that is being prevented and/or treated. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.
In the present invention, the RNASE4 or an effective dose thereof is administered to the subject at a daily dose of 10 to 60mg/kg body weight/time for 10 to 60 consecutive days. Specifically, the effective dose is 10-20 mg/kg body weight/time per day, and the administration is continuous for 20-40 days.
Treatment as used herein refers to slowing, interrupting, arresting, controlling, stopping, alleviating, reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
Prevention as used herein refers to any action that inhibits symptoms or delays the tone of a particular symptom by administering the product of the present invention.
The second aspect of the invention protects the use of an RNASE4 gene or protein as a drug target in the development or screening or in the manufacture of a medicament for the prevention and/or treatment of inflammatory bowel disease.
In the second aspect, the use specifically refers to: candidate substances are screened by taking the RNASE4 gene or protein as an action target to find an RNASE4 agonist which is used as an alternative inflammatory bowel disease therapeutic drug.
The third aspect of the invention protects the use of an RNASE4 protein or an RNASE4 agonist in the manufacture of a product for inhibiting proliferation of helicobacter (paraasutterella).
In the third aspect, the RNASE4 protein is an IC of helicobacter (Parasutterella) 50 At 0.37. Mu.M, 1. Mu.MRNASE 4 can cause serious damage to the bacterial membrane of the species helicobacter (Parasutterella).
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, in vivo experiments show that ribonuclease 4 (RNASE 4) can effectively improve the symptoms of weight reduction, disease activity index increase, colon shortening, intestinal epithelial destruction and inflammatory related factor expression level increase caused by inflammatory bowel disease; through in vitro experiments, the IC of RNASE4 to PARA bacteria is found 50 About 0.37. Mu.M, 1. Mu.M NASE4 can cause serious damage to the pari bacteria wall membrane structure, and the PI positive rate is nearly 100% when the pari bacteria are treated by 5. Mu.M. Comprehensive results show that RNASE4 can treat inflammatory bowel disease, provides a new treatment means for treating inflammatory bowel disease, and also provides a reference for application and development of RNASE 4.
Drawings
FIG. 1 shows the sequencing and electrophoresis patterns after knocking out the RNase4 gene in C57BL/6 mice.
Figure 2 shows a graph of the change in body weight of mice in each group during DSS induction.
Figure 3 shows a graph of disease activity index change in each group of mice during DSS induction.
Fig. 4 shows the results of colon length of mice after DSS induction, representing P <0.01.
Fig. 5 shows the results of the observation of colon tissue structure and histomorphology scoring for HE staining, representing P <0.05.
Fig. 6 shows a graph of the detection of inflammatory factor expression levels, representing P <0.05; * Represents P <0.01; * Represents P <0.001.
FIG. 7 shows a graph of the results of the bactericidal activity of RNASE4 against PARA bacteria at various concentrations.
FIG. 8 shows a graph of Propidium Iodide (PI) staining of PARA bacteria after treatment with RNASE4 at different concentrations.
FIG. 9 shows a scanning electron microscope image of cell surface morphology following treatment with RNASE4 at different concentrations.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
In the examples described below, dextran sodium sulfate solution was dissolved in sterile water using DSS drug powder at a final concentration of 2.5% (i.e. 2.5g/100 mL).
Example 1
In this example, a mouse enteritis model was constructed and mice body weight, disease activity index, colon length and expression level of inflammatory factors in the colon were observed by gavage administration of recombinant RNASE4 protein, comprising the steps of:
1.1 constructing a mouse enteritis model
1) Construction of Rnase4 knockout mice Using TALEN technology (Rnase 4) -/- ) The test mice are identified and then used as test mice, the identification results are shown in the right graph of fig. 1, and the test mice are all C57BL/6 background.
The principle of the construction of the mouse by using TALEN technology by the entrusted Sai technology limited company is that a stop codon is designed in advance, so that the translation of RNASE4 protein is stopped in advance, and finally, the RNASE4 protein cannot be expressed in the mouse. The effect of the external immunoblotting was used to verify the effect of Rnase4 on the Rnase4 knocked-out mice, and the results are shown in the right panel of FIG. 1. As can be seen from fig. 1, mice (Rnase 4 -/- ) RNASE4 protein is not expressed in vivo.
The experimental procedure of immunoblotting was: harvesting WT (wild-type mice) and RNase4 -/- The colon tissue of the mice is lysed and extracted with protein, after centrifugation for 10min at 16000g, 200 μl of supernatant is added to 50 μl LSDS-PAGE protein loading buffer (5×) (bi yun day, cat No. P0015) and boiled in a boiling water bath for 10min. After polypropylene gel electrophoresis, transferring the membrane to a PVDF membrane, immersing the PVDF membrane in a sealing liquid (5% skimmed milk powder), and sealing for 1h at normal temperature on a shaking table. anti-RNASE 4 antibody (homemade, reference ribonucleotide 4is associated with aggressiveness and progression of prostate cancer.Commun Biol.2022;5:625) was added and incubated overnight at 4 ℃. After washing off the primary antibody, secondary antibody HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (purchased from Proteintech, cat. No. SA 00001-2) was added and incubated for 1H at normal temperature. After washing off the excess secondary antibody, HRP substrate developing solution is added to expose the developing band.
2) Then RNase 4is added -/- Mice were randomly divided into two groups, one of which was supplemented daily with 20mg/kg body weight of recombinant RNASE4 protein (RNASE 4 -/- -RNASE4 group), another group was used as experimental control group (RNASE 4 -/- A group). In addition, a group of Wild Type (WT) mice was added as a blank. Each group was fed normally. Rnase4 -/- The RNASE4 group is a lyophilized RNASE4 pure protein powder, dissolved in sterile PBS aqueous solution. The experimental control group was a mixture of sterile PBS powder and water.
3)Rnase4 -/- group-RNASE 4 and Rnase4 -/- After the groups are continuously filled with stomach for 30 days every day, normal drinking water of the mice is replaced by 2.5% (m/v) aqueous solution of dextran sodium sulfate (Dextran Sulfate Sodium Salt, DSS), after the groups are continuously drunk for 6 days, normal water is replaced for 2 days, a enteritis model of the mice is obtained through induction, and the weights and disease activity indexes of the mice in each group are recorded.
4) Mice were sacrificed after induction, colon tissue of the mice was collected, HE stained colon tissue, and qPCR (Thermo TaqPath TM qPCR Master Mix) to detect the expression level of various inflammatory factors in colon tissue.
DDS induction started to sacrifice, body weight was recorded by weighing daily, and the results are shown in fig. 2.
Disease activity index (Disease activity indexDAI) is a comprehensive score combining body weight score, stool score and hematochezia score, and the total score of 3 results is divided by 3 to obtain DAI value, and the result is shown in figure 3. Wherein, weight score: 0, no weight loss; 1, 1-5% decrease; 2, 6-10% decrease; 3, 11-20% decrease; 4, drop by more than 20%; fecal scoring: 0, solid state stool; 1, solid stool, which is easy to deform; 2, not forming feces; 3, liquid excrement; hematochezia score: 0, occult blood detection is negative; 1, occult blood detection is positive; 2, blood is visible in the feces; 3, severe hematochezia.
The colon part was measured for its length and the results are shown in fig. 4.
The distal colon tissue was formalin-fixed at about 1cm of the intestinal section and subsequently HE stained and histomorphometric scored (pathology scored) according to HE staining, as shown in fig. 5. The histomorphometric score is obtained by comprehensively scoring the two cases of the comprehensive inflammation score and the ulcer score and dividing the total score of 2 results by 2. Wherein, the inflammation score is as follows: 0, normal (normal range); 1. mild (small, focal, or diffuse, limited to the lamina propria); 2. moderate (multifocal or locally extensive, extending to submucosa); 3. severe (transmural inflammation, ulcer coverage > 20 crypts). Ulcer scoring: score 0, normal (no ulcers); 1. mild (1-2 ulcers, involving 20 crypts in total); 2. moderate (3-4 ulcers, involving 20-40 crypts altogether); 3. severe symptoms (more than 4 ulcers or more than 40 crypts).
Tissue RNA was extracted from colon tissue of about 1cm, and qPCR was performed to detect the expression levels of various inflammatory factors in colon tissue, such as CCL2, CCL3, CXCL1, CXCL2, GCSF, IL-6, IL-1. Beta. IL-17A, S A8 and TNF. Alpha. As a result, see FIG. 6.
As can be seen from FIG. 2, RNase4 -/- Group mice lost body weight up to 75% at maximum, while Rnase4 -/- The weight loss ratio of RNASE4 group was significantly reduced by 80% and was similar to that of the blank group.
As can be seen from FIG. 3, RNase4 -/- Group mice have disease activity index up to 3, while Rnase4 -/- The disease activity index of the RNASE4 group was significantly reduced to 2.5, similar to that of the blank group.
As can be seen from FIG. 4, the colon length of the blank is 5.5cm, and Rnase4 -/- Group mice had a colon length of 4.8cm, rnase4 -/- Colon length of 5.1cm for RNASE4 group, blank and Rnase4 -/- The colon length of the RNASE4 group is significantly higher than that of Rnase4 -/- A group.
As can be seen from FIG. 5, rnase4 was seen from HE staining -/- The crypt structure in the colon tissue of group mice was destroyed and a structure resembling cavitation appears, indicating that the integrity of the colon tissue was severely destroyed; and Rnase4 -/- RNASE4 group, similar to the blank group, the colon tissue maintained good integrity. Also demonstrating Rnase4 from histomorphology scoring -/- The RNASE4 group, i.e. the anaplerosis RNASE4 protein, is effective in treating inflammatory enteritis and restoring healthy levels.
As can be seen from FIG. 6, RNase4 -/- Inflammatory-related factor expression was significantly elevated in group mice, whereas Rnase4 -/- -RNASE4 group has significantly reduced expression of inflammatory-related factors, similar to the blank group.
DSS can directly break colorectal epithelial barrier and increase intestinal permeability, thereby inducing enteritis, and Rnase4 knockout mice treated by DDS can find that Rnase4 -/- Group mice have more severe enteritis symptoms such as weight loss, shorter intestinal tracts and higher disease index. And after supplementing RNASE4 protein, the symptoms of enteritis induced by DSS can be obviously improvedIncluding significantly reduced weight loss in mice, significantly reduced disease activity index, longer colorectal length, reduced disruption of intestinal epithelial structures, and significantly reduced expression of inflammatory-related factors.
Example 2
In this example, a strain of helicobacter (Parasutterella, abbreviated as PARA) was selected and isolated from the intestinal tract of mice, and earlier studies demonstrated that the abundance of PARA was correlated with the occurrence and development of inflammatory bowel disease, and in this example, PARA bacteria were treated with recombinant RNASE4 proteins at different concentrations to examine the killing activity of RNASE4 on PARA bacteria.
Document 1 (Parasutterella, in association with irritable bowel syndrome and intestinal chronic Inflamation.J. Gastroentol hepatol.2018nov;33 (11): 1844-1852.Doi:10.1111/jgh.14281.Epub2018Jun 3) discloses that the abundance of helicobacter (Parasutterella, abbreviated as PARA) is positively correlated with the occurrence and development of inflammatory bowel disease, and thus PARA is the subject of investigation.
2.1 in vitro antibacterial experiments
1) The log phase grown PARA bacteria were centrifuged at 3000rpm for 3 min at 1mL and the supernatant was discarded.
2) The bacteria were then diluted in gradient to a final concentration of 10 by resuspension with pH7.210mM sodium phosphate buffer 5 ～10 6 CFU/mL and after incubation for 2 hours at 37 ℃ with concentration gradients of 0 μm, 0.1 μm, 0.5 μm,1 μm, 2 μm, 5 μm, 10 μm, 50 μm, 100 μm RNASE4, RNASE4-K40A (enzyme activity mutein) and BSA, respectively, the incubated bacteria were inoculated into 96-well plates, OD600 readings were detected after 48 hours of incubation and sterilization plots were drawn.
The sterilization curve chart is shown in fig. 7.
As can be seen from FIG. 7, there is a significant inhibition of PARA bacterial growth by low concentration of RNASE4, IC 50 About 0.37 μm.
2.2 propidium iodide staining (PI staining)
PI staining is commonly used for apoptosis detection, which releases red fluorescence after intercalation into double-stranded DNA, and PI can stain the nucleus by penetrating the broken cell membrane. The PI entering the cell is increased, the red fluorescence intensity is enhanced, and the damaged degree of the cell membrane can be judged according to the intensity of fluorescence, namely the PI dyeing rate, so that the damaged state of the cell is indirectly reflected.
1) The log phase grown PARA bacteria were centrifuged at 3000rpm for 3 min at 1mL and the supernatant was discarded.
2) The bacteria were then resuspended in pH7.210mM sodium phosphate buffer and diluted to a final concentration of 10 7 CFU/mL, packed into 4 sterile centrifuge tubes.
3) To the bacterial solutions, 0. Mu.M RNASE4, 1. Mu.M RNASE4, 5. Mu.M RNASE4 and 10. Mu.M RNASE4 were added, respectively, and after incubation at 37℃for 2 hours, centrifugation was carried out at 3000rpm for 10 minutes, and the supernatant was discarded.
4) 500. Mu.L of PBS containing 1. Mu.g/mL of propidium iodide and 1. Mu.g/mL of Hoechst was added, and incubated at room temperature for 20 minutes in the absence of light.
5) Finally, centrifugation at 3000rpm for 10 minutes, discarding the supernatant, re-suspending with an appropriate amount of PBS, observing under a confocal fluorescence microscope and calculating the propidium iodide positive rate. And a pH7.210 mN sodium phosphate buffer was used as a control group (buffer).
The effect of RNASE4 on the integrity of the PARA outer membrane was analyzed by PI staining and the results are shown in FIG. 8.
As can be seen from FIG. 8, the PI positive rate was about 60% when PARA bacteria were treated at 1. Mu.M, and the PI positive rate was nearly 100% when PARA bacteria were treated at 5. Mu.M.
2.3 scanning electron microscope observation
1) The log phase grown PARA bacteria were centrifuged at 3000rpm for 3 min at 1mL and the supernatant was discarded.
2) The bacteria were then diluted to a final concentration of 10 by resuspension with pH7.210mM sodium phosphate buffer 7 CFU/mL, packed into 5 sterile centrifuge tubes.
3) And respectively adding a solvent (pH 7.210mM sodium phosphate buffer) into the bacterial liquid to serve as a Control group, marking as Control, and taking 1 mu M recombinant RNASE4 protein, 5 mu M recombinant RNASE protein, 10 mu M recombinant RNASE4 protein and 50 mu M recombinant RNASE4 protein as four experimental groups, incubating at 37 ℃ for 2 hours, centrifuging at 3000rpm for 10 minutes, and discarding the supernatant to obtain a bacterial sample.
4) Bacterial samples were taken 1.5mL each and centrifuged at 3000rpm for 3 minutes, and the supernatant was discarded and washed once with PBS buffer.
5) Then 1mL of PBS solution containing 2.5% glutaraldehyde is added, and after one hour of fixation at room temperature, the mixture is transferred to 4 ℃ for fixation overnight; centrifugation at 3000rpm for 3 min, the fixative was discarded and washed three times with PBS buffer for 10min each.
6) Then adding a proper amount of 1% osmium acid, and fixing the sample for 2 hours at room temperature; centrifugation at 3000rpm for 3 min, osmium acid was discarded, and washed three times with PBS buffer for 10min each.
7) Subsequently, the samples were dehydrated with 50%, 70%, 90% and 100% ethanol solutions, respectively, for 15 minutes at each concentration; finally, the sample is critical point dried and observed with a scanning electron microscope.
The effect of RNASE4 on PARA bacterial wall membrane was observed by scanning electron microscopy, and the result is shown in FIG. 9.
As can be seen from FIG. 9, after 1. Mu. MRNASE4 treatment, the surface of PARA bacteria has obvious holes, the wall membrane structure is seriously damaged, and after 5. Mu.M, 10. Mu.M and 50. Mu.M treatment, the wall membrane structure of PARA bacteria is seriously damaged. (FIG. 9).
The methods for purifying recombinant RNASE4 protein and RNASE4-K40A protein of example 2 in the examples of the present application are as follows:
transformation of E.coli: transforming the recombinant plasmid pET11 alpha-RNASE 4 or the recombinant plasmid pET11 alpha-RNASE 4-K40A into special expression escherichia coli BL21 (DE 3), and coating a proper amount of bacterial liquid on an LB solid culture plate containing 100 mug/mL ampicillin, and culturing for 18 hours at 37 ℃; then, single colonies were picked and inoculated into 5mL ampicillin-resistant LB medium and shaken at 37 ℃. 1mL of the bacterial liquid is inoculated into 1L of 2 XYT culture liquid overnight, a constant temperature shaking table at 37 ℃ is used for 250rpm culture until the absorbance A600 value is between 0.8 and 1.0, and IPTG is added to the final concentration of 1mmol/L. Expression was induced at 37℃for 3 hours, and then the expressed bacteria were collected by centrifugation at 4000rpm for 20 minutes at 4 ℃.
Inclusion body preparation and dissolution operations: adding pre-chilled lysis buffer (Tris-HCI 50mL,0.5 MPH=8 EDTA4mL, ddH) at 4deg.C to the above collected expressed bacteria pellet 2 O constant volume to 1L) 50mL, vortex shaking to resuspend the bacterial liquid, centrifuging at 4 ℃ for 20 minutes at 4000rpm, resuspend the bacteria with 20mL of lysis buffer, adding 100mg of lysozyme, incubating for 1 hour in ice bath, and then crushing the bacterial body with power of 300 by an ultrasonic instrumentThe pellet was collected by centrifugation at 12000rpm for 20 minutes at 4℃at 5 seconds and 5 seconds apart for 6 minutes. The inclusion bodies were resuspended in 30mL inclusion body wash (0.3 mL Triton-x100 added to 30mL lysate), and the inclusion bodies were collected by centrifugation at 12000rpm at 4℃for 20 minutes and repeated 1 time, and the resulting pellet was the inclusion body of higher purity. The inclusion body precipitate was added to 20mL of inclusion body denatured solution (guanidine hydrochloride 13.38g, reduced glutathione 0.922g, 1.5M, PH8.8Tris-HCI 1.336 mL,0.5M, PH8.0EDTA 80. Mu. L, MQwater8mL, 10M NaOH 280. Mu.L) and dissolved by stirring under nitrogen atmosphere for 2 hours, and centrifuged at 4000rpm for 20 minutes at 4℃to slowly drop the supernatant into 1L of renaturation buffer (L-arginine 87.1g, oxidized glutathione 0.394g to a constant volume of 1LMQ water). After the supernatant was added to the renaturation buffer, stirring was continued for about 1 hour, and the mixture was allowed to stand overnight to allow sufficient time for folding and renaturation of the recombinant RNASE4 protein.
Purifying recombinant protein: firstly, diluting renaturation buffer solution with 2L of ultrapure water, slowly enriching and primarily purifying by cation affinity chromatography (SP-sepharose fast flow), eluting the combined RNASE4 by using elution buffer solution (1M Tris-HCI 10mL, naCI 58.5g constant volume to 1LMQ water, regulating pH to 8.0); and further utilizing an Shimadzu LC-20 high performance liquid chromatograph to separate and purify the high-purity RNASE4 protein by matching with a C18 reversed-phase high performance liquid chromatography column of Beckman Coultee company in U.S.A. Mobile phase for purifying RNASE4 protein: solution A was ultrapure water containing 0.1% TFA, solution B was a mixed solution of isopropanol, acetonitrile and ultrapure water containing 0.08% TFA, the concentration of solution B in the mobile phase rose from 30% to 50% in 0 to 50 minutes, peaks of RNASE4 protein appeared around 26 minutes, and peak proteins were collected. Finally, the RNASE4 concentration is measured and split into 1mg of freeze-dried powder per tube and stored at-80 ℃ for use with ultra-low Wen Bingxiang, thus obtaining recombinant RNASE4 protein.
The recombinant plasmid pET11 alpha-RNASE 4is pET-11 alpha and RNASE4 which can be obtained by recombination in the prior art and is an over-expression plasmid.
The RNASE4-K40A protein is obtained by mutating the 40 th amino acid-lysine on the RNASE4 sequence in the recombinant plasmid pET11 alpha-RNASE 4 to alanine, so that the enzyme activity of the mutant is mutated. The kit used for the mutation was named Mut Express IIFast Mutagenesis Kit V (Vazyme).
Firstly, designing a primer, wherein the sequence of the primer is as follows:
RNASE4 mutation upstream primer: ATCACTGCGCGCGCTTCAACACCTTCATCC
RNASE4 mutation upstream primer: TTGAAGCGCGCGCAGTGATACAAAGTCATC
The recombinant plasmid pET 11. Alpha. -RNASE4 was then amplified using Phanta Max Super-Fidelity DNA Polymerase in the kit. Since the amplification product contains the original template plasmid, in order to prevent the formation of false positive transformants after transformation, dpnI enzymatic digestion must be performed to remove the methylated template plasmid before recombinant circularization is performed. And then, the digestion product is subjected to high-efficiency recombination on the site to be mutated under the catalysis of Exnase II, so that the in-vitro cyclization of the linear DNA is realized. And finally, transforming the recombinant product into competent cells of escherichia coli BL21 (DE 3), coating the competent cells into a flat plate, culturing for 12 hours, screening and identifying positive clones, preparing and dissolving inclusion bodies, and purifying to obtain the recombinant DNA.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
- Use of an RNASE4 protein or an RNASE4 agonist in the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease.
- 2. The use according to claim 1, wherein the inflammatory bowel disease is selected from ulcerative colitis or crohn's disease;and/or, said RNASE4 agonist refers to a substance that increases the level of an RNASE4 gene or protein.
- 3. The use according to claim 1, wherein the inflammatory bowel disease is at least one of the following A1) -A6):a1 Weight loss;a2 A) an increase in disease activity index;a3 Colorectal shortening;a4 Intestinal epithelial structural disruption;a5 Elevated inflammatory factor expression levels;a6 Dysbacteriosis in the intestinal tract.
- 4. The use according to claim 1, wherein the medicament of the pre-treatment or therapy of inflammatory bowel disease medicament has the function of at least one of the following B1) -B6):b1 Increasing body weight;b2 Reducing disease activity index;b3 Colorectal lengthening;b4 Slowing down the disruption of the intestinal epithelial structure;b5 Reduced inflammatory factor expression levels;b6 Maintaining intestinal flora balance.
- 5. The use according to claim 1, wherein RNASE4 or its use alone or in combination with other drugs.
- 6. The use according to claim 5, wherein the RNASE4 or its combination with a pharmaceutically acceptable adjuvant constitutes a pharmaceutical composition.
- 7. The use according to claim 6, wherein the pharmaceutically acceptable excipients are selected from one or more of carriers, diluents, binders, lubricants and wetting agents.
- 8. The use according to claim 6, wherein the pharmaceutical composition is one or more of a solution, an injection, a spray, a nasal drop, an aerosol, a powder spray, a tablet, a capsule and a granule.
- Use of an rnase4 gene or protein as a drug target in the development or screening or in the manufacture of a medicament for the prevention and/or treatment of inflammatory bowel disease.
- Use of RNASE4 protein or RNASE4 agonist for the preparation of a product for inhibiting proliferation of helicobacter (paraasutterella).
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