Application of proteus mirabilis extract

Abstract

The invention discloses application of a proteus mirabilis extract, and belongs to the technical field of biological medicines. The proteus mirabilis extract contains virulence factor antigens with strong specificity and high proteus mirabilis pathopoiesis relevance, and the proteus mirabilis pathopoiesis important characteristic is that high expression of the virulence factors inevitably causes rise of IgM, IgA and IgG antibody levels of the anti-proteus mirabilis extract in blood. Therefore, the antibody level of the anti-proteus mirabilis extract IgM, IgA and IgG in blood can accurately reflect the pathogenic condition of proteus mirabilis. The proteus mirabilis extract closely related to the pathogenicity of proteus mirabilis is prepared through purification for the first time and is named as EPM12176, the extract and the corresponding blood antibody level can be used for diagnosis of the pathogenicity of the proteus mirabilis, and the extract and the antibody thereof can also be used for prevention and treatment of the pathogenicity of the proteus mirabilis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of a Proteus mirabilis extract. Background Art

[0002] Proteus mirabilis (PM) is a Gram-negative bacterium that is widely distributed in nature and on the surface and gastrointestinal tract of humans and animals. It is a common conditional pathogen. The antigens of Gram-negative bacteria mainly include O antigen, outer membrane protein, flagella, pili, capsule, etc. With the increasing use of broad-spectrum antibiotics, Proteus mirabilis has become an important conditional pathogen, which can cause serious infections in humans, including nosocomial infections, and in severe cases, death. Proteus mirabilis has a close relationship with clinical practice. Proteus mirabilis is the main pathogen of urinary tract infections second only to Escherichia coli. The formation of kidney stones and bladder stones may also be related to Proteus mirabilis infection. Proteus mirabilis can also cause bacteremia, otitis media, osteomyelitis, chronic diarrhea and other diseases.

[0003] At present, the detection and diagnosis of diseases related to Proteus mirabilis usually adopts bacterial culture and biochemical identification, serological identification (O antigen detection), polymerase chain reaction (PCR), mass spectrometry technology and other identification and diagnosis. Since Proteus mirabilis is widely present in nature and in the gastrointestinal tract and body surface of humans and animals, false positives are very likely to occur. Therefore, the above detection methods cannot detect whether Proteus mirabilis is pathogenic. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of a Proteus mirabilis extract to solve the problem that the existing detection means cannot detect whether Proteus mirabilis is pathogenic.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: provide an application of a Proteus mirabilis extract in the preparation of a product for detecting the pathogenicity of Proteus mirabilis, the Proteus mirabilis extract comprising extracellular and extracrine composite antigens; wherein the extracellular antigens comprise flagellar antigens and fimbriae antigens; the extracrine antigens comprise proteolytic enzymes, hemolysins, hyaluronidase, and phospholipase.

[0006] Furthermore, the molecular weight of the Proteus mirabilis extract is 12 kDa-176 kDa.

[0007] The present invention provides an application of a Proteus mirabilis extract in preparing a product for detecting the pathogenicity of Proteus mirabilis. The Proteus mirabilis extract is the extract described above, that is, it includes extracellular and extrasecretory composite antigens; wherein the extracellular antigens include flagella antigens and pili antigens; and the extrasecretory antigens include proteolytic enzymes, hemolysins, hyaluronidase, and phospholipase.

[0008] Further, the pathogenicity caused by Proteus mirabilis is at least one of urinary tract infection, nosocomial infection, gastroenteritis (such as diarrhea) and arthritis caused by Proteus mirabilis.

[0009] Furthermore, the hosts that the Proteus mirabilis causes disease to are humans and animals.

[0010] Furthermore, the product is at least one of a kit, a reagent strip and a chip.

[0011] Furthermore, the samples for product testing include at least one of blood, serum, plasma, tissue homogenate, secretion, exudate, transudate, excrement and culture medium.

[0012] Furthermore, the kit includes at least one of a Proteus mirabilis extract, a microplate coated with a Proteus mirabilis extract, a turbidity reaction tube for a Proteus mirabilis extract, anti-Proteus mirabilis extract IgM, IgA and IgG antibodies, anti-Proteus mirabilis extract IgM, IgA, IgG rheumatoid factor and enzyme, fluorescein, biotin, radioactive element markers, a microplate coated with a Proteus mirabilis extract, anti-Proteus mirabilis extract IgM, IgA and IgG antibodies, and anti-Proteus mirabilis extract IgM, IgA, IgG rheumatoid factor.

[0013] Furthermore, the reagent strip includes at least one of a Proteus mirabilis extract, anti-Proteus mirabilis extract IgM, IgA, IgG antibodies, anti-Proteus mirabilis extract IgM, IgA, IgG rheumatoid factor and its enzyme, fluorescein, biotin, and radioactive element markers.

[0014] Further, enzymes include horseradish peroxidase and alkaline phosphatase; and fluoresceins include FITC, PE, and APC.

[0015] Furthermore, the Proteus mirabilis extract is at least one of a Proteus mirabilis fermentation extract and a Proteus mirabilis colony extract.

[0016] Further, the Proteus mirabilis colony extract is prepared by the following method:

[0017] (1) scraping Proteus mirabilis colonies and then suspending them;

[0018] (2) keeping the product obtained in step (1) at 55-60° C.;

[0019] (3) freezing and thawing the product obtained in step (2) 1-3 times;

[0020] (4) ultrasonically melting the product obtained in step (3) and centrifuging it to retain the supernatant;

[0021] (5) subjecting the product obtained in step (4) to ultrahigh-speed centrifugation, removing the supernatant and adding a buffer solution to obtain.

[0022] Furthermore, the insulation time in step (2) is 2-4 hours; the frequency of ultrasonic thawing in step (4) is 10-30 Hz; and the ultracentrifugation in step (5) is 50000-80000 rpm for 10-60 minutes.

[0023] The present invention provides a derivative of a Proteus mirabilis extract, wherein the derivative comprises at least one of a gene corresponding to the Proteus mirabilis extract, a synthetic primer, an antibody, a rheumatoid factor, a marker, a modified product retaining antigenic characteristics, an active virulence factor, an action product of the active virulence factor, and a cross-reactive antigenic substance.

[0024] The present invention provides a method for screening candidate detection products for detecting pathogenicity of Proteus mirabilis. The method adopts a detection product comprising at least one of a Proteus mirabilis extract, anti-Proteus mirabilis extract IgM, IgA and IgG antibodies, and anti-Proteus mirabilis extract IgM, IgA, and IgG rheumatoid factor as a control for detection and evaluation.

[0025] The invention provides a method for preparing a vaccine for preventing and treating Proteus mirabilis. The components of the vaccine include Proteus mirabilis extract, and at least one of anti-Proteus mirabilis extract IgM, IgA and IgG antibodies.

[0026] The present invention provides a method for screening candidate products for treating pathogenicity caused by Proteus mirabilis, comprising the following steps:

[0027] (1) Establish an animal model of Proteus mirabilis pathogenicity;

[0028] (2) treating the model animal obtained in step (1) with the product to be tested;

[0029] (3) detecting the expression level of anti-Proteus mirabilis extract antibodies and / or anti-Proteus mirabilis extract RF;

[0030] (4) Select the products to be tested with relatively low expression levels of anti-Proteus mirabilis extract antibodies and / or anti-Proteus mirabilis extract RF as candidate products.

[0031] The present invention provides a method for screening candidate vaccines for preventing Proteus mirabilis from causing disease, comprising the following steps:

[0032] (1) Treating animals with the vaccine to be tested;

[0033] (2) infecting, colonizing or challenging the animal obtained in step (1) with Proteus mirabilis;

[0034] (3) detecting the expression level of anti-Proteus mirabilis extract antibodies and / or anti-Proteus mirabilis extract RF;

[0035] (4) Selecting the vaccine candidates with relatively low expression levels of anti-Proteus mirabilis extract antibodies and / or anti-Proteus mirabilis extract RF as candidate vaccines.

[0036] The present invention provides an application of a Proteus mirabilis extract in screening candidate detection and prevention products for Proteus mirabilis pathogenicity. The Proteus mirabilis extract is the extract described above, that is, it includes extracellular and exocrine composite antigens; wherein the extracellular antigens include flagella antigens and pili antigens; the exocrine antigens include proteolytic enzymes, hemolysins, hyaluronidase, and phospholipase; the detection product includes at least one of a kit, a reagent strip, and a chip; and the prevention product includes at least one of a medicine, a biological product, and a medical device.

[0037] The present invention has the following beneficial effects:

[0038] (1) The present invention is the first to purify and prepare an extract of Proteus mirabilis that is closely related to the pathogenicity of Proteus mirabilis, named EPM12176. The extract and its corresponding blood antibody level can be used in the diagnosis of the pathogenicity of Proteus mirabilis. The extract and its antibodies can also be used in the prevention and treatment of the pathogenicity of Proteus mirabilis.

[0039] (2) Stronger specificity. The extract of Proteus mirabilis contains virulence factor antigens such as pili, flagella, hemolysin, proteolytic enzyme, etc., which are highly antigenic and specific and highly relevant to the pathogenicity of Proteus mirabilis. The important feature of the pathogenicity of Proteus mirabilis is the high expression of these virulence factors, which will inevitably lead to an increase in the levels of anti-EPM12176 IgM, IgA, and IgG antibodies in the blood. Therefore, the levels of anti-EPM12176 IgM, IgA, and IgG antibodies in the blood can accurately reflect the pathogenicity of Proteus mirabilis.

[0040] (3) Higher accuracy. The current conventional detection methods for Proteus mirabilis are difficult to confirm or exclude its relationship with pathogenicity even if it detects positive or negative results for Proteus mirabilis. For example, patients with Proteus mirabilis invasion and disease may have negative Proteus mirabilis culture results due to the use of antibiotics. Since the levels of anti-EPM12176 IgM, IgA, and IgG antibodies in the blood can accurately reflect the relationship between Proteus mirabilis and whether it causes disease, the diagnosis of Proteus mirabilis disease is more accurate.

[0041] (4) Simpler, faster and cheaper. For example, to detect the levels of anti-IgM, IgA and IgG antibodies in the blood, you only need to provide an ELISA kit pre-coated with EPM12176, add serum to the coated plate wells, wash and add enzyme-labeled antiserum antibodies, and then perform color development and determination. The results will be available within 2 hours. Compared with the current conventional detection methods of Proteus mirabilis, such as bacterial culture identification, PCR, and mass spectrometry, it is simpler, faster and cheaper.

[0042] (5) The important feature of the pathogenicity of Proteus mirabilis is that the virulence factors located on the bacteria, such as pili and flagella, are equally important as the virulence factors secreted outside the cells, such as proteases and hemolysins, and they have a synergistic effect. Therefore, the purification and preparation of Proteus mirabilis extracts, that is, the purification and preparation of the composite antigens of the surface and secretion of Proteus mirabilis, is of great significance for the detection and diagnosis of Proteus mirabilis pathogenicity and the prevention and treatment of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a picture of rabbits with arthritis induced by Proteus mirabilis and the results of their X-ray, pathology, non-specific immune indexes, and specific anti-Proteus mirabilis IgM, IgG, and IgA levels;

[0044] Figure 2 is a flow chart for the preparation of Proteus mirabilis extract;

[0045] Figure 3 This is a diagram of the morphological changes of Proteus mirabilis during the treatment process;

[0046] Figure 4 This is the SDS-PAGE analysis of Proteus mirabilis extract;

[0047] Figure 5 The molecular weight and distribution diagram of Proteus mirabilis extract EPM12176;

[0048] Figure 6 This is a graph showing the antigenic performance analysis of Proteus mirabilis extract EPM12176;

[0049] Figure 7 This is the result of using Proteus mirabilis extract EPM12176 to detect the levels of IgM, IgA and IgG in the serum of rabbits with Proteus mirabilis-induced arthritis;

[0050] Figure 8 This is the result graph of using Proteus mirabilis extract EPM12176 to detect serum anti-EPM12176 IgM, IgA, and IgG levels in clinical patients. DETAILED DESCRIPTION

[0051] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0052] Proteus mirabilis is widely distributed in nature and on the surface of human and animal bodies and in the gastrointestinal tract. When the host's immune function declines or an exposed wound occurs and Proteus mirabilis has suitable growth conditions, Proteus mirabilis can cause disease. Rheumatic diseases are common diseases involving bones, joints, muscles and surrounding soft tissues, and most of the causes are currently unclear. Among them, rheumatoid arthritis (RA) is a common chronic, highly disabling immune disease. The research of the present invention found that feeding Proteus mirabilis mixed with feed can induce a disease in rabbits with redness and swelling of the small joints of the limbs as the main symptoms. Further combined with symptoms, X-rays, pathology, and non-specific immune index detection, the research found that the symptoms such as arthritis caused were consistent with the clinical symptoms of rheumatoid arthritis, and no inconsistency was found between the symptom characteristics of the experimental rabbits and the clinical rheumatoid arthritis (see Figure 1 , rabbit A and rabbit B are male and female rabbits, respectively). Combining the characteristics of Proteus mirabilis-induced rabbit pathogenic symptoms, X-ray, pathology and non-specific immune indicators, the 1958 ARA, 1987 ACR and 2010 ACR / EULAR RA classification standards were used for classification (diagnosis), and all of them can be classified as RA. After multiple blood cultures, no bacterial growth was found, but serum anti-Proteus mirabilis IgM, IgA, and IgG antibodies were detected, which were significantly increased by 1-10 times compared with before the experiment. At the same time, feces were taken for aerobic dominant flora culture, and about 30% of Proteus mirabilis colonies grew dominantly from time to time. It is inferred that the arthritis caused by Proteus mirabilis should be caused by its virulence factors that are free to the extracellular space, and the arthritis caused by Proteus mirabilis should be closely related to the free virulence factors of Proteus mirabilis.

[0053] The present invention utilizes the biological properties of the outer pili and flagellar virulence factors of Proteus mirabilis, such as easy detachment and release, and adopts a series of biophysical techniques to separate and extract the Proteus mirabilis extract (see Figure 2 ), thereby obtaining a composite antigen substance of the extracellular and secreted virulence factors of Proteus mirabilis, so that the biological effects of the composite antigen substance can be further verified in cell lines or animal models, confirming its role in the diagnostic reagent and preventive and therapeutic drug of Proteus mirabilis pathogenicity.

[0054] Example 1: Preparation and analysis of Proteus mirabilis extract

[0055] (1) extracting a Proteus mirabilis extract, comprising the following steps:

[0056] ① Use a sterile L-stick to scrape the Proteus mirabilis colonies cultured at 37°C for 24 hours on the nutrient agar plate, suspend them in 0.02M PBS (pH 7.4) buffer, and shake them evenly by hand.

[0057] ② Place in a 37℃ incubator and reset the incubator temperature to 56℃. When the temperature rises to 56℃, count for 2 hours to kill bacteria and inactivate enzyme activity. After taking out, place at room temperature and keep the temperature constant for 1 hour.

[0058] ③ Freeze in a -20℃ refrigerator for 3 hours, take it out and place it in a 37℃ environment for 2 hours, then shake it vigorously for 3 minutes to release the extracellular toxic factors and inactivate the enzyme activity.

[0059] ④Repeat ③ steps twice to obtain the inactivated freeze-thawed bacterial suspension of Proteus mirabilis.

[0060] ⑤Under 4℃, ultrasonically vibrate at 15Hz for 3min to further release the extracellular virulence factors and inactivate enzyme activity to obtain the ultrasonically vibrated suspension of Proteus mirabilis ( Figure 3 To show the bacterial morphology at different stages under the microscope).

[0061] ⑥ Centrifuge at 4000rpm for 30min at 4℃ to remove residual bacteria and their macromolecules, including O antigen and polysaccharide capsule; the residual bacteria and macromolecules are precipitated at the bottom of the tube, and the supernatant is retained, which is the primary extract solution of Proteus mirabilis extract.

[0062] ⑦Under 4℃ condition, ultra-high speed centrifuge at 60000rpm for 30min. Non-specific small molecules released from bacteria and mixed in culture medium are all in the supernatant. Remove the supernatant, add 0.02M PBS (pH7.4) buffer solution, shake by hand to obtain the purified solution of Proteus mirabilis extract, and store at -20℃ for later use after aliquoting.

[0063] (2) SDS-PAGE analysis of Proteus mirabilis extracts

[0064] Take 30 μL of the purified solution of the extracellular and exocytic composite antigens of Proteus mirabilis, add 30 μL of 2×SDS loading buffer, mix well and take 20 μL for SDS-PAGE electrophoresis analysis, the separation gel concentration is 12%.

[0065] (3) Analysis of molecular weight and distribution of proteins extracted from Proteus mirabilis

[0066] ① Determine by using the double standard molecular weight mean ratio positioning method. Draw a straight line in the center of all the strips, take two standard molecules near the molecular weight molecule to be measured, whose molecular weight mean is exactly the molecular weight of the standard molecule, measure the vertical distance between the mean standard molecular line and the other two standard molecular lines and calculate the ratio. Under the premise that the molecular line to be measured is between the mean molecular line and the other standard molecular line, use the ratio to calculate and determine the position of the mean molecular line and the molecular weight mean line of the other standard molecular line. And so on, until the mean molecular line completely overlaps with the molecular line to be measured, the mean molecular weight is the molecular weight of the molecule to be measured.

[0067] ②Use the pipette tool (I) of PHotoshopCS6 software to measure the grayscale (blue light) of the SDS-PAGE protein map. Click the left mouse button on the map test point to display the grayscale value of the point and record the blue light grayscale value. Calculation formula:

[0068] Grayscale of a molecular substance stripe = (grayscale average of three random points before the stripe - grayscale average of three random points in the stripe) × width of the stripe (mm).

[0069] The higher the gray value of the band, the higher the protein content of the band, thus obtaining the approximate distribution of various major protein substances in the electrophoresis spectrum.

[0070] (4) Results analysis

[0071] SDS-PAGE analysis of Proteus mirabilis extracts showed (see Figure 4 b) The extract of Proteus mirabilis contains antigenic substances with a molecular weight of 12 kDa-176 kDa. Figure 5 It can be seen that the content of substances with a molecular weight of 12kDa-16kDa in the extract of Proteus mirabilis is the highest, reaching 28.15%; followed by substances with a molecular weight of 25-26kDa, 36kDa and 69-71kDa, all of which are above 10%, namely 15.08%, 13.57% and 13.07% respectively, and the above four molecular weight substances account for about 69.87% of the total; the content of substances with a molecular weight of 43kDa, 33kDa and 31kDa is about 5%, namely 6.86%, 5.51% and 4.58% respectively; finally, the content of substances with a molecular weight of 176kDa, 105kDa, 81kDa, 62kDa, 57kDa, 54kDa and 46kDa is relatively low, all of which are below 3%, namely 1.9%, 2.71%, 1.35%, 1.09%, 2.41%, 0.99% and 2.71% respectively.

[0072] Example 2: Analysis of Antigenic Properties of Proteus mirabilis Extracts

[0073] (1) Total antigen performance analysis

[0074] Take the Proteus mirabilis extract solution, dilute it with 0.02M PBS buffer (pH7.4) to 1mg / mL, and inject it into the ear vein of the immunized rabbits. The first injection is 0.5mL, and 1.0mL is injected on the 3rd, 5th, and 7th days respectively, and 1.5mL is injected on the 9th day. About three weeks later, blood is collected from the central ear artery and serum is separated. The serum is diluted with normal saline at a ratio of 1:4×1.0 2 The dilution ratio was 1:5.12×10 4 , and the suspension of Proteus mirabilis (3×10 7 CFU / mL) agglutination reaction titer, and the immune serum of Proteus mirabilis whole bacterial suspension was used as control.

[0075] (2) Antigen specificity analysis

[0076] Take 20 portions (15 μL / portion) of serum from rabbits immunized with Proteus mirabilis extract and add them to clean glass slides respectively. Use another inoculation loop to aseptically take blood plates cultured for 24 hours, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Yersinia, Citrobacter freundii, Serratia liquefaciens, Klebsiella pneumoniae, Enterobacter agglomerans, Enterobacter aerogenes, Pseudomonas alcaligenes, Shigella flexneri, Aeromonas sobria, Proteus vulgaris, Trichoderma arizona, Pseudomonas cepacia, Streptococcus faecalis, Micrococcus luteus, Bacillus subtilis, Streptococcus bovis and Streptococcus equine, and mix them with the serum to observe whether there is agglutination reaction.

[0077] (3) Analysis of specific antigen components and their properties

[0078] ① Analysis of flagellin antigen and its performance: The serum of rabbits immunized with the composite antigens of Proteus mirabilis was incubated in the microwells of the ELISA plate at a ratio of 1:1.0×10 2 The dilution ratio was 1:2.56×10 4 Then, 100 μL of freshly prepared 3×10 7 After gently shaking and mixing the CFU / mL Proteus mirabilis suspension, apply a drop on a glass slide, cover it with a coverslip, and observe under a microscope. If no obvious displacement of the bacteria is observed, the inhibition test is positive.

[0079] ② Analysis of exocytic protein antigens and their performance: Inoculate Proteus mirabilis in broth culture medium, culture at 37℃ for 16h (logarithmic growth period), then centrifuge at 5000rpm for 10min, take the supernatant, filter and sterilize with a 0.22μm water system PES filter membrane, which is the sterile supernatant. Take part of the sterile supernatant, keep it in a 56℃ incubator for 2h, freeze it at -20℃, and repeat freezing and thawing 3 times to inactivate the active enzyme activity, which is the inactivated supernatant. Secondly, take 3 clean transparent small test tubes, add 1.0mL of sterile supernatant, inactivated supernatant, and broth culture medium (negative control) respectively, add 20μL of rabbit serum immunized with exocytic composite antigens of Proteus mirabilis to each tube, shake gently, place at 37℃ for 10min, adjust to zero with broth tube liquid, detect turbidity at 340nm wavelength, and read OD value.

[0080] ③ Analysis of O antigen, capsular antigen and its performance: Take the concentrated suspension of Proteus mirabilis and place it in a 100℃ water bath for 1h, cool it to room temperature and dilute it to about 3×10 7 CFU / mL, take 15μL on a clean glass slide, take another 15μL of Proteus mirabilis extract immune serum and mix it, shake gently to mix, and observe whether there is agglutination reaction.

[0081] (4) Results analysis

[0082] ① The titer of agglutination reaction of rabbit serum immunized with Proteus mirabilis extract was 1:6.4×10 3 (See Figure 6 a) As a control, the titer of rabbit serum immunized with whole bacterial suspension of Proteus mirabilis was 1:3.2×10 3 (See Figure 6 b), indicating that the total antigenic properties of Proteus mirabilis extract are excellent.

[0083] ② There was no agglutination reaction between rabbit serum immunized with Proteus mirabilis extract and 20 other common clinical bacteria, indicating good antigen specificity.

[0084] ③ The inhibitory titer of serum from rabbits immunized with Proteus mirabilis extract to inhibit the displacement of Proteus mirabilis reached 1:6.4×10 3 , indicating that the flagellin antigen performance of Proteus mirabilis extract is excellent.

[0085] ④ The OD values ​​of the reaction between the rabbit serum immunized with Proteus mirabilis extract and the sterile supernatant and inactivated supernatant of Proteus mirabilis culture were 0.1442 and 0.143, respectively. This shows that the exoprotein antigens of Proteus mirabilis contained in the extract of Proteus mirabilis have excellent performance. At the same time, 56℃ preservation and repeated freezing and thawing have no effect on the performance of the exoprotein antigens of Proteus mirabilis, and the immune serum has a good binding reaction with the exoprotein antigens of the active enzyme.

[0086] ⑤ There was no agglutination reaction between the rabbit serum immunized with Proteus mirabilis extract and Proteus mirabilis after being heated in a 100℃ water bath, indicating that the Proteus mirabilis extract did not contain O antigen or capsular antigen.

[0087] Example 3: Application research of Proteus mirabilis extract

[0088] Preparation of coated plates: Take the Proteus mirabilis extract solution, dilute it to a certain concentration, and coat the 96-well ELISA plate. After washing, blocking, washing, sealing and storing at -4°C for future use.

[0089] (1) Animal experiments

[0090] ① Experimental method: First, 1 mL of blood was collected before the animal experiment, and serum was separated for control experiment. Proteus mirabilis was mixed with food and fed to rabbits to make RA model. Blood was collected through the central auricular artery at different times to separate serum for later use. During the test, the coated microplate was taken, and rabbit serum before and after the experiment was added, and then HRP-sheep anti-rabbit IgM, IgG, and IgA antibodies were added, and then color was developed and OD450 was measured. Compared with before the experiment, OD / OD 实验前 >2.0 is positive, and the larger the ratio, the higher the antibody level.

[0091] ② Experimental results: Rabbits A and B showed symptoms such as occasional kicking of the front legs, redness and swelling of the small joints of the limbs, avoidance of touch, and subcutaneous nodules, which are consistent with the symptoms of rheumatoid arthritis (see Figure 1 ). From Table 1 and Figure 7 It can be seen that the serum anti-PM IgM antibody of rabbit A increased significantly on the 32nd day of the experiment, 1.54 times higher than before the experiment, and increased sharply on the 60th day, 13.67 times higher than before the experiment, and then decreased rapidly, and the increase was not obvious on the 72nd day of the experiment; rabbit B increased sharply on the 32nd day compared with before the experiment, with an amplitude of 10.76 times, and then decreased rapidly, and increased significantly by 1.44 times on the 60th day, and still increased on the 72nd day, but not significantly. Anti-PM IgA antibody of rabbit A increased sharply on the 32nd and 60th days of the experiment, 7.28 times and 9.55 times higher than before the experiment, respectively, and still increased significantly on the 72nd day, 2.06 times higher than before the experiment; rabbit B increased more sharply on the 32nd day compared with before the experiment, with an amplitude of 4.61 times, and then the increase gradually decreased, and increased significantly by 2.73 times and 1.18 times on the 60th day and 72nd day, respectively. The anti-PM IgG antibody level of rabbit A showed almost no change on the 32nd day compared with that before the experiment, but increased sharply on the 60th day, increasing by 7.31 times compared with that before the experiment, and then decreased rapidly, falling to close to the level before the experiment on the 72nd day; the anti-PM IgG antibody level of rabbit B increased sharply on the 32nd day compared with that before the experiment, with an amplitude of 5.77 times, and then the increase decreased rapidly, increasing significantly by 1.34 times on the 60th day, and returned to the level before the experiment on the 72nd day.

[0092] Table 1 Levels and changes of each subtype of anti-Pm antibody in experimental rabbits

[0093]

[0094] The above experimental results show that Proteus mirabilis invasion of the gastrointestinal tract can cause diseases such as rheumatoid arthritis symptoms, and can also cause a significant increase in the levels of anti-PM IgM, IgG, and IgA antibodies in the blood. Therefore, the levels of serum anti-PM IgM, IgG, and IgA antibodies can be used as important diagnostic indicators for Proteus mirabilis pathogenicity, including rheumatoid arthritis. Proteus mirabilis extract is an extracellular and extra-secretory composite antigen substance of the virulence factor of Proteus mirabilis. As an antigen reagent for detecting the levels of anti-PM IgM, IgG, and IgA antibodies, it is beneficial to significantly increase the sensitivity and accuracy of the diagnosis of Proteus mirabilis pathogenicity (including RA) compared with the usual Proteus mirabilis whole bacterial antigen or Proteus mirabilis bacterial suspension antigen that is mainly O antigen and has low extracellular and extra-secretory antigen content.

[0095] (2) Clinical testing experiments

[0096] ① Research subjects: 35 patients with rheumatoid arthritis provided by the Department of Traditional Chinese Medicine Rheumatology of a tertiary hospital in Chongqing were collected, blood samples were taken, serum was collected, and 21 patients with rheumatism other than rheumatoid arthritis in the same department, including ankylosing spondylitis, psoriatic arthritis, inflammatory bowel arthritis, reactive arthritis, neuropathic arthropathy, synovial sarcoma, etc., were collected as controls. Blood samples of 20 healthy subjects who excluded various joint diseases were collected as healthy controls. All research subjects were informed of this study and signed the informed consent form, which was approved by the hospital's organizational ethics committee.

[0097] ② Experimental method: Take the coated plate, add the serum of rheumatoid arthritis (RA) patients, non-RA patients and healthy subjects, and add HRP-sheep anti-human IgM, IgG, or IgA antibody after reaction. After reaction, add colorimetric agent, adjust the well to zero with distilled water, and measure OD450. SPSS20.0 software was used for statistical analysis of the measured data, variance analysis was used for comparison of means within the group, and t-test was used for comparison between groups. P < 0.05 indicated that the difference was statistically significant.

[0098] ③ Experimental results: see Table 2 and Figure 8The clinical RA group was compared with the healthy physical examination group and the non-RA rheumatism group, and the serum anti-PM-IgM, IgA, and IgG levels were significantly increased, and the differences were statistically significant (P < 0.0001, P < 0.0001, and P < 0.05, respectively). This shows that the serum anti-PM-IgM, IgA, and IgG levels should have the function of diagnosing rheumatoid arthritis and differentially diagnosing other rheumatic diseases other than rheumatoid arthritis, and the Proteus mirabilis extract provided by the present invention can be used as an antigen reagent for detecting serum anti-PM-IgM, IgA, and IgG levels.

[0099] The above results further indicate that the Proteus mirabilis extract provided by the present invention, as an antigen reagent, will be beneficial to increase the sensitivity and accuracy of Proteus mirabilis pathogenic diagnosis compared to the whole bacterial suspension antigen.

[0100] Table 2 Clinical patient serum anti-PM-IgM, IgA, IgG OD detection analysis (x±SD)

[0101]

[0102]

[0103] The above results indicate that the Proteus mirabilis extract provided by the present invention can be used as an antigen substance for diagnostic reagents to detect the levels of anti-PM-IgM, IgA, and IgG in human or animal serum, thereby playing an important role in the diagnosis and differential diagnosis of diseases such as urinary tract infection, nosocomial infection, gastroenteritis (diarrhea), and rheumatoid arthritis caused by Proteus mirabilis infection and colonization.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of a Proteus mirabilis extract in the preparation of a product for detecting the pathogenicity of Proteus mirabilis, characterized in that: The Proteus mirabilis extract includes extracellular and extracrine composite antigens; wherein the extracellular antigens include flagella antigens and pili antigens; and the extracrine antigens include proteolytic enzymes, hemolysins, hyaluronidase and phospholipase.

2. The use of a Proteus mirabilis extract in the preparation of a vaccine for preventing and treating Proteus mirabilis pathogenicity, characterized in that: The Proteus mirabilis extract includes extracellular and extracrine composite antigens; wherein the extracellular antigens include flagella antigens and pili antigens; and the extracrine antigens include proteolytic enzymes, hemolysins, hyaluronidase and phospholipase.

3. The use of a Proteus mirabilis extract in screening candidate detection and prevention products for pathogenic Proteus mirabilis, characterized in that: The Proteus mirabilis extract includes extracellular and extracrine composite antigens; wherein the extracellular antigens include flagellar antigens and fimbriae antigens; the extracrine antigens include proteases, hemolysins, hyaluronidases and phospholipases; the detection product includes at least one of a kit, a reagent strip, and a chip; the prevention and treatment product includes at least one of a medicine, a biological product, and a medical device.

4. The use according to any one of claims 1 to 3, characterized in that: The pathogenicity caused by Proteus mirabilis is at least one of urinary tract infection, nosocomial infection, gastroenteritis and arthritis caused by Proteus mirabilis.

5. The use according to claim 1, characterized in that: The product is at least one of a test kit, a reagent strip and a chip.

6. The use according to claim 5, characterized in that: The kit comprises at least one of a Proteus mirabilis extract, a microplate coated with a Proteus mirabilis extract, a turbidity reaction tube for a Proteus mirabilis extract, antibodies against IgM, IgA and IgG from a Proteus mirabilis extract, rheumatoid factors against IgM, IgA and IgG from a Proteus mirabilis extract, and enzymes, fluorescein, biotin, radioactive element markers, a microplate coated with a Proteus mirabilis extract, antibodies against IgM, IgA and IgG from a Proteus mirabilis extract, and rheumatoid factors against IgM, IgA and IgG from a Proteus mirabilis extract.

7. The use according to claim 5, characterized in that: The reagent strip comprises at least one of a Proteus mirabilis extract, anti-Proteus mirabilis extract IgM, IgA, IgG antibodies, anti-Proteus mirabilis extract IgM, IgA, IgG rheumatoid factor and its enzyme, fluorescein, biotin, and radioactive element markers.

8. The use according to any one of claims 1 to 3, characterized in that: The Proteus mirabilis extract is at least one of a Proteus mirabilis fermentation extract and a Proteus mirabilis colony extract.

9. The use according to claim 8, characterized in that: The Proteus mirabilis colony extract is prepared by the following method: (1) scraping Proteus mirabilis colonies and then suspending them; (2) keeping the product obtained in step (1) at 55-60° C.; (3) freezing and thawing the product obtained in step (2) 1-3 times; (4) ultrasonically melting the product obtained in step (3) and centrifuging it to retain the supernatant; (5) subjecting the product obtained in step (4) to ultrahigh-speed centrifugation, removing the supernatant and adding a buffer solution to obtain.

10. The use according to claim 9, characterized in that: The insulation time in step (2) is 2-4 hours; the frequency of ultrasonic melting in step (4) is 10-30 Hz; the ultra-high-speed centrifugation in step (5) is 50000-80000 rpm for 10-60 minutes.

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