Enterococcus faecium dihydroorotate oxidase protein and application thereof

Abstract

This invention, applicable to the field of biomedical technology, provides a dihydroorotate oxidase protein from Enterococcus faecium and its applications. This invention, for the first time, identifies the acclimation-inducing activity of Enterococcus faecium dihydroorotate oxidase (DHO) and successfully achieves soluble expression of the recombinant DHO protein, eliminating the risk of pathogenicity from live bacteria. Purification facilitates standardized production, improving safety and stability. Studies have demonstrated that a single acclimation immunization with DHO protein significantly reduces bacterial loads in the major organs and blood of mice infected with SS2, increasing their survival rate. It also significantly enhances resistance to APP and HPS, significantly improves clinical manifestations of infection, and significantly reduces bacterial loads in the lungs. This fundamentally strengthens the animals' natural immunity and disease resistance, achieving a broad-spectrum protective effect with a single shot. This invention provides a research foundation for the discovery of new immune-regulating components and offers new insights for anti-infection and vaccine design.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an Enterococcus faecium dihydroorotate oxidase protein and an application thereof. Background Art

[0002] Pig bacterial disease is a complex disease caused by multiple factors and pathogens. Its difficult to effectively control characteristics have caused huge economic losses to the pig industry. Among them, SS2 (Streptococcus suis type 2, Streptococcus suis type2), APP (Actinobacillus pleuropneumoniae, Actinobacillus pleuropneumoniae ), HPS (Haemophilus parasuis, Haemophilus parasuis ) as the primary pathogen, bacterial respiratory diseases in pigs pose a serious threat to the healthy development of the pig industry. These diseases have complex pathogenic components and often manifest clinically as mixed infections or secondary infections with multiple pathogens, significantly increasing the difficulty of disease prevention and control.

[0003] Currently, the primary strategy for farms to combat these diseases is to administer multiple vaccines. However, this approach has numerous drawbacks. Most vaccines are effective only against a single pathogen. When faced with clinically occurring mixed infections or secondary infections involving multiple pathogens, their effectiveness is poor, resulting in low protection rates. Furthermore, there is a lack of vaccines that can protect against multiple pathogens. This not only significantly increases prevention and control costs but also reduces the economic benefits of farms, making it difficult to meet actual production needs.

[0004] In recent years, with the continuous deepening of immunological research, new strategies for preventing and controlling diseases by enhancing the natural immunity of animals and improving their disease resistance have received widespread attention. As the body's first immune defense barrier against infection by pathogenic microorganisms, the natural immune response plays a key role in the initial stage of animals' resistance to infection, and the "domesticated immunity" characteristics of the natural immune system give it an immune memory function. From the new perspective of natural immune memory, especially domesticated immunity, enhancing animal natural immunity and improving its disease resistance to achieve a broad-spectrum protection effect of "one shot, multiple protections" has become an effective strategy for animal disease prevention and control. Based on this, in order to address the shortcomings of existing strategies for the prevention and control of bacterial diseases in pigs, the present invention proposes a dihydroorotate oxidase (DHO) protein of Enterococcus faecium and its application, in order to provide a new approach for the prevention and control of bacterial diseases in pigs. Summary of the Invention

[0005] The purpose of the present invention is to provide an Enterococcus faecium dihydroorotate oxidase protein and its application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A dihydroorotate oxidase (DHO) protein from Enterococcus faecium with acclimation immunity induction effect, wherein the amino acid sequence of the DHO protein is shown in SEQ ID NO.2, and the nucleotide sequence encoding the DHO protein is shown in SEQ ID NO.1; the DHO protein is prepared by the following steps:

[0008] A prokaryotic expression vector pET-28a containing the nucleotide sequence encoding the DHO protein was constructed; the prokaryotic expression vector pET-28a was transformed into BL21 (DE3) competent cells and induced for expression; and the DHO protein was obtained by Ni column purification and ultrafiltration concentration.

[0009] The use of the DHO protein as described above in the preparation of an immune product for inducing macrophage acclimation is shown to enhance the phagocytic ability of macrophages and improve the cell survival rate after secondary infection.

[0010] Furthermore, the macrophages are selected from RAW264.7 cells or mouse peritoneal macrophages.

[0011] A use of the DHO protein as described above in the preparation of a drug for preventing or treating porcine bacterial respiratory diseases, wherein the pathogens of porcine bacterial respiratory diseases include SS2, APP and HPS.

[0012] The use of the DHO protein as described above in the preparation of a broad-spectrum anti-bacterial infection product can simultaneously produce a protective effect on SS2, APP and HPS.

[0013] A use of the DHO protein as described above as a vaccine adjuvant in the preparation of bacterial vaccines.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention identifies for the first time that dihydroorotate oxidase (DHO) from Enterococcus faecium has an acclimation-inducing effect and successfully achieves soluble expression of the DHO recombinant protein, avoiding the risk of disease caused by live bacteria. Purification of the active ingredient removes ineffective or even harmful substances, facilitating standardized production and improving safety and stability.

[0016] 2. The present invention first demonstrated, through a macrophage acclimation immunization model, that DHO protein pre-stimulation significantly enhanced the phagocytic capacity of macrophages and increased macrophage survival after secondary infection. The efficacy of DHO protein acclimation immunization was subsequently evaluated using a mouse model. The results showed that a single acclimation immunization with DHO protein significantly reduced bacterial loads in the major organs and blood of mice infected with SS2 and increased their survival rates. It also significantly enhanced the mice's resistance to APP and HPS, significantly improved the clinical manifestations of infection, and significantly reduced bacterial loads in the lungs.

[0017] 3. This invention fundamentally enhances an animal's natural immunity and disease resistance, achieving a broad-spectrum protective effect with a single injection. This not only provides a research foundation for the discovery of new immune-regulating components, but also offers new insights into anti-infection and vaccine design. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of SDS-PAGE electrophoresis analysis of expression products; M: Marker; 1: before induction; 2: after induction; 3: supernatant; 4: precipitate; 5: outflow; 6: 20mM imidazole eluent; 7: 50mM imidazole eluent; 8: 500mM imidazole eluent.

[0019] Figure 2 The results of the evaluation of the effect of DHO protein on acclimating immune cells are shown in Figure 2. A is the evaluation of the phagocytic function of RAW264.7 cells using the neutral red method; B is the evaluation of the phagocytic function of mouse peritoneal macrophages using the neutral red method; C is the detection of cell death during the second bacterial attack SS2 after DHO protein-induced acclimation of RAW264.7 by flow cytometry; D is Figure 2 Quantitative analysis of C in the middle; E is the flow cytometry detection of cell death in peritoneal macrophages of acclimated mice after DHO protein induction and secondary infection with SS2; F is Figure 2 Quantitative analysis of E.

[0020] Figure 3 The figures show the weight changes, survival rate of mice challenged with SS2 for the second time, and bacterial load results after DHO protein acclimation and immunization; A shows the weight changes of mice during DHO protein acclimation and immunization; B shows the survival rate analysis of mice challenged with SS2 after acclimation and immunization; C shows the changes in bacterial loads in the main organs and blood of mice challenged with SS2 72 hours after DHO protein acclimation and immunization.

[0021] Figure 4These are the test results of the resistance of DHO protein-induced domesticated immunity to other pathogens; A is the clinical symptom score of mice after APP infection; B is the weight change of mice after APP infection; C is the lung bacterial load of mice after APP infection; D is the clinical symptom score of mice after HPS infection; E is the weight change of mice after HPS infection; F is the lung bacterial load of mice after HPS infection. DETAILED DESCRIPTION

[0022] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0023] The nucleotide sequence encoding the DHO protein in the embodiment is shown in SEQ ID NO.1:

[0024]

[0025] The amino acid sequence of DHO protein is shown in SEQ ID NO.2:

[0026] MSLETTFANHIFANPLMNASGVHCMTTQELDELAHSEAGAFITKSCTINERKGNPEPRYFDVPLGSINSMGLPNLGFSYYLEYALAYEKVQENQNQPLFFSIAGMSVQENLEMLEKIEKSGFNGITELNLSCPNVPGKPQLAYDFEATYETLKEVFSIFSKPLGIK LPPYFDFAHFDQMADILNQFPLTYVNAINSVGNGLYIDTEQEAVVIKPKEGFGGIGGEYIKPTALANVRAFYTRLKPEIQIIGTGGIRTGQDAFEHLLCGASMLQIGTELHKEGPEIFSRIIKELTQIMSEKGYTSIDEFKGKLRTISGSLSIRTDELCKIKSESE. The GenBank accession number is WP_134860114.1.

[0027] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0028] Example 1: Construction of DHO protein expression vector and purification and expression of recombinant protein;

[0029] Based on the target protein gene sequence in GenBank, Shanghai Sangon Biotechnology Co., Ltd. was commissioned to construct a His-tagged pET-28a prokaryotic expression vector, using BamHI and XhoI as restriction sites. The identified positive plasmid was transformed into BL21 (DE3) competent cells. A single colony was picked from the plate and inoculated into a liquid medium containing resistance. The cells were cultured overnight at 37°C, then transferred to fresh culture medium at a 1% ratio and cultured in a shaking incubator at 37°C until the OD value of the culture solution reached 0. 600=0.6-0.8, and a sample was retained as the pre-induction bacterial solution. Expression was then induced by adding 0.1% IPTG and incubated at 37°C for 7-8 hours. After the incubation period, a sample was retained as the post-induction bacterial solution. The bacterial solution was then centrifuged at 8000 rpm for 10 minutes, the supernatant discarded, and the cells resuspended in 30-50 ml of buffer. The resuspended bacterial solution was placed on ice and ultrasonically disrupted (200 W, 4 seconds / cycle) until the solution became clear. After disruption, the solution was centrifuged again at 12000 rpm for 10 minutes, the supernatant collected, and the precipitate retained. Finally, the DHO protein was purified using Ni column resin according to the manufacturer's instructions. The purified protein was concentrated by ultrafiltration, and the concentration was determined using the BCA assay.

[0030] The expression products were analyzed by SDS-PAGE electrophoresis. Figure 1 As shown, in the 500mM imidazole eluate sample, a distinct and relatively single protein band at approximately 40kDa is observed, with relatively few contaminants, consistent with the expected molecular weight of the DHO protein. Comparing the pre- and post-induction samples, the band at this position is significantly enhanced, indicating successful induction of target protein expression. The clear band in the 500mM imidazole eluate sample indicates that the Ni column purification yielded a relatively pure DHO protein suitable for subsequent experiments.

[0031] Example 2: Evaluation of the effect of DHO protein on acclimating immune cells;

[0032] 2.1 Evaluation of macrophage phagocytic ability;

[0033] Macrophages are among the first cells in the immune system to participate in immune responses. They play a crucial role in defending against infection, and their phagocytic capacity is crucial. To evaluate the effect of DHO protein on macrophage phagocytosis, 7,000 RAW264.7 cells were seeded per well of a 96-well plate. After overnight incubation, the supernatant was discarded and 100 μL of 100 μg / mL DHO protein was added to each well. A control group containing only blank culture medium was also set up. Three replicate wells were incubated for 24 hours, and the supernatant was discarded. 100 μL of 0.1% neutral red solution was added to each well and incubated at 37°C for 2 hours. The neutral red solution was then discarded, and the cells were washed three times with PBS to remove residual dye. Subsequently, 100 μL of cell lysis buffer (a 1:1 volume ratio of glacial acetic acid and ethanol) was added to each well and gently shaken at room temperature for 2 hours. The absorbance of the solution was measured at 620 nm, and the neutral red phagocytosis rate was calculated according to the formula.

[0034] Neutral red phagocytosis rate / %=(OD 620 Value / Blank Group OD 620 value) × 100.

[0035] like Figure 2 As shown in A and B, compared with untamed macrophages, the phagocytic ability of RAW264.7 cells and mouse peritoneal macrophages tamed with DHO protein was improved for neutral red dye, indicating that DHO protein can effectively enhance the macrophages' ability to take up exogenous substances and improve their immune defense function.

[0036] 2.2 Macrophage survival rate after secondary challenge;

[0037] After incubating the cells with DHO protein for 1 day (the control group was treated simultaneously with an equal volume of saline), the stimulus was removed and the cells were allowed to rest for 3 days. Macrophages were then infected with SS2 (using SS2 CVCC606) at an MOI of 30 and incubated in a constant temperature incubator for 3 hours. The cells were then rinsed three times with sterile PBS, gently pipetting until all cells were detached, and centrifuged at 1000 rpm / min for 5 minutes to collect them. The cells were resuspended in 1× Annexin-V buffer and stained with PI solution. The cells were then incubated at room temperature in the dark for 15 minutes. The survival of the acclimated macrophages after SS2 infection was assessed by flow cytometry.

[0038] like Figure 2 As shown in Figures CF, the survival rates of RAW264.7 cells and mouse peritoneal macrophages in the trained+SS2 group, after DHO protein acclimation and induction, upon secondary SS2 stimulation were significantly improved compared to the control+SS2 group. This suggests that DHO protein-induced acclimation immunity can enhance macrophage survival under pathogen attack, providing a stronger defense against infection.

[0039] The above results indicate that the domesticated immunity induced by DHO protein not only enhances the phagocytic ability of macrophages, but also improves the survival rate of macrophages after secondary bacterial challenge.

[0040] Example 3: Evaluation of the immune effect of DHO protein on mouse acclimation;

[0041] Mice were female ICR mice, with the experimental animal ethics review number SY202201009, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0042] 3.1 Detection of weight changes and survival rates of mice after acclimation and immunization, and detection of bacterial loads in major organs and blood after secondary challenge;

[0043] Ten ICR mice were randomly divided into two groups, with five mice in each group. Each mouse was given 200 μg of DHO protein by intraperitoneal injection for acclimation and immunization (the control group was treated with 200 μL of normal saline by intraperitoneal injection at the same time). Four days after the completion of acclimation and immunization, each mouse was challenged with 2×LD 50 The mice were inoculated with SS2 (using SS2 CVCC606) bacterial solution. The weight changes and mortality of the mice were observed and recorded. After 72 hours, the bacterial loads in the tissues and organs of the mice in each group were compared.

[0044] like Figure 3 As shown in Figure A, the weight changes of the trained mice after immunization were not significantly different from those of the control group, indicating that DHO protein treatment did not cause physiological abnormalities in mice. Figure 3 As shown in B, the survival rate of the trained+SS2 group reached 60%, which was significantly higher than the 0% of the control+SS2 group, confirming that intraperitoneal injection of DHO protein can significantly increase the survival rate of mice infected with a lethal dose of SS2. Figure 3 As shown in C, compared with the control+SS2 group, the bacterial content in the liver, spleen, lung, kidney and blood of the mice in the trained+SS2 group was significantly reduced, indicating that DHO protein can effectively reduce the bacterial load in various organs and blood of mice after SS2 challenge.

[0045] 3.2 Testing the resistance of DHO protein-induced acclimation immunity to other pathogens;

[0046] The acclimated immune mouse model was constructed in the same manner as in 3.1.

[0047] 20 ICR mice were randomly divided into 4 groups, 5 in each group. The first group was the trained+APP group: the mice were acclimated and immunized, and then 1×LD 50 The second group was the control+APP group: the mice were not acclimated and immunized, but were injected with an equal volume of normal saline, and then 1×LD was dripped into the nose of each mouse. 50 The third group was the trained+HPS group: mice were acclimated and immunized, and then each mouse was intraperitoneally injected with 1×LD 50 The fourth group was the control+HPS group: the mice were not acclimated and immunized, but were injected with an equal volume of normal saline, and then each mouse was intraperitoneally injected with 1×LD 50 HPS.

[0048] Clinical symptoms of mice in each group were observed and scored at 0, 6, 12, and 24 hours after infection. Observation criteria included activity, coat condition, and ocular discharge. Scoring was based on a scale of 0 (normal), 1 (slightly severe), 2 (moderately severe), and 3 (extremely severe). The average value was used. Mouse weight was recorded every 6 hours.

[0049] 24 hours after the infection, 3 mice in each APP and HPS group were euthanized, and their lungs were collected and added into sterile PBS solution to prepare homogenate. After gradient dilution, the homogenate was plated and counted, and the bacterial load in the lungs of mice in each group was compared.

[0050] like Figure 4 As shown in A, the clinical symptom scores of the two groups of mice were the highest 6 hours after APP infection. After 6 hours, the scores gradually decreased over time. The clinical symptoms of the mice in the trained+APP group were significantly milder than those in the control+APP group. This shows that DHO protein domestication immunity can effectively alleviate the clinical symptoms of mice infected with APP. At the same time, the weight loss of mice in the control+APP group was significantly higher than that of mice in the trained+APP group ( Figure 4 Middle B), indicating that DHO protein domestication immunity helps maintain the stable weight of mice after APP infection. The results of the lung bacterial load of mice at 24 hours showed that the lung bacterial load of mice in the trained+APP group was significantly lower than that in the control+APP group ( Figure 4 Middle C), indicating that DHO protein domestication immunization can effectively reduce the bacterial load of APP in the lungs of mice.

[0051] like Figure 4 As shown in Figure D, the clinical symptom scores of both groups of mice were the highest 24 hours after HPS infection. The increase in clinical symptom scores of mice in the trained+HPS group was smaller than that in the control+HPS group, indicating that DHO protein acclimation immunization has a positive effect on alleviating symptoms of mice infected with HPS. At the same time, compared with the control+HPS group, the trained+HPS group had less weight loss ( Figure 4 Middle E), indicating that DHO protein domestication immunity helps reduce the impact of HPS infection on mouse body weight. The results of mouse lung bacterial load at 24 hours showed that the lung bacterial load of mice in the trained+HPS group was significantly lower than that in the control+HPS group ( Figure 4 Middle (F) shows that DHO protein domestication immunization can effectively reduce the bacterial load of HPS in the lungs of mice.

[0052] The above results show that DHO protein domestication immunization can enhance the resistance of mice to APP and HPS, improve the clinical status of mice after infection with pathogens, and significantly reduce the bacterial load in the lungs.

[0053] In summary, the present invention verifies the effectiveness of DHO protein domesticated immunity through cell and animal model systems, laying a solid research foundation for the exploration of new immune regulatory components. It also provides new ideas for anti-infection strategies and vaccine design, and shows potential application value in the prevention and control of porcine bacterial respiratory diseases (such as those caused by SS2, APP, and HPS).

[0054] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A use of DHO protein in the preparation of a drug for preventing or treating porcine bacterial respiratory diseases, characterized in that: The pathogens of the porcine bacterial respiratory disease are Streptococcus suis type 2, Actinobacillus pleuropneumoniae and Haemophilus parasuis; The amino acid sequence of the DHO protein is shown in SEQ ID NO.2, and the nucleotide sequence encoding the DHO protein is shown in SEQ ID NO.

1.

2. A use of DHO protein in the preparation of a broad-spectrum antibacterial infection product, characterized in that: The bacteria are Streptococcus suis type 2, Actinobacillus pleuropneumoniae and Haemophilus parasuis; The amino acid sequence of the DHO protein is shown in SEQ ID NO.2, and the nucleotide sequence encoding the DHO protein is shown in SEQ ID NO.1.

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