MyxoALP1 protein of myxobacteria and its application

Abstract

The application discloses a myxobacterium MyxoALP1 protein and application thereof. The application obtains a myxobacterium MyxoALP1 protein with an ApeC-like domain in bacteria, the ApeC-like domain of the protein has similar characteristics with ApeC in invertebrates, and four conserved cysteines and three DXED motifs are reserved. Research shows that the myxobacterium MyxoALP1 recombinant protein has the ability of binding and coagulating bacteria and fungi, can bind various bacteria well, has broad-spectrum binding and coagulation activity, can be used for coagulation and removal of bacteria and fungi through the binding and coagulation of bacteria, can be used for preparing a drug for treating bacterial infectious diseases, and provides a new selection for the development of a product and a medicament for binding and coagulating bacteria.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology. More specifically, it relates to the MyxoALP1 protein of myxobacteria and its applications. Background Technology

[0002] Bacterial infections are a common category of diseases. Due to the vast number of bacterial species, which can cause various illnesses, there is no unified epidemiological data. Patients with bacterial infections, carriers, and infected animals can serve as sources of infection. The transmission routes of bacterial infections include exogenous and endogenous routes. Exogenous routes refer to infection through other bacteria infecting patients, carriers, or infected animals; endogenous routes mainly refer to the presence of pathogens in the patient's own body, which can cause disease when the body's resistance is lowered. Because of the complexity of bacteria, they can spread between people, between people and the environment, or between animals and humans through various transmission routes. Furthermore, because bacteria in the environment are difficult to detect and eliminate, they can easily infect humans or animals. Therefore, eliminating pathogens carried by the environment, humans, and animals is particularly important.

[0003] In 2014, a domain of approximately 200 amino acids, ApeC (Apextrin C-terminal), was first identified in the genome of the amphioxus. Proteins containing the ApeC domain are called ACP (ApeC Containing Protein). The amphioxus ACP can recognize DAP-type and Lys-type peptidoglycans and also has the function of agglutinating bacteria. This protein has also been found in oysters (Crossostrea gigas), where it acts as a pattern recognition protein to assist the body in generating an immune response. The ACP molecule in the Manila clam (Ruditapes philippinarum) can inhibit bacterial growth, increase nitric oxide activity, and activate the NF-κB signaling pathway. The ACP molecule also participates in the embryonic development of sea urchins (Heliocidaris erythrogramma), being specifically expressed in the ectoderm to cope with the enormous stress generated during embryonic development; therefore, it is also known as apical extracellular matrix protein (Apextrin). Following fertilization, the expression of two ACP molecules, ApelB and ApelP, in mussels (Mytilus galloprovincialis) is rapidly upregulated, but their expression remains relatively stable in adulthood. This indicates that current research on ACP function is primarily focused on invertebrates, with limited coverage of ACP proteins from other sources, particularly bacterial ApeC-like domains, whose functional roles in bacteria remain unknown. Therefore, to provide more products for resisting pathogen invasion and eliminating pathogenic bacteria and fungi, it is necessary to develop more potential functional proteins from diverse bacterial sources for bacterial binding and agglutination, facilitating pathogen clearance by the body and providing more options for the development of products and drugs that bind and agglutinate bacteria. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing research on ApeC-like domains from existing bacterial sources and to provide the MyxoALP1 protein from myxobacteria and its applications.

[0005] The purpose of this invention is to provide a myxobacterial MyxoALP1 protein and its encoded nucleotide sequence.

[0006] Another objective of this invention is to provide applications of the MyxoALP1 protein from myxobacteria and its recombinant protein.

[0007] Another object of the present invention is to provide an agent for binding and / or agglutinating bacteria or fungi.

[0008] Another object of the present invention is to provide a drug for treating bacterial infectious diseases.

[0009] Another object of the present invention is to provide a method for combining and / or agglutinating bacteria for the purpose of non-disease treatment diagnosis.

[0010] Another object of the present invention is to provide a method for combining and / or agglutinating yeast fungi for the purpose of non-disease treatment diagnosis.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] The present invention provides a myxobacterial MyxoALP1 protein, the amino acid sequence of which is shown in SEQ ID NO: 2, and the nucleotide sequence encoding the MyxoALP1 protein is shown in SEQ ID NO: 1.

[0013] This invention yields a myxobacterial MyxoALP1 protein with an ApeC-like domain. This ApeC-like domain shares similarities with ApeC in invertebrates, retaining four conserved cysteine ​​residues and three DXED motifs. Research shows that the recombinant myxobacterial MyxoALP1 protein possesses the ability to aggregate and coagulate bacteria, effectively binding to a variety of bacteria and fungi, such as Gram-positive Staphylococcus aureus and Enterococcus faecalis; Gram-negative bacteria like Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus, and Acinetobacter calcium acetate; and fungi like Saccharomyces cerevisiae. It exhibits broad-spectrum binding and coagulation activity, enabling the aggregation and removal of bacteria. This allows for the preparation of drugs for treating bacterial infectious diseases and provides new options for the development of bacterial binding and coagulation products and pharmaceuticals.

[0014] This invention provides the use of MyxoALP1 protein or recombinant protein of myxobacteria for binding and / or agglutinating bacteria and / or fungi for non-disease diagnostic and therapeutic purposes.

[0015] This invention provides the use of MyxoALP1 protein or recombinant protein of the myxobacterium in the preparation of formulations that bind and / or agglutinate bacteria or fungi.

[0016] This invention provides the use of MyxoALP1 protein or recombinant protein of the myxobacterium in the preparation of medicaments for treating bacterial infectious diseases.

[0017] Preferably, the bacteria are Gram-positive and Gram-negative bacteria.

[0018] More preferably, the bacteria are one or more of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, Vibrio paraheamolyticus, and Acinetobacter caloacetius.

[0019] Preferably, the fungus is *Saccharomyces cerevisiae*.

[0020] The present invention provides an preparation for binding and / or agglutinating bacteria or fungi, containing the MyxoALP1 protein or its recombinant protein as described above.

[0021] This invention provides a drug for treating bacterial infectious diseases, containing MyxoALP1 protein or its recombinant protein.

[0022] This invention provides a method for binding and / or agglutinating bacteria for non-disease treatment and diagnosis purposes, using MyxoALP1 protein or its recombinant protein to bind or agglutinate bacteria.

[0023] Preferably, the bacteria are one or more of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, Vibrio paraheamolyticus, and Acinetobacter caloacetius.

[0024] This invention provides a method for combining and / or agglutinating yeast fungi for non-disease treatment and diagnosis purposes, using MyxoALP1 protein or its recombinant protein, or the above-mentioned preparations to treat the yeast fungi.

[0025] Preferably, the yeast fungus is Saccharomyces cerevisiae.

[0026] In particular, the present invention shows that the MyxoALP1 protein of myxobacteria or its recombinant protein can be used to bind to and eliminate bacteria. Therefore, the present invention is not limited to binding, agglutinating and eliminating bacteria in the environment, but can also be used for the preparation of drugs for treating diseases caused by bacterial infections.

[0027] The present invention has the following beneficial effects:

[0028] This invention provides the MyxoALP1 protein from myxobacteria and its applications. Studies show that the recombinant MyxoALP1 protein can bind well to a variety of bacteria and fungi, exhibiting broad-spectrum binding activity. Through its binding and agglutination abilities, it can be used to agglutinate and remove bacteria and fungi, providing more methods for binding and eliminating bacteria and for the preparation of drugs for treating bacterial infectious diseases. The recombinant MyxoALP1 protein provided by this invention can bind and agglutinate bacteria and fungi, which is of great significance for the preparation of bacterial scavengers or drugs for treating bacterial infectious diseases, providing new options for product and drug development. Attached Figure Description

[0029] Figure 1 Figure showing the results of the predicted structure analysis of the MyxoALP1 protein from myxobacteria.

[0030] Figure 2 The figure shows the comparison results of MyxoALP1 protein from myxobacteria and ACP proteins from other invertebrate species.

[0031] Figure 3 This is a diagram of the restriction sites of plasmid pET32a(+).

[0032] Figure 4 This is a diagram showing the construction of the recombinant expression plasmid pET32a(+)-MyxoALP1 for the MyxoALP1 protein.

[0033] Figure 5 Electrophoresis images of recombinant protein MyxoALP1 after induced expression and purification (lane 1: protein marker; lane 2: uninduced TRX-MyxoALP1 sonicated precipitate; lane 3: induced MyxoALP1 sonicated mixture; lane 4: induced MyxoALP1 sonicated supernatant; lane 5: induced MyxoALP1 sonicated precipitate; lane 6: purified TRX-MyxoALP1).

[0034] Figure 6 The image shows the results of a bacterial binding assay for the recombinant protein MyxoALP1.

[0035] Figure 7 The image shows the results of a bacterial agglutination experiment of the recombinant protein MyxoALP1. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038]

[0039] The amino acid sequence SEQ ID NO: 2 of the MyxoALP1 protein of myxobacteria is: MRNVVRGLVV WALCSTQLGCGQEDPSPSHVEPAIEAVASSSAPLSTNDVGVIAPEGGCPAFAPTVWIHMDNEDSRQSSYVSDWVGATQVNGSGNTTFTFCRVDGNKFKALSTSASPLSNFALLKLGASCPAGAAEFSRYFDNEDRDNMNSNSNSANIWPNIQGRRDTTLTFCLFTGATTPASSL PILEFDYGVFATPYFRFGKGAGVIFTDDEDTRNANAYTVAYDFQWAAKAIVTEGGNTTLYTARAAYVYCGDNTCNAEEGERSCPSDCTVCGNGICGPRETASSCADDCGWCGDGVCSNSEDSRTCGSDCARCGDFACNGGETPQSCPLDCNCNDLTSSPDGLIPTCPGPNG.

[0040] Example 1: Determination and structural analysis of the MyxoALP1 protein gene sequence in myxobacteria

[0041] Current functional studies on ACPs have focused primarily on invertebrates. This application uses the ApeC domain sequence of amphioxus ACP1 as the input sequence and searches bacterial genome databases using PSI-blast. The results show that ApeC-like structures exist in five bacterial phyla: Fibrobacteres, Bacteroidetes, Proteobacteria, Actinobacteria, and Acidobacteria. Further, genomic DNA was extracted and analyzed from myxobacteria of the genus *Myxococcus*. The obtained target fragment was ligated into the pUC57 Simple Vector cloning vector and transformed into *E. coli* DH5α. Recombinant clones were then selected for sequencing. The full-length genome sequence of the CDS region used in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After sequencing analysis, the full-length genome sequence was obtained, and subsequent SMART prediction analysis results are shown below. Figure 1 The results showed that the protein encoded by this gene is composed of an N-terminal signal peptide and a C-terminal ApeC domain, and it was named MyxoALP1. Analysis revealed that the MyxoALP1 gene is 1068 bp in length and encodes 355 amino acids. Its gene DNA sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0042] Further analysis using MEGA software compared the homologous sequences of ACP proteins from different known invertebrate species, and the results are as follows: Figure 2 As shown, the ApeC-like domain of myxobacteria retains four conserved cysteine ​​residues and three DXED motifs.

[0043] Example 2: Construction of recombinant pET32a-MyxoALP1 expression plasmid

[0044] Using the UC57 Simple plasmid containing the MyxoALP1 gene from Example 1 as a template, a full-length prokaryotic recombinant protein of MyxoALP1 was prepared at Abmart. The pET32a(+) vector containing a thioredoxin (TRX) fusion system was used, as shown in the figure. Figure 3 As shown, the recombinant expression vector pET32a(+)-MyxoALP1 was purified on NTA chelate agarose magnetic beads using its contained 6×His tag. The construction process is as follows: Figure 4 As shown, the exogenous gene sequence in the constructed expression vector pET32a(+)-MyxoALP1 was confirmed to be correct by sequencing.

[0045] Example 3: Expression and purification of the myxobacterial MyxoALP1 fusion protein

[0046] The pET32a(+)-MyxoALP1 plasmid constructed in Example 2 was transformed into Escherichia coli BL21(DE3). 1 μL of the culture was inoculated into 40 mL of LB broth containing ampicillin and incubated overnight at 37°C and 220 rpm. The next day, 10 mL of the bacterial culture was inoculated into 400 mL of LB broth containing ampicillin and incubated at 37°C and 220 rpm until OD (dose elapsed). 600 =0.6; 1M IPTG was added to the culture medium at a ratio of 1:1000, resulting in a final concentration of 1mM. Expression was induced at 30℃ for 4 hours; after expression, the cells were centrifuged at 6000 rpm for 5 min. Then, sterile PBS at 4℃ was added at a ratio of 1g bacteria to 5mL PBS, the bacteria were resuspended by shaking, and centrifuged at 4℃ for 6000 rpm for 5 min, washing twice. The supernatant cells were collected and resuspended in 20mL of 20mM Tris buffer (pH 8.0).

[0047] Target protein staining and purification: Small amounts of supernatant and inclusion bodies were collected and stained with Coomassie Brilliant Blue to detect protein expression. The majority of the target protein was found in the supernatant. The supernatant was purified using PuriMag Magarose-NTA-Ni magnetic beads. 4 mL of the magnetic bead suspension was placed on a magnetic separator. After the solution became clear, the supernatant was discarded, and sterile ddH2O was added for three consecutive washes. Binding buffer was added at a volume ratio of 1:2.5, and this process was repeated twice. The supernatant bacterial sonication solution was collected, and magnetic beads were added at a volume ratio of 5:1. The mixture was inverted and incubated at 4°C for 30 min. Washing was performed twice with Wash Buffer at a volume ratio of 1:5. Then, Elution Buffer was added at a volume ratio of 1:2.5. The collected supernatant was the target protein fraction. This process was repeated three times, and the eluent was collected for analysis.

[0048] Concentration and determination of the target protein: Rinse the newly purchased Millipore ultrafiltration centrifuge tubes several times with pre-cooled distilled water. Add the purified protein solution to the ultrafiltration centrifuge tubes and centrifuge at 4000g for 15 min at 4°C. Collect the supernatant (approximately 1 mL). Repeat the above steps until all liquid is concentrated. Store briefly in distilled water at 4°C, and store long-term in 20% ethanol at -20°C. Finally, determine the protein concentration using the Pierce BCA Protein Assay Kit. The final protein concentration is determined by BCA gradient protein electrophoresis grayscale analysis.

[0049] The results of SDS-PAGE electrophoresis analysis of the ultrasonic lysis and precipitation of genetically engineered bacteria are as follows: Figure 5 As shown, the strain exhibits a distinct band of specific expression product in the ultrasonic lysis supernatant and precipitate after induction, yielding purified myxobacterium-MyxoALP1 recombinant protein, which is present in both the supernatant and inclusion bodies, with the majority existing in the supernatant form.

[0050] Example 4: Bacterial binding activity analysis of recombinant protein MyxoALP1 from myxobacteria

[0051] Add approximately 2 × 10⁻⁶ to 1 mL of PBS. 7The sample was incubated with bacteria or fungi and 5 μg of MyxoALP1 protein purified in Example 3. The bacteria included Gram-positive Staphylococcus aureus and Enterococcus faecalis; Gram-negative Escherichia coli, Vibrio anguillarum, Vibrio paraheamolyticus, and Acinetobacter caloacetius; and the fungus Saccharomyces cerevisiae. The mixture was incubated at 4°C for 1 h. The sample was then centrifuged at 15000×g for 10 min, washed four times with PBST (0.1% Tween-20, v / v), resuspended in 80 μL PBS and 20 μL 5× loading buffer, and then boiled at 100°C for 10 min. Western blot analysis of the binding protein was performed using mouse anti-6×His mAb (sigma), with the pET32(a+) empty vector containing the thioredoxin (TRX) fusion system as a blank control.

[0052] Bacterial binding activity of recombinant MyxoALP1 protein from myxobacteria, such as Figure 6 As shown, the MyxoALP1 recombinant protein can bind to bacteria such as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus, and Acinetobacter calcifera, as well as fungi such as Saccharomyces cerevisiae, indicating that the MyxoALP1 recombinant protein has a broad spectrum of binding to microorganisms and can bind to a variety of different bacteria or fungi.

[0053] Example 5: Bacterial agglutination activity analysis of recombinant protein MyxoALP1 from myxobacteria

[0054] 2 μL of the preservative cultures of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, and Saccharomyces cerevisiae from Example 4 were inoculated into 5 mL of suitable liquid culture medium and cultured overnight under suitable conditions. Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were cultured overnight at 37°C on LB medium. The next day, 1.5 mL of bacterial cells (OD) were collected. 600 >1.5), 6000g, centrifuged for 5 min, washed 3 times with sterile PBS; Vibrio anguillarum was cultured overnight at 28℃ on seawater medium and Saccharomyces cerevisiae on YPD medium, then 50 μL of 10 mg / mL FITC was added to each bacterial culture, and the volume was made up to 1 mL with PBS. The cultures were incubated at room temperature in the dark with shaking for about 3 h; the bacterial cells were washed 3-6 times with sterile PBS until the solution was colorless, and the bacteria were resuspended in 1 mL of PBS solution for later use; the bacterial concentration was adjusted to OD0.05. 600=2.0, take 50 μL of FITC-labeled bacteria (final concentration approximately 1 × 10⁻⁶). 7 CFU / ml of bacteria or 1×10 6 CFU / ml yeast) and 10 μg of target protein were co-incubated in a 96-well plate, and PBS was added to 100 μL. After thorough mixing, the mixture was incubated at room temperature in the dark for 2 h. Agglutination activity was observed and photographed under a fluorescence microscope. The pET32(a+) empty vector containing the thioredoxin (TRX) fusion system was used as a blank control.

[0055] The results are as follows Figure 7 As shown, the MyxoALP1 recombinant protein can effectively agglutinate various bacteria and fungi, including Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, and Saccharomyces cerevisiae, and can be used to agglutinate and remove bacteria and fungi.

[0056] In summary, the MyxoALP1 protein obtained in this invention possesses an ApeC domain, retains four conserved cysteine ​​residues and three DXED motifs, exhibiting similar characteristics to ApeC in invertebrates. This invention demonstrates that the recombinant MyxoALP1 protein possesses the ability to aggregate and coagulate bacteria, effectively binding to a variety of bacteria and fungi, such as Gram-positive Staphylococcus aureus and Enterococcus faecalis; Gram-negative bacteria like Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus, and Acinetobacter calcifera; and the fungus Saccharomyces cerevisiae. It exhibits broad-spectrum binding and coagulation activity, enabling the aggregation and removal of bacteria through binding and agglutination. Therefore, the recombinant MyxoALP1 protein can be used to bind and eliminate bacteria, enabling the development of drugs for treating bacterial infectious diseases and providing new options for the development of products and agents that bind and agglutinate bacteria.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a Myxobacterium MyxoALP1 protein or a recombinant protein thereof for binding and / or agglutination of bacteria and / or fungi for purposes other than diagnosis or treatment of disease, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO: 2, and the nucleotide sequence encoding the protein is shown as SEQ ID NO: 1; the bacteria are one or more of Staphylococcus aureus ( Staphylococcus aureus ), Enterococcus faecalis ( Enterococcus faecalis ), Escherichia coli ( Escherichia coli ), Vibrio anguillarum ( Vibro anguillarum ), Vibrio parahaemolyticus ( Vibro paraheamolyticus ), Acinetobacter calcoaceticus ( Acinetobacter caloacetius ); and the fungus is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

2. Use of a Myxobacterium MyxoALP1 protein or a recombinant protein thereof for the preparation of a preparation for binding and / or agglutination of bacteria or fungi, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO: 2, and the nucleotide sequence encoding the protein is shown as SEQ ID NO: 1; the bacteria are one or more of Staphylococcus aureus ( Staphylococcus aureus ), Enterococcus faecalis ( Enterococcus faecalis ), Escherichia coli ( Escherichia coli ), Vibrio anguillarum ( Vibro anguillarum ), Vibrio parahaemolyticus ( Vibro paraheamolyticus ), Acinetobacter calcoaceticus ( Acinetobacter caloacetius ); and the fungus is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

3. Use of a Myxobacterium MyxoALP1 protein or a recombinant protein thereof in the preparation of a medicament for the treatment of a bacterial infectious disease, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO: 2, and the nucleotide sequence encoding the protein is shown as SEQ ID NO: 1; and the bacteria are one or more of Staphylococcus aureus ( Staphylococcus aureus ), Enterococcus faecalis ( Enterococcus faecalis ), Escherichia coli ( Escherichia coli ), Vibrio anguillarum ( Vibro anguillarum ), Vibrio parahaemolyticus ( Vibro paraheamolyticus ), Acinetobacter calcoaceticus ( Acinetobacter caloacetius ).

4. A method of binding and / or agglutinating bacteria for purposes other than disease treatment or diagnosis, characterized in that, The bacteria are one or more of Staphylococcus aureus (Staphylococcus aureus), Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Vibrio anguillarum (Vibrio anguillarum), Vibrio parahaemolyticus (Vibrio parahaemolyticus), and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus). Staphylococcus aureus Enterococcus faecalis Escherichia coli Vibro anguillarum Vibro paraheamolyticus Acinetobacter caloacetius < / s>​​​​​ 5. A method of binding and / or agglutinating yeast fungi for purposes other than disease treatment or diagnosis, characterized in that, The yeast fungus is treated by using myxobacterium MyxoALP1 protein or its recombinant protein; the amino acid sequence of the protein is shown as SEQ ID NO: 2, and the nucleotide sequence of the coding protein is shown as SEQ ID NO: 1.

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