MyxoALP1 protein of myxobacteria and application thereof

Abstract

The invention discloses a MyxoALP1 protein of myxobacteria and an application of the MyxoALP1 protein. The myxobacteria MyxoALP1 protein with an ApeC-like structural domain is obtained through research in bacteria, the ApeC-like structural domain of the protein has similar characteristics with ApeC in invertebrates, and four conservative cysteines and three DXED motifs are reserved. Research shows that the myxobacteria MyxoALP1 recombinant protein has the capability of combining and coagulating bacteria and fungi, can be well combined with various bacteria, has broad-spectrum combining and coagulating activity capability, can be used for agglutinating and removing bacteria and fungi by combining and agglutinating bacteria, can be used for preparing medicines for treating bacterial infectious diseases, and has wide application prospects. And a new choice is provided for the development of combination and agglutination bacteria products and medicaments.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and more specifically, relates to a myxobacterium MyxoALP1 protein and an application thereof. Background Art

[0002] Bacterial infection is a relatively common disease. There are many types of bacteria that can cause different diseases, so there is no unified epidemiological data. Patients with bacterial infection, carriers, sick livestock, and carrier animals can serve as sources of infection. The transmission routes of bacterial infection include exogenous and endogenous routes. The exogenous route refers to infection through other bacterial infection patients, carriers, or sick livestock and carrier animals; the endogenous route mainly refers to the presence of pathogens in the patient himself. Generally, when the body's resistance decreases, pathogenic bacteria can cause disease. Bacterial species are complex, and bacteria can cause transmission between people, between people and the environment, or between animals and humans through different transmission routes. Because bacteria in the environment are difficult to detect and remove, they can easily infect humans or animals. Therefore, it is particularly important to remove pathogens carried by the environment, humans, and animals.

[0003] In 2014, a domain ApeC (Apextrin C-terminal) with about 200 amino acids was first identified in the genome of amphioxus. The protein containing the ApeC domain is called ACP (ApeC Containing Protein). The ACP of amphioxus can recognize DAP-type and Lys-type peptidoglycans and has the function of agglutinating bacteria. This protein has also been found in oysters (Crossostrea gigas), as a pattern recognition protein that assists the body in producing immune responses. The ACP molecules of the Philippine curtain clam (Ruditapes philippinarum) can inhibit the growth of bacteria, increase the activity of nitric oxide and activate the NF-κB signaling pathway. The ACP molecule is also involved in the embryonic development of sea urchins (Heliocidaris erythrogramma), and is specifically expressed in the ectoderm to cope with the huge tension generated during embryonic development, so it is also called the apical extracellular matrix protein (Apextrin). After fertilization, the expression of two ACP molecules, ApelB and ApelP, of mussels (Mytilus galloprovincialis) is rapidly upregulated, but their expression is relatively stable after adulthood. It can be seen that the current research on the function of ACP is only concentrated in invertebrates, and there are few reports on ACP proteins from other sources, especially the ApeC-like domain from bacteria, and its functional role in bacteria is also unknown. Therefore, in order to provide more products that can be used to prepare and produce products that resist pathogen invasion and eliminate pathogenic bacteria and fungi, it is necessary to develop more potential functional proteins in bacteria from different sources for bacterial binding and agglutination, to facilitate the body's elimination of pathogens, and to provide more options for the development of products and agents that bind and agglutinate bacteria. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the existing research on ApeC-like domains derived from bacteria, and to provide a myxobacterium MyxoALP1 protein and an application thereof.

[0005] The purpose of the present invention is to provide a myxobacterium MyxoALP1 protein and a nucleotide sequence encoding the protein.

[0006] Another object of the present invention is to provide the application of the myxobacterium MyxoALP1 protein and its recombinant protein.

[0007] Still another object of the present invention is to provide an agent that binds and / or agglutinates bacteria or fungi.

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

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

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

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0013] The present invention obtains a myxobacterium MyxoALP1 protein with an ApeC-like domain in bacteria. The ApeC-like domain of the protein has similar characteristics to ApeC in invertebrates, retaining four conserved cysteines and three DXED motifs. The present invention shows that the recombinant protein of the myxobacterium MyxoALP1 has the ability to aggregate and agglutinate bacteria, and can well bind to a variety of bacteria and fungi, such as Gram-positive Staphylococcus aureus and Enterococcus faecalis; Gram-negative bacteria Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus and Acinetobacter calcoaceticus; and fungi Saccharomyces cerevisiae, etc. It has a broad spectrum of binding and agglutination activity, can be used to agglutinate and remove bacteria through binding and agglutination of bacteria, can be used to prepare drugs for treating bacterial infectious diseases, and provides new options for its binding, agglutination of bacterial products and drug development.

[0014] The present invention provides the use of a myxobacterium MyxoALP1 protein or a recombinant protein thereof in binding and / or agglutinating bacteria and / or fungi for non-disease diagnosis and treatment purposes.

[0015] The present invention provides use of a myxobacterium MyxoALP1 protein or a recombinant protein thereof in preparing a preparation for binding and / or agglutinating bacteria or fungi.

[0016] The present invention provides use of a myxobacterium MyxoALP1 protein or a recombinant protein thereof in preparing a medicine for treating bacterial infectious diseases.

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

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

[0019] Preferably, the fungus is Saccharomyces cerevisiae.

[0020] The present invention provides a preparation for binding and / or agglutinating bacteria or fungi, comprising the MyxoALP1 protein or a recombinant protein thereof.

[0021] The invention provides a medicine for treating bacterial infectious diseases, comprising MyxoALP1 protein or a recombinant protein thereof.

[0022] The present invention provides a method for binding and / or agglutinating bacteria for the purpose of non-disease treatment and diagnosis, wherein MyxoALP1 protein or a recombinant protein thereof is used to bind or agglutinate bacteria.

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

[0024] The present invention provides a method for binding and / or agglutinating yeast fungi for the purpose of non-disease treatment and diagnosis, wherein MyxoALP1 protein or its recombinant protein, or the above-mentioned preparation is used to treat yeast fungi.

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

[0026] In particular, the present invention shows that the myxobacterium MyxoALP1 protein or its recombinant protein can be used to bind and eliminate bacteria, so the present invention is not limited to being used for 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] The present invention provides a myxobacterium MyxoALP1 protein and an application thereof. Studies have shown that the myxobacterium MyxoALP1 recombinant protein can bind to a variety of bacteria and fungi well, has a broad-spectrum binding activity, and can be used to agglutinate and remove bacteria and fungi through the ability to bind and agglutinate bacteria and fungi, providing more methods and means for binding and removing bacteria and for preparing drugs for treating bacterial infectious diseases. The myxobacterium MyxoALP1 recombinant protein provided by the present invention can bind and agglutinate bacteria-binding fungi, and is of great significance for preparing bacterial removers or drugs for treating bacterial infectious diseases, providing new options for the development of its products and agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of protein structure prediction and analysis of Myxobacterium MyxoALP1.

[0030] Figure 2 A comparison diagram of the MyxoALP1 protein from myxobacteria and ACP proteins from other invertebrate species.

[0031] Figure 3 This is the restriction site map of plasmid pET32a(+).

[0032] Figure 4 The diagram shows the construction of the recombinant expression plasmid pET32a(+)-MyxoALP1 of the MyxoALP1 protein.

[0033] Figure 5 This is the electrophoresis diagram of the induced expression and purification of the recombinant protein MyxoALP1 (Lane 1: protein Marker; Lane 2: uninduced TRX-MyxoALP1 ultrasonic precipitate; Lane 3: induced MyxoALP1 ultrasonic mixture; Lane 4: induced MyxoALP1 ultrasonic supernatant; Lane 5: induced MyxoALP1 ultrasonic precipitate; Lane 6: purified TRX-MyxoALP1).

[0034] Figure 6 This is a graph showing the results of a bacterial binding test of the recombinant protein MyxoALP1.

[0035] Figure 7 This is the result of bacterial agglutination experiment of recombinant protein MyxoALP1. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the 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 of the gene sequence and structural analysis of the myxobacterium MyxoALP1 protein

[0041] At present, the functional research on ACP is only concentrated in invertebrates. This application uses the ApeC domain sequence of amphioxus ACP1 as the input sequence and searches the bacterial genome database through PSI-blast. The results show that ApeC-like exists in 5 bacterial phyla, namely Fibrobacteres, Bacteroidetes, Proteobacteria, Actinobacteria and Acidobacteria. Further, the genomic DNA of the myxobacteria of the genus Myxococcus was extracted and analyzed, and the obtained target fragment was connected to the pUC57 Simple Vector cloning vector and transformed into Escherichia coli DH5α, and the recombinant clones were selected for sequencing. The full gene sequence of the CDS region used in the present invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After sequencing analysis, the full-length sequence of the gene was obtained, and then a SMART prediction analysis result was performed. Figure 1 It was shown 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. After analysis, it was found that the full length of the MyxoALP1 gene of Myxobacterium was 1068bp, encoding 355 amino acids. Its gene DNA sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0042] The MEGA analysis software was further used to compare the homologous sequences of ACP proteins from different invertebrate species. Figure 2 As shown, the ApeC-like domain of myxobacteria retains four conserved cysteines and three DXED motifs.

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

[0044] The full-length prokaryotic expression recombinant protein of MyxoALP1 was prepared at Abmart using the UC57 Simple plasmid containing the MyxoALP1 gene in Example 1 as a template. The pET32a(+) vector containing the thioredoxin (TRX) fusion system was used, such as Figure 3 As shown, the 6×His tag contained therein was used for purification on NTA chelated agarose beads. The recombinant expression vector pET32a(+)-MyxoALP1 was obtained, and its construction process is as follows Figure 4 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 myxobacterium MyxoALP1 fusion protein

[0046] The pET32a(+)-MyxoALP1 plasmid constructed in Example 2 was transformed into Escherichia coli BL21(DE3), 1 μL of the inoculum was inoculated into 40 mL of LB culture medium containing ampicillin, and the culture was incubated at 37°C, 220 rpm, overnight; the next day, 10 mL of the culture was inoculated into 400 mL of LB culture medium containing ampicillin, and the culture was incubated at 37°C, 220 rpm, until the OD 600 =0.6; 1M IPTG was added to the culture medium at a ratio of 1:1000, with a final concentration of 1mM, and the expression was induced at 30℃ for 4 hours; after the expression, the mixture was centrifuged at 6000rpm for 5min. Then, sterile PBS at 4℃ was added at a ratio of 1g bacteria: 5mL PBS, the bacteria were resuspended by shaking, and the mixture was centrifuged at 4℃ and 6000rpm for 5min, and the mixture was washed twice. The supernatant was collected and resuspended in 20mL of 20mM Tris buffer (PH8.0).

[0047] Target protein staining and purification: A small amount of supernatant and inclusion body was taken separately, and the expression of protein was detected by Coomassie Brilliant Blue staining technique. Most of the target protein was present in the supernatant. The supernatant was purified using PuriMag Magarose-NTA-Ni magnetic beads. 4 mL of magnetic bead suspension was placed on a magnetic separator for separation. After the solution was clarified, the supernatant was discarded and sterilized ddH2O was added for repeated washing 3 times. Binding Buffer was added at a volume ratio of 1:2.5, and repeated 2 times; the supernatant bacterial ultrasonic disruption solution was taken, and magnetic beads were added at a volume ratio of 5:1, and mixed by inversion, and incubated at 4°C for 30 minutes; Wash Buffer was added at a volume ratio of 1:5 for washing 2 times, and then Elution Buffer was added at a volume ratio of 1:2.5. The supernatant was the target protein component. This was repeated 3 times, and the eluate was collected for detection.

[0048] Concentration and concentration determination of target protein: Wash the newly purchased Millipore ultrafiltration centrifuge tube several times with pre-cooled distilled water, add the purified protein solution to the ultrafiltration centrifuge tube, centrifuge at 4000g for 15 minutes at 4°C, collect the upper liquid (about 1mL); repeat the above steps until all liquids are concentrated. Seal in a 4°C refrigerator with distilled water for a short term, and seal in a -20°C refrigerator with 20% ethanol for a long term. Finally, use Pierce BCA Protein Assay Kit to determine the protein concentration. Finally, use BCA gradient protein electrophoresis grayscale analysis to determine the protein concentration.

[0049] The results of SDS-PAGE electrophoresis analysis of the ultrasonic lysis precipitation of genetically engineered bacteria are as follows Figure 5 As shown, it indicates that after induction, the strain has obvious specific expression product bands in the ultrasonic lysis supernatant and precipitate, and the purified myxobacterium-MyxoALP1 recombinant protein is obtained in both the supernatant and inclusion bodies, and most of it exists in the supernatant.

[0050] Example 4 Analysis of Bacterial Binding Activity of Myxobacterium MyxoALP1 Recombinant Protein

[0051] Add ~2×10 7Bacteria or fungi and 5 μg of MyxoALP1 protein purified in Example 3, wherein the bacteria are Gram-positive Staphylococcus aureus, Enterococcus faecalis; Gram-negative bacteria Escherichia coli, Vibroanguillarum, Vibro paraheamolyticus and Acinetobacter caloacetius; fungi Saccharomyces cerevisiae, incubated at 4°C for 1 hour. The sample was then centrifuged at 15000×g for 10 minutes, the precipitate was washed four times with PBST (0.1% Tween-20, v / v), suspended in 80 μL PBS and 20 μL 5× loading buffer, and then boiled at 100°C for 10 minutes. The bound proteins were analyzed by Western blot using mouse anti-6×His mAb (Sigma), and the pET32(a+) empty vector containing the thioredoxin (TRX) fusion system was used as a blank control.

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

[0053] Example 5 Analysis of bacterial agglutination activity of recombinant protein of myxobacterium MyxoALP1

[0054] 2 μL of the seed-keeping bacteria of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibrio anguillarum, and Saccharomyces cerevisiae in Example 4 were inoculated into 5 mL of appropriate liquid culture medium and cultured overnight under appropriate conditions. Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were cultured in LB medium at 37° C. overnight; 1.5 mL of cells (OD 600 >1.5), 6000g, centrifuged for 5min, washed 3 times with sterile PBS; cultured Vibrio anguillarum with seawater medium and Saccharomyces cerevisiae with YPD medium at 28°C overnight, then added 50μL of 10mg / mL FITC to each bacterium, added PBS to the volume of 1mL, incubated at room temperature with shaking for about 3h in the dark; washed the bacteria 3-6 times with sterile PBS until the solution was colorless, and suspended the bacteria in 1mL PBS solution for later use; adjusted the bacterial concentration to OD 600= 2.0, take 50 μL of FITC-labeled bacteria (final concentration is about 1×10 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, supplemented with PBS to 100 μL, mixed thoroughly, incubated at room temperature in the dark for 2 h, and then the agglutination activity was observed under a fluorescence microscope and photographed. 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 a variety of bacteria and fungi such as 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 myxobacterium MyxoALP1 protein obtained in bacteria by the present invention has an ApeC domain, retains four conserved cysteines and three DXED motifs, and has similar characteristics to ApeC in invertebrates. The present invention shows that the recombinant protein of the myxobacterium MyxoALP1 has the ability to aggregate and agglutinate bacteria, and can well bind to a variety of bacteria and fungi, such as Gram-positive Staphylococcus aureus and Enterococcus faecalis; Gram-negative bacteria Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus and Acinetobacter calcoaceticus; and the fungus Saccharomyces cerevisiae, and has a broad spectrum of binding and agglutination activity, and is used to agglutinate and remove bacteria through binding and agglutination of bacteria. Therefore, the recombinant protein of the myxobacterium MyxoALP1 can be used to bind and remove bacteria, can be used to develop and prepare drugs for the treatment of bacterial infectious diseases, and provides new options for its binding, agglutination of bacterial products and the development of medicaments.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A myxobacterium MyxoALP1 protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

2. A nucleotide sequence encoding the protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

1.

3. Use of the MyxoALP1 protein or its recombinant protein according to claim 1 in binding and / or agglutinating bacteria and / or fungi for non-disease diagnosis and treatment purposes.

4. Use of the MyxoALP1 protein or its recombinant protein according to claim 1 in preparing a preparation for binding and / or agglutinating bacteria or fungi.

5. Use of the MyxoALP1 protein or its recombinant protein according to claim 1 in the preparation of a drug for treating bacterial infectious diseases.

6. The use according to any one of claims 3 to 5, characterized in that: The bacteria are one or more of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibro anguillarum, Vibro paraheamolyticus, and Acinetobacter caloacetius; and the fungus is Saccharomyces cerevisiae.

7. A preparation for binding and / or agglutinating bacteria or fungi, characterized in that Containing the MyxoALP1 protein or its recombinant protein according to claim 1.

8. A drug for treating bacterial infectious diseases, characterized in that: Containing the MyxoALP1 protein or its recombinant protein according to claim 1.

9. A method for binding and / or agglutinating bacteria for purposes other than disease treatment and diagnosis, characterized in that: The MyxoALP1 protein or its recombinant protein according to claim 1 is used to bind or agglutinate bacteria; the bacteria are one or more of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Vibro anguillarum, Vibro paraheamolyticus, and Acinetobacter caloacetius.

10. A method for binding and / or agglutinating yeast fungi for non-disease treatment and diagnosis purposes, characterized in that: The MyxoALP1 protein or its recombinant protein according to claim 1, or the preparation according to claim 7 is used to treat yeast fungi.

Images