CN116410969B - Phage, phage lyase and application thereof - Google Patents
Phage, phage lyase and application thereof Download PDFInfo
- Publication number
- CN116410969B CN116410969B CN202310466379.4A CN202310466379A CN116410969B CN 116410969 B CN116410969 B CN 116410969B CN 202310466379 A CN202310466379 A CN 202310466379A CN 116410969 B CN116410969 B CN 116410969B
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- phage
- staphylococcus aureus
- lyase
- infection
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Classifications
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Abstract
The invention relates to the technical field of microorganisms, in particular to phage, phage lyase and application thereof. The phage lyase provided by the invention has an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having substantially the same enzymatic activity; the invention discovers that the staphylococcus aureus phage has the preservation number of CGMCC No.45450, and the phage lyase is derived from the staphylococcus aureus phage, can inhibit staphylococcus aureus in a broad spectrum, can prevent or treat diseases caused by staphylococcus aureus, such as skin infection, respiratory tract infection, digestive system infection, urinary system infection or blood infection, and can inhibit staphylococcus aureus in environment, production facilities, food or animal feed, and prevent and treat biofilm generated by staphylococcus aureus.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to phage, phage lyase and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus), also known as staphylococcus aureus, is a bacteria that is widely found in the environment, usually colonized in the skin and nasal cavities of humans. Under normal conditions, the staphylococcus aureus does not cause harm to health. However, when it enters the human body, it may cause infection, especially on the human body with immunosuppression or other diseases, the light person causes skin infection, the heavy person causes systemic diseases such as pneumonia, systemic infection, etc., which causes threat to human life. The staphylococcus aureus becomes one of 6 antibiotics resistant bacteria in the world through self evolution or gene level transfer mechanisms, so that the antibiotics resistant to methicillin, vancomycin and the like can cause great clinical burden, increase the recurrence of infection and increase the clinical cost and even the death rate of infected patients, so that the development of a clinical treatment scheme capable of effectively combining antibiotics or replacing antibiotics is imperative.
A bacteriophage is a virus parasitic to bacteria that is able to invade bacterial cells and replicate using its metabolic mechanisms. During replication, the phage will use a lytic enzyme to disrupt the cell wall of the host cell, releasing the nascent phage. Phages have been used for the prevention and treatment of human and animal diseases of bacterial origin since the late 1910 s. Lytic enzymes are the primary tool for phage lysis of host cells, which breaks down the glucan of the bacterial cell wall and breaks the cell wall, thereby disrupting the bacterial cell releasing the internal material and the nascent phage. Because of the special structure of bacterial cell walls, it is often difficult for common antibiotics to penetrate the cell walls and act directly on bacterial cells. Therefore, the use of phage or its lytic enzymes to disrupt bacterial cell walls has become a new antimicrobial strategy. Compared with antibiotics, the phage and the lyase thereof can break through the limitation of primary drug resistance of bacteria (such as biological film and the like), thereby killing the bacteria better. Due to its specificity for the host, the phage or lyase protects other microorganisms from damage while the pathogenic bacteria are clarified, thus maintaining the balance of micro-ecology.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a phage, phage lyase and application thereof.
For this purpose, the invention provides the following technical scheme:
a phage lyase having an amino acid sequence as shown in SEQ ID NO. 1; or an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having substantially the same enzymatic activity.
Optionally, the phage lyase is derived from staphylococcus aureus phage, and the preservation number of the phage lyase is CGMCC No.45450.
A staphylococcus aureus phage has a preservation number of CGMCC No.45450, a preservation unit of China general microbiological culture Collection center, a preservation address of North Star Xiyu No. 1,3 in the Korean area of Beijing, and a preservation date of 2023, 03 and 23 days.
Biological material, comprising any one of the following:
(1) A nucleic acid molecule encoding said phage lyase;
(2) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines expressing said phage lyase;
(3) An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of (1);
(4) A recombinant vector, recombinant microorganism or transgenic cell line comprising the expression cassette of (2) or (3);
(5) A recombinant microorganism or a transgenic cell line comprising the recombinant vector described in (2) or (3) or (4).
Alternatively, the nucleic acid molecule of the phage lyase has a nucleotide sequence as shown in SEQ ID NO. 2; or a nucleotide sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 and capable of expressing substantially the same enzyme activity.
The phage lyase, the staphylococcus aureus phage or the biomaterial has the use of any one of the following:
a. use in the inhibition of staphylococcus aureus in the treatment of non-disease;
b. use in the preparation of a product for inhibiting staphylococcus aureus;
c. use in the preparation of a product for the prevention or treatment of a disease caused by staphylococcus aureus;
d. The application of the composition in preparing a product for preventing and treating biofilm generated by staphylococcus aureus.
Optionally, in the use of the composition for preparing a product for inhibiting staphylococcus aureus, the product comprises a bacteriostatic agent, a disinfectant, a cleaning agent, a feed additive or a food additive;
and/or, the staphylococcus aureus comprises drug-resistant staphylococcus aureus; the drug-resistant staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.
Optionally, in the preparation of a product for preventing or treating a disease caused by staphylococcus aureus, said product comprising a drug; and/or
In the preparation of a product for preventing or treating a disease caused by staphylococcus aureus, wherein the disease comprises skin infection, respiratory tract infection, digestive system infection, urinary system infection or blood infection;
And/or, the staphylococcus aureus comprises drug-resistant staphylococcus aureus; the drug-resistant staphylococcus aureus comprises methicillin-resistant staphylococcus aureus;
Optionally, the skin infection comprises skin abscess, eczema, or folliculitis.
Optionally, in the application of preparing a product for preventing and treating a biofilm generated by staphylococcus aureus, the product comprises a bacteriostatic agent, a disinfectant or a cleaning agent;
and/or, the staphylococcus aureus comprises drug-resistant staphylococcus aureus; the drug-resistant staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.
A bacteriostatic agent or drug comprising said phage lyase, said staphylococcus aureus phage or said biological material as an active ingredient, with or without a carrier or excipient.
The technical scheme of the invention has the following advantages:
1. The phage lyase provided by the invention has an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having substantially the same enzymatic activity; the invention discovers that the staphylococcus aureus phage has the preservation number of CGMCC No.45450, the phage lyase is derived from the staphylococcus aureus phage, can inhibit staphylococcus aureus in a broad spectrum, can be used for preventing or treating diseases caused by staphylococcus aureus, such as skin infection, respiratory tract infection, digestive system infection, urinary system infection or blood infection, and can also be used for inhibiting staphylococcus aureus in environments (such as food processing environments or animal culture environments), production facilities, foods or animal feeds, and can be used for preventing and treating biofilms generated by staphylococcus aureus.
2. The staphylococcus aureus phage provided by the invention has the preservation number of CGMCC No.45450, and is found through the research of the invention, the staphylococcus aureus phage can inhibit staphylococcus aureus in a broad spectrum, can be used for preventing or treating diseases caused by staphylococcus aureus, such as skin infection, respiratory tract infection, digestive system infection, urinary system infection or blood infection, and can also be used for inhibiting staphylococcus aureus in environments (such as food processing environments or animal culture environments), production facilities, foods or animal feeds, and preventing and treating biofilms generated by staphylococcus aureus;
In addition, the staphylococcus aureus phage also has the following advantages:
The heat stability is high, the phage titer is still very high after the phage is acted for 1h at 40-70 ℃, and the phage titer is slightly reduced after the phage is acted for 1h at 70 ℃, but still is more than 10 8 PFU/ml;
The pH application range is wider, and the potency is stable within the pH range of 5-11;
No toxicity and high safety.
The staphylococcus aureus phage has a preservation number of CGMCC No.45450, a preservation unit of China general microbiological culture Collection center, a preservation address of North Star Xiyu No.1 and No.2 in the Korean region of Beijing city, and a preservation date of 2023, 3 and 23.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plaque in example 1 of the present invention;
FIG. 2 is a phage electron microscope image (scale bar 100 nm) in example 1 of the present invention;
FIG. 3 is the result of the thermostability of phage BCSAP-005 in example 2 of the present invention;
FIG. 4 is the pH stability results of phage BCSAP-005 in example 3 of the present invention;
FIG. 5 is the result of a safety test for phages BCSAP-005 in example 4 of the invention;
FIG. 6 shows the result of PCR product electrophoresis of Lys-SA001 gene in example 6 of the present invention; 3 lanes in the figure are 3 replicates;
FIG. 7 shows the result of SDS PAGE analysis in example 6 according to the present invention;
FIG. 8 shows the protective effect of phage BCSAP-005 and lyase Lys-SA001 on Staphylococcus aureus infected mice in example 7 of the present invention;
FIG. 9 is a graph showing the effect of phage BCSAP-005 and lyase Lys-SA001 on blood Staphylococcus aureus loading in Staphylococcus aureus infected mice in example 9 of the present invention;
FIG. 10 is a graph showing the effect of phage BCSAP-005 and lyase Lys-SA001 on the lung tissue Staphylococcus aureus load of Staphylococcus aureus infected mice in example 9 of the present invention;
FIG. 11 is a graph showing the effect of phage BCSAP-005 and lyase Lys-SA001 on Staphylococcus aureus loading in liver tissue of Staphylococcus aureus infected mice in example 9 of the present invention;
FIG. 12 is a graph showing the effect of phage BCSAP-005 and lyase Lys-SA001 on Staphylococcus aureus loading in spleen tissue of Staphylococcus aureus infected mice in example 9 of the present invention;
FIG. 13 is a graph showing the effect of phage BCSAP-005 and lyase Lys-SA001 on Staphylococcus aureus load in kidney tissue of Staphylococcus aureus infected mice in example 9 of the present invention;
FIG. 14 shows the effect of phage BCSAP-005 and lyase Lys-SA001 on the skin of a Staphylococcus aureus infected mouse in example 10 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Staphylococcus aureus RN4220 and LB medium (Bio-engineering, A507002-0250) involved in the following examples are all commercially available products.
EXAMPLE 1 acquisition of Staphylococcus aureus phage BCSAP-005
1L of domestic sewage is collected from a sewage treatment center of a people hospital in the Dragon-Hua region, centrifugal tubes are arranged in the device, centrifugation is carried out for 15 minutes at 10000r/min, the supernatant is taken, and filtering sterilization is carried out by a 0.45 mu M filter.
Preparing strain suspension: staphylococcus aureus RN4220 (purchased from ATCC) was inoculated into 5ml of LB medium and cultured overnight at 37 ℃.
Culturing and proliferating, namely adding 4mL of 3xLB culture medium into 8mL of centrifugally filtered sewage, adding 500 mu L of logarithmic phase (OD 600 -0.3) strain suspension and 12 mu L of CaCl 2 solution with concentration of 1mol/L, uniformly mixing, culturing for 12h at 37 ℃, centrifuging for 20min at 10000r/min of a centrifuge, taking supernatant, filtering and sterilizing by a 0.45 mu M filter to obtain co-culture solution;
Separating phage, namely taking 500 mu L of filtered co-culture solution, adding 200 mu L of corresponding strain suspension and 3.7 mu L of CaCl 2 solution with the concentration of 1mol/L, uniformly mixing, adding 3mL of upper culture medium, pouring into a bottom culture medium to prepare a double-layer LB plate (double-layer plate method), standing upside down after solidification, culturing in a 37 ℃ incubator for more than 5 hours, if plaques exist on the plate, picking typical and transparent single plaques by a gun head, adding into 500 mu L of SM Buffer (purchased from SCIENTIFIC PHYGENE, PH 1845), and placing in a refrigerator at 4 ℃ for overnight to obtain SM mixed solution;
After the co-culture solution and the host bacteria solution are mixed and cultured by a double-layer flat plate method, a large amount of plaque with regular shape and uniform size can be observed on the double-layer flat plate, and as shown in figure 1, a large plaque can be formed on the double-layer flat plate containing the host bacteria, and the average diameter size is 2.5mm. The plaque is formed, the edge of the plaque is neat and has no halo, the shape is uniform and circular, and the plaque is clear and transparent.
Phage first purification: taking 300 mu L from the SM mixed solution of 500 mu L in the last step, uniformly mixing the 300 mu L with the corresponding strain suspension, adding the mixture into 5mL of LB culture medium, adding 5.6 mu L of CaCl 2 solution with the concentration of 1mol/L, co-culturing for more than 4 hours at 37 ℃, centrifuging for 20min at 10000r/min of a centrifuge, filtering and sterilizing the supernatant by using a 0.45 mu M filter, taking 500 mu L of the filtered co-culture solution, adding 200 mu L of corresponding methicillin-resistant staphylococcus aureus (MRSA, purchased from ATCC) logarithmic phase (OD 600 -0.3), adding 3.7 mu L of CaCl 2 solution with the concentration of 1mol/L into 3mL of upper culture medium, pouring into the lower culture medium, preparing a double-layer LB plate again after solidification, inverting the double-layer LB plate after solidification, culturing for more than 5 hours in a 37 ℃ incubator, and taking typical and bright single plaques by using a gun head and adding the single plaques into SM Buffer of 500 mu L; repeating the purification step for 2 times, further purifying the phage, finally obtaining a culture solution of staphylococcus aureus phage, adding 20-30% glycerol, and standing at-20 ℃ for preservation.
100 Mu L of purified phage (10 7 PFU/mL) is dripped on a clean membrane, a copper mesh is put into phage liquid, and the phage liquid is stood for 20min and taken out, and the residual liquid is naturally absorbed for 20 s. And (3) dyeing with uranium acetate dye liquor, taking out after 2min, sucking the residual dye liquor, washing and drying. Then the phage is put into another 1 drop of uranium acetate dye solution for counterstaining, the residual dye solution is sucked out after 2 minutes, and the phage is observed by a transmission electron microscope after drying, and the morphology is shown in figure 2. As can be seen from the figure, the phage has a distinct fibrous structure, and the head has a polyhedral structure. Belonging to the order of the cauda, family of the myophagiidae. The length and width of the head were 54.40nm and 53.56nm, respectively. The tail length is 106.80nm, the claw width is 41.60nm, and the length is 28.40nm. The phage is named BCSAP-005 and preserved with the preservation number of CGMCC No.45450, the preservation unit is the common microorganism center of the microbial strain preservation management committee, the preservation address is the No. 3 of the West-road No. 1 of the Chaoyang area North Star of Beijing city, and the preservation date is 2023, 03 and 23.
Example 2 thermal stability detection
Proliferation of phage BCSAP-005 obtained in example 1, namely taking 300. Mu.L of purified phage solution, uniformly mixing with 300. Mu.L of corresponding logarithmic phase (OD 600 -0.3) staphylococcus aureus RN4220 (purchased from ATCC) strain suspension, adding into 5mL of LB medium, adding 5.6. Mu.L of CaCl 2 solution with concentration of 1mol/L, co-culturing for more than 4h at 37 ℃, centrifuging for 20min at 10000r/min of a centrifuge, taking supernatant, filtering and sterilizing by a 0.45. Mu.M filter, and obtaining phage BCSAP-005 proliferation solution. The obtained phage BCSAP-005 proliferation solution of 5.3X10 9 PFU/ml was subjected to the action at 40℃at 50℃at 60℃at 70℃at 80℃in a water bath for 20min, 40min, 60min, two parallel groups were set for each temperature, and the titer of phage was determined by the double-layer plate method (see phage isolation in example 1).
As shown in FIG. 3, it was found that phage BCSAP-005 was still high in phage titer after 1h at 40℃to 70℃and slightly decreased in phage titer after 1h at 70℃but still above 10 8 PFU/ml. After 20min at 80℃the phage titer was reduced by 4 titers and the phage was completely inactivated for 1 h. As can be seen, the phage BCSAP-005 of the present invention has a higher thermostability.
Example 3pH stability detection
4.5Ml of LB broth (purchased from the manufacturing industry, A507002-0250) with different pH values (2, 3, 4,5, 6, 7,8, 9, 10, 11, 12 and 13) is added into a sterile test tube, three of the broth are placed into a water bath kettle with the temperature of 37 ℃, after the temperature is stable, 500 mul of phage proliferation liquid with the concentration of 5.3 multiplied by 10 9 PFU/ml is added, and the mixture is uniformly mixed and then subjected to water bath with the temperature of 37 ℃ for 1h, 2h and 3h. After the completion of the reaction, a proper amount of HCl or NaOH was added to the mixture to give a pH of about 7, and the phage titer was measured by the double-plate method.
As a result, it can be seen from FIG. 4 that the phage BCSAP-005 titer was stable and substantially unchanged in the pH range of 5 to 11. The phage has a wide pH application range.
Example 4 Security detection
Healthy mice (model Balb/c) with the weight of 18-20 g are selected, and each half of the healthy mice is divided into a test group and a control group, wherein each half of the healthy mice is a female half of the test group, 200 mu l of 2X 10 9 PFU/ml phage BCSAP-005 proliferation liquid is filled into the test group, 200 mu l of sterile physiological saline is filled into the control group, 7d is filled continuously, the behavior of the mice is observed, and the change of organs of the mice is observed by section inspection (optical microscope CX22 OLMPUS Japanese; leica microscopic imaging system DM1000 Leica Germany).
During the experimental process, the mice in the experimental group and the control group have no abnormal behaviors. The observation results of organs such as liver, lung, heart, kidney in the section test group are shown in fig. 5:
The endocardium, the myocardium and the epicardium of heart tissue have clear structures, and the heart wall and the heart cavity are not obviously abnormal; the myocardial fiber is uniformly colored, the cell demarcation is clear, the shape of the cells is consistent, the transverse lines of the myocardial cells are clear, the bright and dark phases are separated, and the interstitium is not abnormal; no significant inflammatory changes were seen;
The rare extramedullary hematopoetic foci in liver tissue (black arrow); the liver cells have normal morphological structure and no obvious inflammation;
The surface of the lung tissue is covered with a smooth serosa without obvious abnormality; the lung parenchyma is a large number of alveoli at each level of branches and the end of bronchi in the lung, the bronchus structure at each level is not obviously abnormal, the alveoli wall consists of a single-layer epithelium, and the structure is clear; the interstitium comprises connective tissues, blood vessels and the like in the lung, and has no obvious abnormality; no significant inflammatory changes were seen;
The kidney tissue surface envelope is composed of dense connective tissue with uniform thickness; kidney essence is superficial cortex and deep medulla, and the cortex medulla demarcation is obvious; the glomeruli in the cortex are uniformly distributed, the cell number in the glomeruli and the matrix are uniform, the tubular epithelial cells are round and full, the brush-shaped edges are orderly and regularly arranged, and the medulla is not obviously abnormal; connective tissue between urinary tubules is renal interstitium, and the interstitium has no obvious hyperplasia; no significant inflammatory changes were seen.
In summary, the experimental group has normal organs such as liver, lung, heart, kidney and the like, and has no obvious difference from the control group, which indicates that the phage BCSAP-005 is nontoxic and has high safety.
Example 5 phage optimal multiplicity of infection (MOI) assay
Phage BCSAP-005 and the host bacteria of the phage were propagated according to the conventional method, the titers of phage and host bacteria were determined, and phage BCSAP-005 and host bacteria were diluted appropriately. 100 mu lBCSAP-005 and host bacteria were added to LB broth (from Producer, A507002-0250) at a ratio of the multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and cultured in a shaker at 37℃and 170rpm until the liquid became clear, and the liquid clear time was recorded. After clarification, the mixture was centrifuged at 11000rpm for 5min, and the centrifuged solution was diluted appropriately, and the titer of phage was measured by a double-layer plate method, and the results are shown in Table 1.
TABLE 1 determination of optimal multiplicity of infection (MOI) of phages
| Multiplicity of infection | Titer PFU/ml | Shaking time |
| 10 | 6.5×1010 | 1.5 |
| 1 | 2.4×1010 | 1.5 |
| 0.1 | 3.5×1010 | 2 |
| 0.01 | 6.45×1010 | 2 |
| 0.001 | 1.2×1010 | 2.5 |
| 0.0001 | 2.5×1010 | 3 |
| 0.00001 | 5.4×1010 | 4 |
As a result, it was found that the optimum multiplicity of infection of phage BCSAP-005 was 10, and that the titer of phage produced by phage infection of the host bacteria as a progeny was 6.5X10 10 PFU/ml, and that the phage titer was highest among 7 multiplicity of infection.
Example 6 identification of phage BCSAP-005 lyase
1. Phage BCSAP-005 genome sequencing
Phage BCSAP-005 were subjected to whole genome sequencing using IlluminaMiseq (San Diego, CA, USA) sequencer. A MISEQ REAGENT KIT V-2 kit (BD, MS-102-2002) is used for constructing a 600bp sequencing library, and the main process is that ultrasonic disruption is carried out on genome DNA, terminal filling is carried out, and after a specific joint is added, amplified, purified and screened DNA is obtained, thus obtaining the constructed sequencing library. Phage raw sequencing data were assembled and spliced using software newbler 2.9. The phage BCSAP-005 obtained had a genome size of 141881bp, was double-stranded DNA, and had a G+C content of 30% and contained 223 predicted open reading frames.
The 223 ORFs protein sequences of phages BCSAP-005 were aligned using the online tool BLASTP and the Conserved Domain Database (CDD), respectively, where phages BCSAP-005 contained 167 known encoded functional proteins and the remaining ORFs were hypothetical proteins. Furthermore, it is further available from the result of genome sequencing that the amino acid sequence of the cleavage enzyme (PHAGE LYSIN) related to the cleavage ability is shown in SEQ ID No.1 and the encoding gene thereof is shown in SEQ ID No.2 (hereinafter abbreviated as Lys-SA 001).
2. Amplification of coding Gene of phage BCSAP-005 lyase
According to the data of the lyase of BCSAP-005 phage, PRIMER PREMIER 5.0.0 software is used for designing primers of the lyase Lys-SA001 respectively, sacI and XhoI cleavage sites are added to two ends of the primers respectively, the nucleotide sequences of the Lys-SA001-F and the Lys-SA001-R are shown as SEQ ID NO.3 and SEQ ID NO.4, and the primers are synthesized by Shanghai bioengineering Co Ltd.
Using phage BCSAP-005DNA as template, respectively establishing 25 μL PCR reaction system: taking 1 mu L of phage DNA; 1. Mu.L of each of the upstream and downstream primers was 10pmol; DNA polymerase 0.5. Mu.L, enzyme activity 0.04U/. Mu.L; dNTP Mix 0.5. Mu.L, 200. Mu. Mol/L of each base concentration; 12.5. Mu.L of 2 Xmix buffer; ddH 2 O8.5. Mu.L. Reaction conditions: pre-denaturation at 94 ℃ for 5min, starting the cycle: denaturation at 94℃for 1min, annealing at 61.4℃for 1min, extension at 72℃for 2min,30 cycles, and final extension at 72℃for 10min. The PCR amplification product was checked by 1% agarose gel electrophoresis to give a band of interest corresponding to the expected size of 1488bp (Lys-SA 001), see FIG. 6. The target fragment amplified by PCR was recovered using SanPrep column type DNA gel recovery kit.
3. Construction of recombinant expression plasmid containing coding Gene of phage BCSAP-005 lyase
The expression vector pCold TF plasmid (purchased from Takara) was subjected to double cleavage reaction in a cleavage system of 10. Mu.L of pCold TF plasmid at a concentration of 100ug/ml; sac1 enzyme activity was (SigmaAldrich 81295-35-4), 1. Mu.L; xho1 enzyme activity was (SigmaAldrich 81295-43-4), 1. Mu.L; 10X MBuffer, 2. Mu.L; ddH 2 O, 6. Mu.L. And (3) carrying out water bath for 4 hours at 37 ℃, verifying an enzyme digestion system through 1% agarose gel electrophoresis, and recovering the gel to obtain the carrier with the sticky end. Determining a 10 mu L connecting system according to the concentration of the target gene PCR gel recovery product and the pCold TF plasmid double enzyme digestion recovery product, wherein 7 mu L of the gel recovery product is obtained; 1 mu L of plasmid double enzyme cutting product; 10×ligation Buffer 1 μl; t4 DNA ligase 1. Mu.L. The connection is carried out at 50℃for 10min. mu.L of the ligation product was added to 100. Mu.L of E.coli DH 5. Alpha. Competent cells, gently mixed, and ice-bathed for 30min. And (5) carrying out heat shock for 90s in a water bath at the temperature of 42 ℃ and rapidly placing the mixture on ice for 2-3min. 890. Mu.L of LB broth was added and incubated at 37℃for 90min. After centrifugation at 12000 Xg for 5min, 800. Mu.L of the supernatant was discarded, the bacterial suspension was resuspended. 100 mu L of bacterial liquid is coated on a nutrient agar plate containing ampicillin (Amp), and after the liquid is completely absorbed, the liquid is subjected to inversion culture at a constant temperature of 37 ℃ for overnight. Randomly picking a single colony on an Amp flat plate, inoculating the single colony into LB broth medium containing Amp for culture to obtain bacterial suspension, and identifying positive bacterial liquid by PCR; extracting the PCR positive bacterial liquid plasmid to carry out SacI and XhoI double enzyme digestion identification, checking enzyme digestion products by 1% agarose gel electrophoresis, simultaneously delivering the PCR positive bacterial liquid to Shanghai Pacific Biotechnology Co-Ltd for sequencing, and comparing the MegAlign tool in a Lasergene software package with a target gene sequence according to the sequencing result, wherein the result shows that the gene inserted into the recombinant plasmid is consistent with the target gene sequence Lys-SA 001.
4. Expression of phage BCSAP-005 lyase
Transferring the recombinant expression plasmid containing Lys-SA001 constructed in the step 3 into BL21 competent cells, adding 1 mu L of the recombinant plasmid into 100 mu L of escherichia coli BL21 competent cells, uniformly mixing, and carrying out ice bath for 30min; heat shock in a water bath at a temperature of 42 ℃ for 90s, rapidly placing on ice for 3min, adding 900 mu L of LB broth, and shake culturing at a constant temperature of 37 ℃ for 90min; 100. Mu.L of the bacterial liquid was spread on a kanamycin-containing nutrient agar plate and incubated overnight at 37 ℃. Expression was performed using LB broth containing 60. Mu.g/mL kanamycin, and Lys-SA001 was cloned using isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) as inducer. Transformed E.coli BL21 cells containing the recombinant expression plasmid of Lys-SA001 were propagated overnight at 37 ℃. Overnight grown inoculum (3 mL) was added to 300mL of expression LB broth and incubated at 37 ℃. When OD 600nm reached 1.5, the cells were induced with 0.5mM IPTG and incubated for an additional 2-3 hours in a shaking incubator at 37 ℃. Cells were collected at 30000Xg for 10min and the pellet obtained was resuspended in 50mM Tris-Cl buffer (pH 6.5) and then sonicated using a UP400S Ultraschall processor (Dr. Heilscher GmbH, teltow, germany). The sonicated mixture was then centrifuged at 30000Xg for 10 minutes. The supernatant obtained by SDS PAGE analysis, results are shown in FIG. 7, lane M: protein electrophoresis molecular weight standard; lane 1: uninduced samples; lanes 2-6: the supernatant (5 replicates) was subjected to Western blot (western blot) assay on the samples of lane 3, and the result showed that the molecular size of the lyase Lys-SA001 was about 55 Kda. The percentage of expressed Lys-SA001 fraction in total protein of cell lysates was determined by densitometric analysis using the gel file system (Syngene, UK). Expression levels were further confirmed by GelQuan and Image J software.
EXAMPLE 7 identification of phage BCSAP-005 and lytic enzyme Lys-SA001 bacteriostatic Activity
The lysis profile of Staphylococcus aureus of phage BCSAP-005 was determined by the double plate method, and 32 strains of Staphylococcus aureus bacterial suspensions (isolated source samples were provided by Shanghai Renjin Hospital) in Table 2 below were prepared, including methicillin-resistant Staphylococcus aureus (MRSA) standard strains USA300 and RN4220. The double-layer plate method is used for detecting whether the agar is cracked or not, after the agar on the upper layer is solidified, the agar is placed in a constant temperature box at 37 ℃ for inversion culture for 6-8 hours, and the observation result is shown in Table 2.
Cell lysate supernatant containing Lys-SA001 (obtained in example 6) was assayed for antibacterial activity against 32 clinically isolated MRSA and methicillin-sensitive staphylococcus aureus (MSSA), and standard strains USA300 and RN4220 by well diffusion on a Muller Hinton agar plate. Overnight grown bacterial suspension was plated on MH agar plates (0.5 MACFARLAND). 5 wells of 6mm diameter were aseptically produced on each agar plate, and 40-50. Mu.l of the culture supernatant containing Lys-SA001 (supernatant obtained in example 6 and corresponding to lane 3 in FIG. 7) was poured and incubated overnight at 37 ℃. Lysates of pET28a transformed BL21 cells were used as negative controls, and Lys-SA001 culture supernatants were also used for activity assays. Clear inhibition zones around the wells on the agar plates were measured in millimeters and the results are shown in table 2.
TABLE 2 identification of bacteriostasis Activity of phages and lytic enzymes
In the above table "+" indicates cleavage and "-" indicates no cleavage.
Example 8 Activity of phage BCSAP-005 and lyase Lys-SA001 against Staphylococcus aureus biofilm
The USA300 strain was inoculated into 5ml of sterile TSB medium and grown at 37 ℃ for 16 hours. A1:100 (v/v) dilution of the USA300 strain culture was transferred to sterile TSB medium and then incubated to an OD 600 of 0.4. The cultures were then centrifuged, resuspended in an equal volume of TSB medium and mixed, 100. Mu.l of the culture was added to each well of a 96-well microtiter plate and divided into five groups. 100. Mu. l BCSAP-005 phage (2X 10 9 PFU/ml) and 100. Mu.l of culture supernatant containing Lys-SA001 (supernatant obtained in example 6 and corresponding to lane 3 in FIG. 7) were added to the different wells. Sterile TSB medium (100 μl) was added to the staphylococcal cultures as a control. Sterile TSB medium (200 μl) was used as a negative control. Each group contained triplicate samples. The plate was covered and bacteria were allowed to adhere and grow at 37℃for 24 hours and 72 hours without stirring. After incubation, the residual biofilm was subjected to Crystal Violet (CV) staining and OD 590 was measured and the ratio of OD 590 values was calculated for the experimental and control groups. The results showed that phage BCSAP-005 and lyase Lys-SA001 had lytic activity against Staphylococcus aureus biofilm (Table 3).
TABLE 3 phage BCSAP-005 and lytic Activity of lytic enzyme Lys-SA001 against Staphylococcus aureus biofilm
| BCSAP-005(％) | Lys-SA001(％) | |
| USA300 | 82.74±4.58 | 59.54±13.47 |
Example 9 phage BCSAP-005 and lytic Lys-SA001 protective animal experiments
Three groups of mice (10 mice each, balb/c gauge) were inoculated intraperitoneally with different doses of Staphylococcus aureus USA300 bacterial suspension (10 7、108 and 10 9 CFU/mouse), and the minimum dose (minimum lethal dose [ MLD ]) that resulted in 100% mortality in the infected mice model after 7 days was determined. The number of dead mice was recorded daily. Once the MLD was determined, 2x MLD was used as the infectious challenge dose, and the USA300 bacterial suspension with MLD of 10 8 CFU was experimentally determined. Mice were intraperitoneally injected with 200. Mu. L BCSAP-005 phage (10 9 PFU) and 200. Mu.L of Lys-SA 001-containing culture supernatant (obtained in example 6 and corresponding supernatant to lane 3 in FIG. 7) 1 hour (n=10 per group) after injection of 2X MLD (2X 10 8 CFU) USA 300. The control group was treated with an equal volume of PBS under the same conditions. The number of dead mice was recorded daily for 30 days.
To measure bacterial load in blood and vital organs. Bacterial counts were measured in 10 μl of peripheral blood taken from mouse tail veins treated with phage, lyase or PBS at predetermined time intervals. To determine bacterial load in organs, lungs, liver, spleen and kidneys were collected from euthanized mice (ketamine and xylazine, 100mg/kg body weight) 1, 12 or 24 hours after USA300 infection. The organs were then weighed and homogenized in sterile PBS on ice. Bacterial load in tissues was measured using serial dilution and plating methods. The data processing method is T-test, where P < 0.05 is shown in comparison to the PBS group and P < 0.01 is shown in comparison to the PBS group.
As shown in FIGS. 8 to 13, it was found that phage BCSAP-005 and lyase Lys-SA001 have a protective effect on Staphylococcus aureus-infected mice and reduce the amount of Staphylococcus aureus in the blood, liver, spleen, lung, kidney and other parts.
Example 10 skin infected mice experiments
To study the skin antibacterial effect of phage BCSAP-005 and lyase Lys-SA001 in vivo, a subcutaneous abscess model (n=8) was established in male BALB/c mice (6-8 weeks old; body weight 20-25 g). Mice were anesthetized with 1% pentobarbital 50mg kg -1, shaved on the backs and rinsed with 75% alcohol. Subsequently, 100 μl of MRSA suspension (1×10 8CFU mL-1) was subcutaneously injected to the right and left of the shaved back of each test mouse. After 24 hours, a focal MRSA infection forms a subcutaneous abscess. Phage BCSAP-005 group 200 μ L BCSAP-005 phage (10 9 PFU) were subcutaneously injected into infected mice; lysase Lys-SA001 group was subcutaneously injected into infected mice with 200 μl of Lys-SA 001-containing culture supernatant (supernatant obtained in example 6 and corresponding to lane 3 in fig. 7. Subcutaneous injection of 100mL PBS was used as a control treatment. Phage BCSAP-005 group, lysase Lys-SA001 group, and PBS control group were administered once every 24 hours for 10 days. Mice were photographed using a standard round paper (blue) with a diameter of 6mm as a reference and observations were recorded daily. Simultaneously abscess areas were analyzed by Image J (NIH) software calculation, and curves of mouse back skin infection abscess areas were drawn. Data treatment method was T-test, where P < 0.05 represents P < 0.01 compared to PBS group.
As shown in FIG. 14, it can be seen from the results that phage BCSAP-005 and lyase Lys-SA001 were effective in alleviating skin abscesses caused by Staphylococcus aureus.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (5)
1. A staphylococcus aureus phage has a preservation number of CGMCC No.45450.
2. The staphylococcus aureus phage of claim 1 having a use of any one of:
a. use in the inhibition of staphylococcus aureus in the treatment of non-disease;
b. use in the preparation of a product for broad-spectrum inhibition of staphylococcus aureus;
c. use in the preparation of a product for the prevention or treatment of a disease caused by staphylococcus aureus; the diseases are staphylococcus aureus infection of blood, liver, spleen, lung or kidney or skin abscess;
d. The application of the composition in preparing a product for preventing and treating biofilm generated by staphylococcus aureus.
3. Use according to claim 2, wherein in the manufacture of a product for the treatment of a disease caused by staphylococcus aureus, said product comprises a medicament;
and/or, the staphylococcus aureus comprises drug-resistant staphylococcus aureus; the drug-resistant staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.
4. Use according to claim 2 or 3, wherein in the manufacture of a product for controlling biofilm produced by staphylococcus aureus, said product comprises a bacteriostatic, pharmaceutical, disinfectant or cleaning agent.
5. A bacteriostatic agent or medicament comprising the staphylococcus aureus phage of claim 1 as an active ingredient, with or without the addition of a carrier or excipient.
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