CN118109445A - Methicillin-resistant staphylococcus aureus phage lyase LysLP-122, and preparation method and application thereof - Google Patents
Methicillin-resistant staphylococcus aureus phage lyase LysLP-122, and preparation method and application thereof Download PDFInfo
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- CN118109445A CN118109445A CN202410332715.0A CN202410332715A CN118109445A CN 118109445 A CN118109445 A CN 118109445A CN 202410332715 A CN202410332715 A CN 202410332715A CN 118109445 A CN118109445 A CN 118109445A
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Abstract
The invention discloses a methicillin-resistant staphylococcus aureus phage lyase LysLP-122, a preparation method and application thereof, wherein recombinant plasmid pET-30a-LysLP-122 is constructed, transformed and induced to express in escherichia coli BL21 to obtain a bacterial precipitate, the bacterial precipitate is subjected to ultrasonic disruption to obtain a precipitate inclusion body protein, washing, dissolving, renaturation and dialysis are carried out, finally endotoxin is removed, filtering and sterilization are carried out, and the methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is obtained, wherein the nucleotide sequence of the LysLP-122 lyase is shown as SEQ ID NO. 2. The LysLP-122 lyase obtained for the first time by the method of the invention, lysLP-122 lyase has the effect of inhibiting and killing methicillin-resistant staphylococcus aureus at a certain concentration, and can be applied to treating diseases caused by clinical methicillin-resistant staphylococcus aureus infection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to methicillin-resistant staphylococcus aureus phage lyase LysLP-122, and a preparation method and application thereof.
Background
Methicillin-resistant staphylococcus aureus (Methicillin-resistantStaphylococcus aureus, MRSA), which belongs to the staphylococci family, is a spherical gram-positive bacterium with a diameter of about 1 μm, forming grape-like clusters. MRSA is a super drug-resistant pathogenic bacterium, has a series of virulence factors and the capability of generating drug resistance to most antibiotics, can cause serious clinical infection such as septicemia, pneumonia, endocarditis and the like, is frequently infected by patients with immunodeficiency or low grade, and old and weak patients, and is also common pathogenic bacterium causing wound infection clinically, thereby forming chronic wound surfaces which are difficult to heal, prolonging the healing period and greatly aggravating personal and social medical burden. Therefore, research and development of new antibacterial agents for the prevention and control of MRSA infection are urgently needed.
Phage (Bacteriophage) is taken as a natural enemy of a bacterium naturally occurring in nature, and phage and derivative lyase thereof have specific sterilization and strong lytic activity, can destroy bacterial biomembrane and are not easy to cause bacteria to generate drug resistance, so that phage and derivative lyase thereof have great potential of novel antibacterial preparations, and are highly valued in the research of treating drug-resistant bacterial infection at present.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide methicillin-resistant staphylococcus aureus phage lyase LysLP-122, which aims to overcome the defect that MRSA bacteria are easy to generate drug resistance to the existing antibiotics and the curative effect of the existing antibiotics on the MRSA bacteria is poor.
The invention also aims to provide a preparation method of the methicillin-resistant staphylococcus aureus phage lyase LysLP-122, which aims to rapidly and simply prepare the methicillin-resistant staphylococcus aureus phage lyase LysLP-122 by a biotechnology means.
The invention also aims to provide an application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122.
In order to achieve the aim, the invention provides methicillin-resistant staphylococcus aureus phage lyase LysLP-122, wherein the nucleotide sequence of the lyase LysLP-122 is shown as SEQ ID NO. 2, and the nucleotide sequence of the SEQ ID NO. 2 is specifically as follows:
5'-ATGGCGCGTAAAGCGCGTATTGTTACCATCAACGACAAACCGTA CCGCTTCTCCAAATTCGAGATGGAGCTGATCGAAAGCCACGGTATTACCGCGGGTATGGTTAGCAAACGCGTTAAAGACGGTTGGGAACTGCACGAAGCAATGGACGCACCGGAAGGTACCCGTCTGTCTGAATATCGCGAGAAAAAGACCATCGAGCGCCTGGAACAGGCACGTCTGGAACGCAAACTGGAACGCAAACAGAAAAAAGAGGCGGAACTGCGTCGCAAAAAACCGCACCTGTTCAACGTTCCGCAAAAACATCCGCGCGGTCGTTACGCTTGCTATCTGATGGAGAACGACATCTTCGTCAAAGTCAAAAAATAG-3'.
A method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122, said method comprising the steps of:
S1, obtaining a preset phage lyase sequence from methicillin-resistant staphylococcus aureus, optimizing and synthesizing the preset phage lyase sequence, constructing the preset phage lyase sequence on an expression vector to obtain a recombinant plasmid pET-30a-LysLP-122, wherein the nucleotide sequence of the preset phage lyase sequence is shown as SEQ ID NO. 1, and the SEQ ID NO. 1 is specifically as follows:
5'-ATGGCGAGAAAAGCAAGAATTGTAACAATAAACGATAAACCTT ATAGGTTCAGTAAATTTGAAATGGAATTAATAGAAAGTCACGGTATAACCGCTGGAATGGTTTCTAAGAGAGTAAAAGACGGTTGGGAACTACATGAAGCAATGGACGCACCAGAAGGTACGCGTTTAAGCGAGTACAGAGAAAAGAAAACAATAGAAAGACTGGAACAAGCTAGACTCGAACGCAAATTGGAAAGAAAGCAAAAGAAAGAGGCAGAGCTAAGAAGAAAGAAGCCACATTTGTTTAATGTGCCACAGAAACATCCAAGAGGACGTTATGCGTGCTACCTGATGGAAAACGACATATTCGTGAAAGTTAAGAAGTAG-3';
S2, transforming and inducing expression are carried out on the recombinant plasmid pET-30a-LysLP-122, and bacterial precipitate is obtained;
S3, performing ultrasonic crushing treatment on the bacterial precipitate to obtain inclusion body protein, and washing, dissolving, purifying and renaturating the inclusion body protein by a column to obtain pure LysLP-122 lyase;
S4, removing endotoxin and bacteria of the pure LysLP-122 lyase, and subpackaging to obtain phage lyase LysLP-122, wherein the nucleotide sequence of the lyase LysLP-122 is shown as SEQ ID NO. 2.
Preferably, in the above technical scheme, in step S1, the expression vector is a prokaryotic expression vector pET-30a.
Preferably, in the above technical scheme, the specific steps of transforming and inducing expression of recombinant plasmid pET-30a-LysLP-122 to obtain bacterial precipitate are as follows:
(1) Transferring the recombinant plasmid pET-30a-LysLP-122 into escherichia coli BL21 (DE 3) to obtain escherichia coli colony containing pET-30a-LysLP-122 plasmid;
(2) Selecting single colony, inoculating the single colony into LB culture solution containing 50 mug/mL kanamycin, and culturing overnight to obtain bacterial solution;
(3) Transferring the bacterial liquid to 100mL of LB culture liquid containing 50 mug/mL kanamycin according to the inoculation amount of 1%, shake-culturing to reach an OD 600 preset value, adding an inducer, shaking overnight to induce fusion protein expression, and obtaining the induced bacterial liquid;
(4) Centrifuging the induced bacterial liquid, and discarding the supernatant to obtain bacterial precipitate.
Preferably, in the above technical scheme, the bacterial liquid is transferred to 100mL of LB culture liquid containing kanamycin with the final concentration of 50 mug/mL according to the inoculation amount of 1%, shake culture is carried out until the OD 600 is preset, after inducer is added, shake is carried out overnight to induce fusion protein expression, and in the step of obtaining the post-induction bacterial liquid, the OD 600 is preset to be 0.6-0.8, and the inducer is IPTG.
Preferably, in the above technical scheme, the steps of performing ultrasonic disruption treatment on the bacterial precipitate to obtain inclusion body protein, washing, dissolving, purifying by column and renaturation to obtain pure LysLP-122 lyase are as follows:
(1) Re-suspending the bacterial precipitate in a lysate, ultrasonically crushing, centrifuging, and collecting precipitate to obtain inclusion body protein;
(2) Washing the inclusion body with an inclusion body washing agent for 3-4 times to obtain a washed inclusion body;
(3) Placing the washed inclusion body in inclusion body dissolving solution for overnight dissolution, centrifuging and taking supernatant to obtain dissolved inclusion body protein;
(4) And (3) purifying and renaturating the inclusion body after dissolution by a column to obtain the pure LysLP-122 lyase.
Preferably, in the above technical scheme, in the step of taking the bacterial precipitate to resuspend in a lysate, performing ultrasonic disruption and centrifugation, and taking the precipitate to obtain inclusion bodies, the lysate is pH=8.0, and the concentration is 20mM Tris-HCl.
Preferably, in the above technical scheme, in the step of washing the inclusion body 3 to 4 times with inclusion body detergent made of pH= 8.050mM Tris, 300mM NaCl containing 1% Triton X-1141% Triton X-100, 2mM EDTA and 5mM DTT to obtain the washed inclusion body.
Preferably, in the above technical scheme, in the step of dissolving inclusion bodies in inclusion body dissolving solution overnight after washing, centrifuging to obtain supernatant, and obtaining the dissolved inclusion bodies, the inclusion body dissolving solution is prepared from ph= 8.050mM Tris, 300mM NaCl, 8M Urea and 20mM Imidazole.
The methicillin-resistant staphylococcus aureus phage lyase LysLP-122 prepared by the preparation method is applied to inhibiting or killing methicillin-resistant staphylococcus aureus infection.
Compared with the prior art, the invention has the following beneficial effects:
(1) The methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is prepared for the first time;
(2) The protein bands of the substances obtained in each stage are detected through SDS-PAGE and Western blo in the preparation process, so that the substances obtained in each stage are determined to contain target protein fragments, the preparation and detection are carried out synchronously, experimental errors can be reduced, target protein loss can be avoided, and the finally obtained protein is LysLP-122 lyase;
(3) A large number of experiments prove that LysLP-122 lyase with a certain concentration has the characteristic of inhibiting or killing methicillin-resistant staphylococcus aureus, so that LysLP-122 lyase can be used for treating serious infection caused by methicillin-resistant staphylococcus aureus, and a new idea is provided for clinically treating diseases caused by methicillin-resistant staphylococcus aureus infection.
Drawings
FIG. 1 is a diagram of a pET-30a-LysLP-122 recombinant plasmid constructed and obtained according to an embodiment of the present invention and a double restriction enzyme identification electrophoresis;
FIG. 2 is a schematic diagram of a recombinant plasmid pET-30a-LysLP-122 obtained according to one embodiment of the present invention;
FIG. 3 is a diagram of SDS-PAGE and Western blot to identify prokaryotic expression of phage lyase LysLP-122 according to an embodiment of the invention;
FIG. 4 is a diagram of the spatial structure of LysLP-122 lyase obtained in one embodiment of the invention;
FIG. 5 is a graph (A diagram) showing the antibacterial effect of LysLP-122 lyase on MRSA bacteria and a graph (B diagram) showing the data analysis of the diameter of the antibacterial zone, which are detected by a double-layer flat transparent zone method;
FIG. 6 is a graph of the color development results (A graph) and a histogram of OD 485 values statistics (B graph) of LysLP-122 lyase against MRSA bacteria obtained in a certain embodiment of the invention using a 96-well assay;
FIG. 7 is a plate coating method to explore the minimum bactericidal concentration of LysLP-122 lyase on MRSA bacteria;
FIG. 8 is a graph of the effect of various concentrations LysLP-122 of lyase on MRSA bacterial growth curves;
FIG. 9 is a graph of scanning electron microscopy (A, B) and a graph of transmission electron microscopy (C and D) after treatment of MRSA bacteria with PBS and LysLP-122 lyase;
FIG. 10 is a graph showing the results of the hemolytic activity of the lytic enzyme of some examples LysLP-122 of the present invention (A graph) and a histogram of the OD 450 values (B graph).
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Experimental agent:
lysate: 20mM Tris-HCl, pH 8.0;
Inclusion body wash: prepared from 50mM Tris (pH 8.0), 300mM NaCl with 1% Triton X-1141% Triton X-100, 2mM EDTA and 5mM DTT;
Inclusion body dissolution liquid: made from 50mM Tris (pH 8.0), 300mM NaCl, 8M Urea and 20mM Imidazole;
Buffer solution: made from 1 XPBS (pH 7.4), 4mM GSH, 0.4mM GSSG, 0.4M L-Arginine and 1M Urea;
and (3) storing liquid: 1 XPBS, pH 7.4.
The raw material sources are as follows:
Methicillin-resistant staphylococcus aureus ATCC43300 (Methicillin-RESISTANT STAPHYLOCOCCUS AUREUS, MRSA) was purchased from Ningbo biotechnology limited; coli BL21 (DE 3) competent cells were purchased from Biotechnology (Shanghai) Co., ltd; prokaryotic expression vector pET-30a is a preserving plasmid for university of Guangxi traditional Chinese medicine laboratory.
Example 1
A preparation method of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 comprises the following steps:
S1, obtaining a preset phage lyase sequence from methicillin-resistant staphylococcus aureus, optimizing, synthesizing and editing the preset phage lyase sequence, constructing the sequence on an expression vector, and obtaining a recombinant plasmid pET-30a-LysLP-122, wherein the nucleotide sequence of the preset phage lyase sequence is shown as SEQ ID NO. 1;
S2, transforming and inducing expression are carried out on the recombinant plasmid pET-30a-LysLP-122, and bacterial precipitate is obtained;
s3, performing ultrasonic crushing treatment on the bacterial precipitate to obtain inclusion body protein, and washing, dissolving, purifying and dialyzing the inclusion body protein to obtain pure LysLP-122 lyase;
S4, removing endotoxin and bacteria in the pure LysLP-122 lyase, and subpackaging to obtain phage lyase LysLP-122.
In step S1, the specific steps for preparing the recombinant plasmid pET-30a-LysLP-122 are as follows: obtaining a preset phage lyase sequence (genome sequencing is carried out on MRSA in the earlier stage of the subject group to obtain MRSA phage lyase nucleotide sequence information) from methicillin-resistant staphylococcus aureus MRSA, wherein the nucleotide sequence of the preset lyase sequence is shown as SEQ ID NO.1, carrying out codon optimization on the preset lyase sequence for optimizing the expression of the preset lyase sequence in escherichia coli, then chemically synthesizing a coding gene, and constructing the coding gene on a prokaryotic expression vector pET-30a to obtain a recombinant plasmid pET-30a-LysLP-122, as shown in figure 1;
In step S2, the specific steps of transformation and prokaryotic expression of recombinant plasmid pET-30a-LysLP-122 are as follows: (1) Transferring the recombinant plasmid pET-30a-LysLP-122 into competent cells of escherichia coli BL21 (DE 3) by using a heat shock method, incubating in LB culture solution without resistance, then coating the culture solution on an LB solid plate containing kanamycin with the final concentration of 50 mug/mL, and culturing overnight at 37 ℃ to obtain escherichia coli single colony containing the plasmid pET-30 a-LysLP-122; (2) Inoculating single colony into LB culture solution containing 50 mug/mL kanamycin, and culturing at 37 ℃ at 200rpm overnight to obtain bacterial solution; (3) Transferring the bacterial liquid into 100mL of LB culture liquid containing 50 mug/mL kanamycin with the inoculation amount of 1%, carrying out shake culture for 3-4h until the OD 600 is 0.6-0.8, adding IPTG with the final concentration of 0.2mM, and carrying out shake overnight at 37 ℃ at 200rpm to induce fusion protein expression, thus obtaining induced bacterial liquid; (4) Centrifuging the induced bacterial liquid at 4000rpm at 4deg.C for 10min to collect residual bacterial liquid, and discarding supernatant to obtain bacterial precipitate; (5) The bacterial precipitate is subjected to bacterial precipitation re-suspension by PBS to obtain re-suspension; after ultrasonic disruption (ultrasonic 4sec, intermittent 8sec, whole time 20min, power 400W) was performed on the heavy suspension, the suspension was centrifuged at 10000rpm at 4℃for 10min, and the precipitate was collected to obtain inclusion bodies. Phage lyase LysLP-122 prokaryotic expression was detected at each stage by 12% SDS-PAGE gel electrophoresis and protein bands were visualized by Coomassie blue staining;
In the step S3, the thallus sediment is subjected to ultrasonic crushing treatment to obtain inclusion bodies, and washing, dissolving, column purification and dialysis are carried out to obtain the pure LysLP-122 lyase, wherein the specific steps are as follows: (1) The bacterial precipitate is resuspended in 20mL of lysate, broken by ultrasonic waves (4 sec, 8sec intermittently, 20min in whole course, 400W in power), centrifuged at 10000rpm at 4deg.C for 10min, and the precipitate is collected to obtain inclusion body protein; (2) Washing the inclusion body protein for 3 times by using inclusion body washing liquid, and removing impurities adhered to the surface of the inclusion body to obtain a washed inclusion body; (3) Dissolving the washed inclusion body with inclusion body dissolving solution at 4 ℃ overnight, centrifuging at 10000rpm at room temperature for 15min, and collecting supernatant solution to obtain the dissolved inclusion body; (4) Loading the dissolved inclusion body into a Ni-IDA gravity affinity chromatographic column with pre-balanced inclusion body dissolution liquid, eluting target proteins by using balanced buffers with different concentrations of imidazole, collecting each eluting component for SDS-PAGE analysis and detection, collecting eluting components with relatively high purity, adding the eluting components into a dialysis bag, dialyzing the eluting components into the buffer solution at 4 ℃ for renaturation, and finally dialyzing the renatured proteins into a storage solution to obtain pure LysLP-122 lyase;
In the step S4, endotoxin and bacteria in the pure LysLP-122 lyase are removed, and the specific steps for subpackaging to obtain phage lyase LysLP-122 are as follows: removing endotoxin of pure LysLP-122 lyase by using PMB affinity medium, filtering and sterilizing the collected protein by using 0.22 μm filter, detecting protein concentration by BCA protein concentration determination kit, subpackaging, and finally obtaining subpackaged phage lyase LysLP-122 and freezing at-80 ℃, wherein the nucleotide sequence number of phage lyase LysLP-122 is shown as SEQ ID NO. 2.
The recombinant plasmid pET-30a-LysLP-122 obtained by the preparation is identified and a structural schematic diagram is constructed, and the results are respectively shown in fig. 1 and fig. 2. Wherein M in FIG. 1 is Marker,1 is recombinant plasmid pET-30a-LysLP-122,2 is Xho I/Sph I double enzyme-cut strip. As can be seen from FIG. 1, the size of the recombinant plasmid pET-30a-LysLP-122 band prepared by the invention is between 5000bp and 6000bp, the length of the recombinant plasmid pET-30a-LysLP-122 band is consistent with that of the recombinant plasmid pET-30a-LysLP-122 in the expectation, the Xho I/Sph I double enzyme digestion band is between 3000bp and 4000bp, and the enzyme digestion product is about 1000bp smaller than that of the recombinant plasmid pET-30 a-LysLP-122; as can be seen by referring to FIG. 2, the Xho I enzyme and the Sph I enzyme are located at both ends of pET-30a-LysLP-122, and after the fixed-point enzyme digestion treatment is performed on pET-30a-LysLP-122 by using the Xho I enzyme and the Sph I enzyme, the digestion product should be about 1000bp smaller than the fragment of recombinant plasmid pET-30 a-LysLP-122. In summary, the construction of the recombinant plasmid pET-30a-LysLP-122 was successful.
The results of identifying the materials obtained at each stage of transformation and prokaryotic expression of recombinant plasmid pET-30a-LysLP-122 are shown in FIG. 3, wherein FIG. 3 consists of A, B, C and D. Panel A shows SDS-PAGE analysis LysLP-122 of the expression of the lyase in BL21 (DE 3), in which M is a protein molecular mass standard, lane 1 is the supernatant collected after cell disruption after overnight induction at 37℃and lane 2 is the precipitate collected after cell disruption after overnight induction at 37 ℃; from panel A, it can be seen that there is a distinct protein band at 15-20kDa in lane 2. Panel B shows SDS-PAGE analysis LysLP-122 of the results of lyase purification, in panel B M is the protein molecular mass standard, lane 1 is the supernatant collected after centrifugation of inclusion body solubilization, lane 2 is the flow-through after incubation of the supernatant with Ni-IDA, lanes 3-5 are the 50mM imidazole eluate fraction, and lanes 6-11 are the 500mM imidazole eluate fraction; lanes 3-5 have a distinct protein band at 15-20 kDa; lane 6 has distinct protein bands at 15-20kDa and 30-40 kDa; lanes 7-9 have a distinct protein band at 15-20 kDa. Panel C shows SDS-PAGE to remove endotoxin, dialysis to PBS and filter sterilization of LysLP-122 lyase, in the C panel M is protein molecular weight standard, lane 1 is 1.00 u g bovine serum albumin, lane 2 is 0.80 u g LysLP-122 lyase, lane 2at 15-20kDa with obvious protein band. Panel D is a diagram of Westernblot identification LysLP-122 lyase, M is a protein molecular mass standard, lane 1 is a target protein, lane 1 has a distinct protein band at 15-20 kDa. As described above, when SDS-PAGE and Westernblot were used to examine whether LysLP-122 lyase was contained in the material obtained at each stage of transformation and prokaryotic expression of recombinant plasmid pET-30a-LysLP-122, the apparent LysLP-122 lyase band was detected at 15-20kDa, indicating that the inclusion body target protein obtained by transformation and prokaryotic expression of recombinant plasmid pET-30a-LysLP-122 in example 1 was LysLP-122 lyase. In summary, it was demonstrated that during transformation and prokaryotic expression of recombinant plasmid pET-30a-LysLP-122, each of the stages contained LysLP-122 components of 15-20kDa in length, indicating that each of the stages contained the target protein fragment.
The physiological index and the spatial structure of LysLP-122 lyase obtained by the method are explored, and the physicochemical data of LysLP-122 lyase are analyzed by using ProtParam online software (https:// web. Expasy. Org/ProtParam /) as shown in the following table 1; the spatial three-dimensional structure of LysLP-122 lyase was predicted using an I-TASSER (ITERATIVE THREADING assembly refinement) server and visualized using PyMOL software, as shown in FIG. 4.
TABLE 1 physical and chemical indicators of MRSA phage lyase LysLP-122
As can be seen from Table 1, the LysLP-122 lyase obtained in the present invention has 122 amino acids and is characterized by good stability and hydrophilicity, and as can be seen from FIG. 4, the LysLP-122 lyase has an alpha-helix spatial three-dimensional structure from N-terminal to C-terminal.
Example 2
An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is provided. The antibacterial effect of LysLP-122 lyase on methicillin-resistant staphylococcus aureus is detected by a double-layer flat transparent ring method, and the specific method is as follows:
(1) Preparing a bacterial suspension: scraping Methicillin-resistant staphylococcus aureus (Methicillin-RESISTANT STAPHYLOCOCCUS AUREUS, ATCC 43300) on the inclined surface of the test tube, inoculating to 4mL of LB culture medium, shaking and culturing overnight at 37 ℃ and 180rpm, diluting the bacterial liquid for 6 times, wherein the purpose of dilution is to make OD 600 be 0.3-0.8, and measuring OD 600 value after dilution. The original bacterial liquid OD 600 is equal to the dilution times the diluted OD 600 value. Dilution was performed by ten-fold dilution, specifically using LB-diluted bacterial suspension of 1X 10 6 CFU/mL (1 OD 600 is about equal to 1X 10 9 CFU/mL).
(2) Adopts a double-layer flat transparent ring method, and comprises the following specific steps: pouring 10mL of 1.5% agar sterilized under high pressure into a 90mm sterile culture dish under sterile conditions, and preparing a first layer of culture medium after the agar is solidified; rapidly placing 4 sterilized oxford cups (with the inner diameter of 6+/-0.1 mm, the outer diameter of 7.8+/-0.1 mm and the height of 10+/-0.1 mm) on the first layer of culture medium, pouring 15mL of culture medium mixed with MRSA bacteria outside the oxford cups, taking out the oxford cups after the culture medium is solidified to obtain a second layer of culture medium with 4 holes, and numbering the second layer of culture medium as holes 1,2, 3 and 4; 100. Mu.L of 50. Mu.g/mL ampicillin (AMPICILLIN, amp) was added to well 1, 100. Mu.L of negative control PBS was added to well 2, and 100. Mu.L of 0.8mg/mL phage lyase LysLP-122 was added to wells 3 and 4, respectively. The flat plate is pre-diffused for 2-4 hours at 4 ℃, then is placed in a constant temperature box at 37 ℃ for culture for 16-20 hours, the diameter of the inhibition zone is observed and measured, each inhibition zone is measured for 3 times, 3 independent repeated experiments are carried out, and the average value is obtained.
The result of detecting the antibacterial effect of LysLP-122 lyase on MRSA by the double-layer plate transparent circle method is shown in FIG. 5, wherein FIG. 5 consists of a graph A and a graph B, the graph A is a antibacterial plate for MRSA, and the graph B is a data analysis graph of LysLP-122 lyase on MRSA antibacterial circle diameter.
As can be seen from the A graph of FIG. 5, the bacterial group growth is normal near the PBS of the hole 1 and the hole 2 to which the ampicillin is applied, and the bacterial group growth is aseptic near the hole 3 and the hole 4 to which the phage lyase LysLP-122 is applied, and a transparent annular antibacterial ring appears; as can be seen by combining the graph B of fig. 5, the antibacterial zone of Amp and PBS is 0mm, the antibacterial zone of LysLP-122 lyase is 20.69±0.56mm, which shows that ampicillin does not inhibit the growth of methicillin-resistant staphylococcus aureus, while LysLP-122 lyase obtained by the invention has an inhibitory effect on the growth of methicillin-resistant staphylococcus aureus, and LysLP-122 lyase hydrolyzes glycan skeletons on the cell wall of methicillin-resistant staphylococcus aureus, so that methicillin-resistant staphylococcus aureus cannot grow and reproduce, and LysLP-122 lyase has the function of inhibiting the growth and reproduction of methicillin-resistant staphylococcus aureus, so that the methicillin-resistant staphylococcus aureus can be used for treating or relieving diseases or wound surfaces caused by methicillin-resistant staphylococcus aureus infection.
Example 3
An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is provided. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of LysLP-122 lyase were determined using a 96-well assay, and the specific steps are as follows:
(1) The method for preparing MRSA bacterial suspension is the same as in the step (1) in the example 2; taking a sterile 96-well plate, adding 100 mu L of sterile LB culture medium plus 100 mu L of PBS (phosphate buffer solution) into each row of 1-well plates as a blank control, adding 100 mu L of freshly prepared 1X 10 6 CFU/mL MRSA bacterial suspension into each row of 2-12-well plates, adding 100 mu L of PBS into each row of 2-well plates as a negative control, adding 100 mu L of LysLP-122 lyase into each row of 3-12-well plates respectively, wherein the final concentration of LysLP-122 lyase in each row of 3-12-well plates is 256, 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 mu g/mL, and setting three repeats.
(2) The 96-well plate was placed in a 37 ℃ incubator for 18 hours, 1 μl of 1% 2,3, 5-triphenyltetrazolium chloride (TRIPHENYL TETRAZOLIUM CHLORIDE, TTC) was added to each well, the minimum dilution at which no red change was seen by the naked eye was MIC, the absorbance of OD 485 was measured by a microplate reader, 20 μl of the culture was spread on the plate from the wells at which no red change was seen by the naked eye, incubated at an appropriate temperature for 24 hours, and the growth of colonies in the plate was observed as MBC at a minimum concentration of the corresponding drug of no colonies or less than 5 grown colonies, each set with 3 independent replicates.
Experiments were performed according to the method of example 3, and after 1. Mu.L of 1% 2,3, 5-triphenyltetrazolium chloride was applied to each well plate, the results are shown in FIG. 6, panel A. As can be seen from panel A of FIG. 6, wells 1,3, 4 and 5, respectively, in which no red change was seen with the naked eye after TTC application, demonstrate that the Minimum Inhibitory Concentrations (MIC) of LysLP-122 enzymes were 256 μg/mL, 128 μg/mL and 64 μg/mL. A portion of the solution was aspirated from each well and the absorbance at OD 485 was measured with a microplate reader, and the results are shown in Panel B of FIG. 6. As can be seen from FIG. 6, panel B, the absorbance at OD 485 for both the control and LysLP-122 enzyme concentrations of 256 μg/mL, 128 μg/mL and 64 μg/mL were below 0.2, and the values for the absorbance of 256 μg/mL were similar to those of the control; the absorbance of LysLP-122 lyase concentration of 32 μg/mL and 16 μg/mL is about 0.4, and the absorbance of LysLP-122 lyase concentration of 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL and 0.54 μg/mL is 0.8 and above 0.8.
The minimum inhibitory concentrations 256. Mu.g/mL, 128. Mu.g/mL and 64. Mu.g/mL described above were used as inoculum for plate coating to obtain the minimum inhibitory concentration (MBC) of the corresponding drug with no colony growth or with the number of colony growth below 5, and the results are shown in FIG. 7. As can be seen from FIG. 7, large-area colonies appeared on plates coated with LysLP-122 lyase at a concentration of 64. Mu.g/mL, 30-40 punctate colonies appeared on plates coated with LysLP-122 lyase at a concentration of 128. Mu.g/mL, and 1-2 punctate colonies appeared on plates coated with LysLP-122 lyase at a concentration of 256. Mu.g/mL, indicating that the MBC of LysLP-122 enzyme was 256. Mu.g/mL.
While MIC is the lowest effective concentration of antibacterial drugs against specific microorganisms in vitro experiments, MBC is an important parameter for measuring antibacterial effects of antibacterial drugs against specific pathogens, and in summary, the experiments and results of example 3 of the present invention teach that LysLP-122 lyase can be used to prepare a bacteriostatic agent for clinically inhibiting wound problems caused by methicillin-resistant staphylococcus aureus infection when the concentration of LysLP-122 lyase is 256 μg/mL, 128 μg/mL, and 64 μg/mL; when the concentration of LysLP-122 lyase is 256 mug/mL, lysLP-122 lyase can be used for preparing a bactericide for clinically killing wound infection or other diseases caused by methicillin-resistant staphylococcus aureus infection.
Example 4
An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is provided. The influence of LysLP-122 lyase on MRSA growth curve is detected, and the specific steps are as follows:
The procedure for preparing 1X 10 8 CFU/mL MRSA bacterial suspension was the same as in step (1) of example 2, and sterile 96-well plates were used, with 100. Mu.L of sterile broth medium and 100. Mu.L of PBS as blank Control added to column 1, 100. Mu.L of sterile PBS and 100. Mu.L of 1X 10 8 CFU/mL of MRSA bacterial suspension added to column 2, as negative Control, and LysLP-122 lyase 100. Mu.L and 100. Mu.L of 1X 10 8 CFU/mL of MRSA bacterial suspension added to columns 3-7, respectively, with final concentrations of LysLP-122 lyase of 16, 32, 64, 128 and 256. Mu.g/mL, respectively, each set with 3 replicates; the samples were placed in an ELISA reader, absorbance at OD 600 was measured every 2 hours, and the measurements were continued for 24 hours, and a growth curve was drawn from the obtained data as shown in FIG. 8.
As can be seen from fig. 8, the number of MRSA bacteria tended to flatten at OD 600 =1.1 after 10 hours, indicating that the number of MRSA bacteria remained stable at OD 600 =1.1 due to environmental factors. Whereas the application of LysLP-122 lyase at 16 μg/mL, 32 μg/mL, 64 μg/mL, 128 μg/mL and 256 μg/mL, the number of MRSA bacteria in the solution was significantly less than that in the negative control, demonstrating that the application of LysLP-122 lyase at 16 μg/mL, 32 μg/mL, 64 μg/mL, 128 μg/mL and 256 μg/mL significantly inhibited MRSA growth; in particular, 128. Mu.g/mL and 256. Mu.g/mL LysLP-122 enzyme almost completely inhibited the growth of MRSA bacteria. The experimental exploration and experimental results further demonstrate that LysLP-122 lyase with a certain concentration can inhibit the growth of MRSA bacteria, and that LysLP-122 lyase can be used for treating wound infection and diseases caused by MRSA bacteria, and can be applied to preparing bacteriostats and bactericides of MRSA bacteria.
Example 5
An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is provided. The microscopic morphological change of MRSA under the action of LysLP-122 lyase is observed by a scanning electron microscope and a transmission electron microscope, and the method comprises the following specific steps:
Preparing a 1X 10 8 CFU/mL MRSA bacterial suspension, wherein the preparation process is the same as the process (1) in the example 2; the experimental group is that 1X 10 8 CFU/mL MRSA bacterial suspension is added into LysLP-122 lyase liquid, the final concentration of LysLP-122 lyase liquid is 64 mug/mL, a negative control group is arranged, PBS is added into 1X 10 8 CFU/mL MRSA bacterial suspension; shaking culture at 37deg.C and 100rpm for 6 hr, centrifuging at 4000rpm for 5min, removing supernatant to obtain precipitate, washing with sterile physiological saline for 3 times, centrifuging at 4000rpm for 10min, removing supernatant to obtain thallus; the cells were fixed at 4℃overnight with 1mL of 2.5% glutaraldehyde, and the results were shown in FIG. 9.
FIG. 9 is a view showing the effect of negative control PBS on MRSA bacteria microscopic morphology by a scanning electron microscope, B view showing the effect of LysLP-122 lyase at a final concentration of 64. Mu.g/mL on MRSA bacteria microscopic morphology by a scanning electron microscope, C view showing the effect of negative control PBS on MRSA bacteria microscopic morphology by a transmission electron microscope, and D view showing the effect of phage lyase LysLP-122 at a final concentration of 64. Mu.g/mL on MRSA bacteria microscopic morphology. LysLP-122 lyase treatment of MRSA bacteria compared with PBS treatment of MRSA bacteria, it was found that the number of MRSA bacteria in panel B was significantly smaller than that in panel A, and that obvious deletion marks were present at the vacancies, probably because LysLP-122 lyase completely lyse the cell walls of MRSA bacteria, resulting in the disappearance of bacteria after lysis into fragments.
Example 6
An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is provided. The hemolytic activity of LysLP-122 lyase on animal erythrocytes is detected, and the experiment comprises the following steps:
Hemolytic activity assays were performed using 4% porcine blood erythrocytes. 100 μl PBS was added to well 1 as a negative control; 1.0% Triton X-100 was added to well No. 2 as a positive control; respectively diluting LysLP-122 lyase into 1024, 512, 256, 128, 64, 32, 16, 8, 4 and 2 mug/mL by PBS according to a multiple ratio dilution method, respectively taking 100 mu L of LysLP-122 lyase solution with each concentration, and placing the solution into 3-12 holes of a 96-well plate for 10 experimental groups; then 100 mu L of 4% red blood cell suspension is added into the wells 1-12, and the final concentration of LysLP-122 lyase in the wells 3-12 is as follows: 512. 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg/mL; after incubation in an incubator at 37℃for 3h, centrifugation at 1500rpm for 10min, 150. Mu.L of the supernatant was placed in a new 96-well plate, absorbance was measured at 450nm, and the hemolysis rate was calculated, and the results are shown in FIG. 10. Wherein, the calculation formula of the hemolysis rate is as follows:
the hemolysis ratio= [ experimental set OD 450 -negative control set OD 450 ]/[ positive control set OD 450 -negative control set OD 450 ] ×100%.
Panel A of FIG. 10 is a hemolytic manifestation of 4% porcine blood erythrocytes in different treatments in 96-well plates, where 1 is a PBS negative control; 2 is a 1.0% triton X-100 positive control; 3-12 are phage lytic enzymes LysLP-122 at final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg/mL, respectively, and when viewed as a histogram of OD 450 values at various treatments in conjunction with panel B of FIG. 9, it was found that the hemolytic activity was comparable to the positive control at a concentration of LysLP-122 of 512 μg/mL and was approximately 25.58% compared to the positive control at a concentration of LysLP-122 lytic enzyme of 256 μg/mL. In summary, it is demonstrated that LysLP-122 lyase does not cause severe hemolysis with animal erythrocytes at a concentration of 256 μg/mL or less, and that LysLP-122 lyase has a similar hemolysis rate to negative control PBS at a concentration of 128 μg/mL or less, and does not cause severe hemolysis with animal erythrocytes, meaning that a concentration of 256 μg/mL or less of LysLP-122 lyase can be used for clinical treatment of methicillin-resistant Staphylococcus aureus without causing severe hemolysis.
All experimental data described above were analyzed using the application statistics software IBM SPSS STATISTICS 19.0 and statistically plotted using GRAPHPAD PRISM software. All experiments were repeated 3 times and the quantitative results were expressed as mean ± standard deviation (X ± S). The quantitative numerical comparison between the two groups adopts independent sample t test, and the quantitative numerical comparison between the multiple groups adopts single factor analysis of variance. In all assays, P <0.05 considered significant differences, statistically significant.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. The methicillin-resistant staphylococcus aureus phage lyase LysLP-122 is characterized in that the nucleotide sequence of the lyase LysLP-122 is shown as SEQ ID NO. 2.
2. A method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122, which is characterized by comprising the following steps:
S1, obtaining a preset phage lyase sequence from methicillin-resistant staphylococcus aureus, optimizing and synthesizing the preset phage lyase sequence, and constructing the sequence on an expression vector to obtain a recombinant plasmid pET-30a-LysLP-122, wherein the nucleotide sequence of the preset phage lyase sequence is shown as SEQ ID NO. 1;
S2, transforming and inducing expression are carried out on the recombinant plasmid pET-30a-LysLP-122, and bacterial precipitate is obtained;
S3, performing ultrasonic crushing treatment on the bacterial precipitate to obtain inclusion body protein, and washing, dissolving, purifying and renaturating the inclusion body protein by a column to obtain pure LysLP-122 lyase;
S4, removing endotoxin and bacteria in the pure LysLP-122 lyase, and subpackaging to obtain phage lyase LysLP-122.
3. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 as claimed in claim 1, wherein in step S1, the expression vector is prokaryotic expression vector pET-30a.
4. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 according to claim 1, wherein the specific steps of transforming and inducing expression of recombinant plasmid pET-30a-LysLP-122 to obtain bacterial precipitate are as follows:
(1) Transferring the recombinant plasmid pET-30a-LysLP-122 into escherichia coli BL21 (DE 3) to obtain escherichia coli colony containing pET-30a-LysLP-122 plasmid;
(2) Selecting single colony, inoculating the single colony into LB culture solution containing 50 mug/mL kanamycin, and culturing overnight to obtain bacterial solution;
(3) Transferring the bacterial liquid to 100mL of LB culture liquid containing 50 mug/mL kanamycin according to the inoculation amount of 1%, shake-culturing to reach an OD 600 preset value, adding an inducer, shaking overnight to induce fusion protein expression, and obtaining the induced bacterial liquid;
(4) Centrifuging the induced bacterial liquid, and discarding the supernatant to obtain bacterial precipitate.
5. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 as claimed in claim 4, wherein the bacterial liquid is transferred to 100mL LB culture liquid containing 50 mug/mL kanamycin according to 1% inoculum size, shake-cultured to OD 600 preset value, adding inducer, shaking overnight to induce fusion protein expression, and obtaining induced bacterial liquid, wherein the OD 600 preset value is 0.6-0.8, and the inducer is IPTG.
6. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 according to claim 2, wherein the bacterial precipitate is subjected to ultrasonic disruption treatment to obtain inclusion body protein, and the inclusion body protein is washed, dissolved, purified by column chromatography and renatured to obtain pure LysLP-122 lyase, which comprises the following specific steps:
(1) Re-suspending the bacterial precipitate in a lysate, ultrasonically crushing, centrifuging, and collecting precipitate to obtain inclusion body protein;
(2) Washing the inclusion body with an inclusion body washing agent for 3-4 times to obtain a washed inclusion body;
(3) Placing the washed inclusion body in inclusion body dissolving solution for overnight dissolution, centrifuging and taking supernatant to obtain a dissolved inclusion body;
(4) And (3) purifying and renaturating the inclusion body after dissolution by a column to obtain the pure LysLP-122 lyase.
7. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 as claimed in claim 6, wherein in the steps of taking bacterial precipitate, re-suspending in lysate, ultrasonic crushing, centrifuging, taking precipitate, and obtaining inclusion body, the lysate is pH=8.0, and the concentration is 20mM Tris-HCl.
8. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 as claimed in claim 6, wherein in the step of washing inclusion bodies 3-4 times with inclusion body detergent, the inclusion body detergent is prepared from pH= 8.050mM Tris, 300mM NaCl containing 1% Triton X-1141% Triton X-100, 2mM EDTA and 5mM DTT.
9. The method for preparing methicillin-resistant staphylococcus aureus phage lyase LysLP-122 as claimed in claim 6, wherein in the step of dissolving inclusion bodies after washing in inclusion body dissolving solution overnight, centrifuging to obtain supernatant, the inclusion body dissolving solution is prepared from pH= 8.050mM Tris, 300mM NaCl, 8M Urea and 20mM Imidazole.
10. An application of methicillin-resistant staphylococcus aureus phage lyase LysLP-122 prepared by the preparation method according to any one of claims 2-8 in inhibiting or killing methicillin-resistant staphylococcus aureus infection.
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