NL2034252B1

NL2034252B1 - Phage lytic enzyme with fluorescent marker and application thereof

Info

Publication number: NL2034252B1
Application number: NL2034252A
Authority: NL
Inventors: Deng Xianyu; Tang Zhongqiong; Wang Feng; Lin Lianbing; Ji Xinyu
Original assignee: Univ Kunming Science & Technology
Priority date: 2022-07-22
Filing date: 2023-03-02
Publication date: 2024-01-29
Also published as: CN115820616A

Abstract

Provided are a phage lytic enzyme with a ﬂuorescent marker and an application thereof. By constructing a recombinant vector and expressing the phage lytic enzyme PGTphg With the ﬂuorescent marker in a genetically engineered strain Escherichia coli BL21 (DE3), the phage lytic enzyme can serve as a ﬂuorescence dye having a long-term ﬂuorescent activity and will not be quenched, so that a sample is not kept in dark place, the cost is saved, the work efﬁciency can be greatly improved, and the research time can be shortened greatly. The phage lytic enzyme PGTphg with the ﬂuorescent marker can be used for preventing and treating a bioﬂlm of Staphylococcus aureus, and has a good bioactivity for inhibiting formation of a bioﬂlm of Staphylococcus aureus, so as to improve prevention and control of drug-resistant bacteria from a common bacterial level (state of planktonic bacteria) to a microbial community level (state of bioﬂlm).

Description

PHAGE LYTIC ENZYME WITH FLUORESCENT MARKER AND

APPLICATION THEREOF

TECHNICAL FIELD

[01] The present invention belongs to the technical field of control of pathogenic microorganisms, and particularly discloses a phage lytic enzyme with a fluorescent marker and an application thereof.

BACKGROUND ART

[02] A Bacterial Biofilm (BBF) is a type of microbial community completely different from planktonic bacteria in growth pattern where bacteria adhere to the surface of an object or an active tissue to adapt to the living environment and are coated in compositions such as exo-polysaccharide matrixes, fibrin and lipoproteins generated by the bacteria in the growth process. The BBF is a special group survival form of bacteria. Owing to the protective effects of the compositions such as exo-polysaccharide matrixes, phages (biofilm bacteria) in the biofilm can escape from the effect of the immune system of the body and predation of phagocytes, and show extremely high resistance to antibiotics and antibacterial drugs. Researches show that the biofilm can improve the drug resistance of bacteria by 10-1,000 times, resulting in a protracted course of wound infection. When the defense system of the body cannot control them, the bacteria may further emigrate from the biofilm and may be disseminated outwards to arrive at other parts to induce new infections, so that the wound 1s easy to relapse and is very difficult to be cured. In addition, some pathogenic bacteria, particularly Staphylococcus aureus, will form the biofilm inside or outside a human body or on the surface of a medical material, which is extremely harmful.

Although the pathogenic bacteria in a planktonic state can be killed by using antibiotics, it is hard to eliminate the bacteria deep in the biofilm, which becomes a great difficulty to solve a problem of super drug-resistant bacteria.

[03] Drug resistance of the BBF may result from combined actions of multiple factors. At present, there have been few researches on formation of the BBF, molecular mechanisms for elimination and related prevention and control technologies, which is an important entry point of the technology in the present invention. Because nearly all of the bacteria can form the BBF under certain conditions, and in addition to corroding the surfaces of pipelines and metals and polluting the medical materials, the bacteria in the BBF can further induce occurrence of various diseases of animals and plants and human being, it is an urgent need to seek for a novel biofilm bacteriostatic agent capable of replacing antibiotics.

[04] A lyase (lysin or endolysin), a kind of cell wall hydrolase expressed and released in the later period where phages infect with host bacteria, lyses the bacteria by hydrolyzing whole cell peptidoglycan, and releases progeny phages. At present, the antibacterial activity of the phage lytic enzyme has been widely accepted, and the phage lytic enzyme is expected to replace conventional antibiotics to become a novel bactericide. However, there is a scarcity of researches on the anti-biofilm activity of the phage lytic enzyme and its inhibition on formation of the BBF. In practice, there have been no reports on using the phage lytic enzyme and adding a fluorescent protein tag for marking the process that the lyase acts on the BBF of Staphylococcus aureus and preventing and treating its formation. Further, owing to special three-dimensional spatial structure, special compositions (polysaccharides, lipids and proteins and the like) and the barrier effect of the BBF different from those of common planktonic bacteria, development of enzymic preparations capable of being used for BBF prevention and control is quite slow now.

[05] It is worth noting that the common planktonic bacteria and the BBF bacteria are two completely different types. The two are entirely different in features such as growth pattern, existence form and drug resistance. For example, even common non-drug-resistant bacteria will generate extremely high drug resistance (generally speaking, the drug resistance can be improved by 10-1,000 times) in case of formation of the BBF.

SUMMARY

[06] The first objective of the present invention is to provide an application of a phage lytic enzyme with a fluorescent marker in preparing a preparation for preventing and treating formation of a biofilm of Staphylococcus aureus, which mainly aims to solve the technical problem of a shortage of an antibacterial agent for inhibiting formation of the biofilm of Staphylococcus aureus at present.

[07] The second objective of the present invention is to provide an application of the phage lytic enzyme with the fluorescent marker in preparing an antibacterial agent for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonellae,

Shigellae and/or Bacillus subtilis, which can solve the technical problem that there are few bactericides about the phage lytic enzyme at present.

[08] The third objective of the present invention is to provide a preparation for preventing and treating formation of a biofilm of Staphylococcus aureus, which can further mark and stain the biofilm of Staphylococcus aureus while effectively killing

Staphylococcus aureus, thereby not only greatly saving the cost, but also improving the work efficiency and shortening the research time.

[09] Compared with the prior art, the present invention has the following advantages and positive effects:

[10] The present invention provides a phage lytic enzyme with a fluorescent marker and an application thereof. Transgenic Escherichia coli which specifically produces the phage lytic enzyme PGTphg with the fluorescent marker is constructed by applying a genetic engineering technology, and combined expression of the fluorescent activity fused phage lytic enzyme is completed by using a single plasmid, so that there is no non-synchronous problem of stability and genetic expressions of multiple plasmids in engineering bacteria. The present invention features simple operation, low cost and high feasibility, and lays a foundation for industrial production application of lyase. By constructing a recombinant vector and expressing the phage lytic enzyme PGTphg with the fluorescent marker in a genetically engineered strain

Escherichia coli BL21 (DE3), the phage lytic enzyme can serve as a fluorescence dye which has a long-term fluorescent activity and will not be quenched, so that a sample is not kept in a dark place, the cost is saved, the work efficiency can be greatly improved, and the research time can be shortened greatly. The phage lytic enzyme

PGTphg with the fluorescent marker provided by the present invention can be used for preventing and treating a biofilm of Staphviococcus aureus, and has a good bioactivity for inhibiting formation of a biofilm of Staphylococcus aureus, so as to improve prevention and control of drug-resistant bacteria from a common bacterial level (state of planktonic bacteria) to a microbial community level (state of biofilm). The present invention has certain innovativeness and is worth being further popularized and applied.

BRIEF DESCRIPTION OF THE DRAWINGS

[11] In order to describe the technical solutions of the embodiments of the present invention more clearly, the drawings needed to be used in the embodiment will be briefly introduced below. It is to be understood that the drawings described below are merely some embodiments of the present invention, and therefore, shall not be viewed as limitation to the scope. Those of ordinary skill in the art can further obtain other relevant drawings according to the drawings without making creative efforts.

[12] FIG. 1 is an electrophoretogram of gel extraction of a PCR product of a lyase gene of a phage TSP4, where M represents a Marker, and channels 2 and 3 show the electrophoretogram of gel extraction of the PCR product of the lyase gene of the phage

TSP4, where the size of a DNA fragment of the PCR product is 501 bp.

[13] FIG. 2 is a PCR detection electrophoretogram of a linking experiment, where

M represents a Marker, the channel 2 shows negative control, and a channel 3 shows a

PCR result of a T7 promoter primer of a recombinant vector PET28a-PGTphg after linking.

[14] FIG. 3 is a detection diagram of protein-induced expression of a lyase

PGTphg in the present invention, where a channel 1 represents a Marker, a channel 2 shows a non-induced fragmentized solution of PGTphg/BL2(DE3), a channel 3 shows that a supernate is taken after PET28a-PGTphg/BL2(DE3) subjected to lactose induction is fragmentized, a channel 4 shows a total fragmentized solution of

PET28a-PGTphg/BL2(DE3) subjected to lactose induction, a channel 5 shows a fragmentized supernate of PET28a-PGTphg/BL21(DE3) IPTG subjected to induction, 5 and the channel 6shows a total fragmentized supernate of PET28a-PGTphg/BL21(DE3)

IPTG subjected to induction, where the size of a target induced product is about 48 kDa.

[15] FIG. 4 is a detection diagram of protein purification of the lyase PGTphg with the fluorescent marker, where M represents a protein Marker, and the channel 1 shows a 500 mM imidazole eluent after the lyase is subjected to a chromatographic column, where the size of a target product obtained by purification is about 48 kDa.

[16] FIG. 5 shows that the lyase PGTphg with the fluorescent marker in the present invention adsorbs and stains a biofilm of Staphylococcus aureus. The fluorescent staining activity of the lyase is detected by means of a fluorescence microscope, i.e., the effect of PGTphg as a “fluorescence dye” is observed.

[17] FIG. 6 shows analysis of an inhibitory effect of the lyase PGTphg with the fluorescent marker in the present invention on formation of the biofilm of

Staphylococcus aureus ATCC6538.

[18] FIG. 7 shows analysis of an inhibitory effect of the lyase PGTphg with the fluorescent marker in the present invention on formation of the biofilm of multi-drug resistant Staphylococcus aureus (1606BL 1486).

[19] FIG. 8 shows fluorescence observation of a process of the lyase PGTphg with the fluorescent marker in the present invention acting on the formation of the biofilm of multi-drug resistant Staphylococcus aureus (1606BL 1486), where A acts for 0 min (control), B acts for 30 min, and C acts for 60 min.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[20] In order to make purposes, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and intactly described below. Embodiments with unmarked specific conditions below generally follow conventional conditions or conditions suggested by manufacturers. The used reagents or instruments not indicated by manufacturers are conventional products which can be purchased in the market.

[21] It shall be noted that in the absence of conflict, the embodiments of the present disclosure and features in the embodiments can be combined with one another.

The present invention will be described in detail below with reference to specific embodiments.

[22] The present invention provides an application of a phage lytic enzyme with a fluorescent marker in preparing a preparation for preventing and treating formation of a biofilm of Staphviococcus aureus. The phage lytic enzyme with the fluorescent marker is mainly used for preparing a preparation for preventing and treating formation of a biofilm of food-borne multi-drug-resistant Staphviococcus aureus, and has a good antibacterial effect on formation of the biofilm of multi-drug-resistant

Staphylococcus aureus with high drug resistance.

[23] The phage lytic enzyme with the fluorescent marker includes a lyase derived from a phage TSP4, Siphoviridae, Thermus and a GFP green fluorescent protein tag.

The molecular mass of the phage lytic enzyme is about 48 kDa. The phage lytic enzyme further has the fluorescent staining activity while having the activity of the lyase. The nucleotide sequence of the lyase is shown in SEQ ID NO. 1, and the amino acid sequence of the lyase is shown in SEQ ID NO. 4.

[24] The present invention further provides an application of the phage lytic enzyme with the fluorescent marker in preparing an antibacterial agent for

Staphvlococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonellae,

Shigellae and/or Bacillus subtilis.

[25] The present invention further provides a preparation for preventing and treating formation of a biofilm of Staphylococcus aureus, including the phage lytic enzyme with the fluorescent marker.

[26] The concentration of the phage lytic enzyme with the fluorescent marker is not smaller than 50 ug/ml.

[27] The preparation further includes 8-12 mg/ml NaCl, 0.15-0.25 mg/ml KCl, 1.5-2.0 mg/ml Na;HPO, and 0.2-0.4 mg/ml KH:PO:.

[28] The pH value of the preparation is 7.3-7.5. The pH value is preferably adjusted with HCI to prevent introduction of other novel ions into the preparation.

[29] A method for preparing the preparation includes the following steps: sterilizing a buffer solution; and uniformly mixing the buffer solution with the phage lytic enzyme with the fluorescent marker to obtain the preparation. The sterilizing method is preferably high pressure steam sterilization and filtration sterilization.

Parameters of high pressure steam sterilization are 15 psi and 20 min, to guarantee the sterilization speed and the sterilization effect.

[30] Examples

[31] Molecular cloning of a phage lytic enzyme PGTphg and construction of a recombinant expression vector

[32] Amplification of a gene of the phage lytic enzyme (a genomic DAN of the phage TSP4 of Thermus TC16 is taken as a template)

[33] Primer sequences used for amplification of the gene of the lyase of the phage

TSP4 of thermus are shown in Table 1:

Table 1 Sequence table

S | Se Sequence e | qu q | en u | ce e | na n | m ce | e

N

0.

S | N | ATGCGTCTACCGACTAAGACTTCCCGCTTTGGTTATGTGCACGGCC

Q | le | ACGGCCCTACGCCTACTAGCGACCTTGGTCAGCCTGTGTATGCCCC

I | ot | TGAGGATGGCGTGGTGGTCTATGCCCGGACTGGGTCAGGTACCTGG

D | id | GGTGGGCTGGTGGTGGTCTTGGGCAAAAGCGGCTTTGCCCATCGG

N | e | CTAGGCCATGTGCGCAACATTCGGGTCAAAGAGGGACAGGAGGTG

S| F 5'-ggaattcatggcaatgcgtctaccgactaagac-3'

E | or

Ql w

I ar

Did

N | pr

Oli . | m 2 | er

SIR S'-atttgcggccgctttacctcctagcaacttgg-3'

E | ev

Q er

I se

D pr

Ni

Om [er 3

S | A | MRLPTKTSRFGYVHGQRNHEGIPHPGYDLNNGPTPTSDLGQPVYAPE

E | m | DGVVVYARTGSGTWGGLVVVLGKSGFAHRLGHVRNIRVKEGQEVKE

Q | in | GQQVAEIGEFVKGLPHLHYDMVEPKVIHTISILIKAPY VRWDFWHVNF

Iio PKLFEHMYVDPARFHPELAKLLGGK

D | ac

N id

O | se [qu 4 | en ce of ly as e

[34] The underlined parts of the forward primer and the reverses primer represent

EcoRI and Notl restriction enzyme cutting sites, respectively.

[35] (2) A PCR amplification system is as follows:

Table 2: Compositions of the PCR amplification system

[36] (3) Amplification conditions are as follows:

[37] The reaction system was uniformly mixed, and the mixture was pre-degenerated at 94°C for 10 min first and then degenerated at 94°C for 45 s, annealed at 58°C for 45 s, and extended at 72°C for 28 s, and after 30 circles, the mixture was extended at 72°C for 10 min. After the reaction, 3 ul of a product was subjected to electrophoretic analysis in a 1% agarose gel.

[38] 2. Gel extraction and purification for the PCR product

[39] (1) The 1% agarose gel was poured into an electrophoresis apparatus;

[40] (2) The PCR product to be separated and purified was subjected to spot electrophoresis, and electrophoresis was stopped in a proper position;

[41] (3) The gel containing the target fragment was cut off under an ultraviolet lamp and was transferred to a 1.5 ml Ep tube;

[42] (4) The target fragment was extracted with a gel extraction kit purchased from BIOTEKE CORPORATION; and

[43] (5) A gel extraction result was electrophoretically detected in the 1% agarose gel (see FIG. 1), and as shown in the result of FIG. 1, extraction was successful, and the size of the DNA fragment of the lyase was 501 bp.

[44] 3. Construction of the recombinant expression vector

[45] (1) Preparation of a linear vector pET28a-EGFP with a sticky end

[46] In order to link the target gene fragment to the expression vector pET28a-EGFP, the fragment with the sticky end, i.e, the restriction enzyme cutting site, of the target fragment is needed. Similarly, in order to insert the target fragment into the vector, the vector needs to have the sticky end, and the restriction enzyme cutting sites of the target fragment and the vector are the same.

[47] A. With a plasmid extraction kit (BIOTEKE), plasmid extraction includes the following operating steps:

[48] (1)Culture activation: a sterile inoculating loop was dipped with a culture preserving fluid cryopreserved at -80°C, was inoculated to a Kanamycin LB plate by means of a gas-liquid-solid method, and was cultivated at 37°C for 12-16 h;

[49] (2) Bacteria enrichment and phage collection: 5 ul of kanamycin (the final concentration is 100 pg/ml) was added into a 5 ml LB medium; a positive clone was picked with the inoculating ring and was inoculated to a KNa”-LB medium; then the

KNa’-LB medium was put in a 37°C incubator for shake cultivation and was left overnight; and 3 ml of cultivated bacteria solution was centrifugalized at 5,000 rpm at room temperature for 5 min to precipitate the phages, and a supernate was abandoned;

[50] (3) The phages were resuspended with 250 ul of a solution P1 (containing an

RNA enzyme) to precipitate, and the phages were subjected to vortex oscillation till the phages were suspended thoroughly;

[51] (4) 250 pl of a solution P2 was added, and the medium was turned mildly up and down for 8 times to fully lyse the phage until the solution became clear;

[52] (5) 400 pl of a solution P3 was added, and the medium was immediately turned mildly up and down for 8 times, the medium was put at room temperature for 5 min, the mixture was centrifugalized at 13,000 rpm at room temperature for 10 min, and a supernate was taken carefully;

[53] (6) An adsorption column was placed on a collecting tube, the supernate obtained in the previous step was added into the adsorption column AC (the adsorption column was placed on the collecting tube, and the solution could be added in two times as the amount of the solution was great), the mixture was centrifugalized at 13,000 rpm for 1 min, and a filtrate was abandoned;

[54] (7) 500 ul of a deproteinized solution PE was added, the mixture was centrifugalized at 13,000 rpm for 60 s, and a filtrate was abandoned,

[55] 500 ul of a rinsing solution WB was added, the mixture was centrifugalized at 13,000 rpm for 60 s, and a filtrate was abandoned;

[56] (9) The step (7) was repeated once, the mixture was centrifugalized at 13,000 rpm for 60 s, a filtrate was abandoned, the mixture was centrifugalized at 13,000 rpm for 2 min in avoid column, the mixture was put at room temperature for 3-5 min, and residual ethanol was removed; and

[57] The adsorption column AC was taken out and put in a clean centrifuge tube, 70 ul of an eluting buffer solution EB (preheated at 65°C) was added to the middle part of an adsorption film, the centrifuge tube was placed at room temperature for 1 min, and the mixture was centrifugalized at 13,000 rpm for 1 min to elute the plasmid.

[58] (2) A double enzyme digestion system for the genetic fragment of the lyase of TSP4 and the PET28a-EGFP plasmid is as follows:

Table 3: Compositions of an enzyme digestion reaction system

Enzyme digestion Dosage Enzyme digestion Dosage of the TSP4 PCR of the product PET28a-EGFP plasmid

Ce

[59] Enzyme digestion reaction conditions: the reaction was performed at 37°C for 2 h to extract the genetic fragment of TSP4 and the PET28a-EGFP plasmid after enzyme digestion.

[60] (3) Linkage and transformation and sequencing validation for the recombinant expression vector

[61] The linear vector pET28a-EGFP with the sticky end and the genetic fragment of the lyase of TSP4 obtained in the previous experiment are linked and transformed, and are subjected to colony PCR identification and PCR identification by the universal primers of the T7 promoter (See FIG. 2) and sequencing validation to obtain the recombinant expression vector PET28a-PGTphg. The result in FIG. 2 shows successful identification and transformation.

[62] Test example 1

[63] Induced expression and affinity column purification of the phage lytic enzyme PGTphg with the fluorescent makers in Escherichia coli

[64] 1. Induced expression of a recombinant protein of the phage lytic enzyme

PGTphg with the fluorescent maker in an Escherichia coli engineered strain

[65] The constructed recombinant vector PET28a-PGTphg was transformed into the Escherichia coli BL21 (DE3), the strain containing the recombinant plasmid was cultivated overnight, the bacterial solution was inoculated to an LB liquid medium of

Kan'(the final concentration was 50 pg/ml) at 1%, and the mixture was subjected to shake cultivation at 37°C till the ODsoo value of the mixture was 0.6-0.8; 4 ml of the bacteria solution was taken out for a control experiment; IPTG (the final concentration was 0.5 mM) and lactose (the final concentration was 0.7 g/L) were added into the residual bacteria solution, and the mixture was put in a table concentrator for induced cultivation at 20°C at 150 rpm for 12 h, and 5 ml of a sample was taken for detection.

[66] 2. Detection of an over-expression condition of PGTphg after induction by

SDS-PAGE

[67] The 5 ml of bacteria solution taken out was centrifugalized at 8,000 rpm for 10 min, a supernate was abandoned, an imidazole solution with the final concentration of 30 mM was added to suspend the phages, the phages were ultrasonically fragmentized (the power was 25%, and the device was operated for 3 s and stopped for 4 s, totally 3 min), and the phages were subjected to thermolysis at 98°C for 10 min to fragmentize the phages so as to release proteins in the phages; a SDS-PAGE gel was prepared, with 5% of spacer gel and 12% of separation gel; a sample was fed sequentially for electrophoresis (80 V, 30 min for the spacer gel and 120 V, 120 min for the separation gel), after electrophoresis, the SDS-PAGE gel was stained and then taken out, a R250 Coomassie brilliant blue staining solution was added, and the mixture was oscillated and left overnight for decoloring and photographing analysis (see FIG. 3). The result of FIG. 3 shows that the size of the target induced product is about 48 kDa.

[68] 3. Affinity column purification of the phage lytic enzyme PGTphg with the fluorescent maker

[69] The BL21 (DE3) strains containing the recombinant plasmid

PET28a-PGTphg were massively induced by means of the above method, and the bacteria solution was centrifugalized to collect the Escherichia coli phages (4°C, 8000 rpm, 10 min). The phages suspended by a PBS solution were ultrasonically fragmentized and were centrifugalized at 13,000 rpm at 4°C for 10 min, a supernate was manually purified with a nickel column, the column was first cleaned with ddH:0 which was 10 times of the column in volume and was then balanced with 30 mM imidazole which was 10 times of the column in volume, the sample was fed to the column, the column was eluted with 150 mM imidazole which was 10 times of the column in volume and was then eluted with 500 mM imidazole which was 10 times of the column in volume, the column was cleaned with ddH>O which was 10 times of the column in volume, and finally, the column was filled with 20% anhydrous ethanol, an expression product of the lyase PGTphg purified was subjected to SDS-PAGE detection (see FIG. 4), and the result of FIG. 4 shows that the size of the purified target product is about 48 kDa.

[70] Test example 2

[71] Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC6538,

Escherichia coli K88 CGMCC1.2385, Salmonella CMCC (B) 50094, Bacillus subtilis

CMCC (B) 63501 and Shigella CMCC (B) 51105 cultured overnight were diluted to 5x10°, and EDTA was added, so that the final concentration was 1 mM; after being uniformly mixed, the mixture was reacted at 37°C for 30 min, the product was centrifugalized and resuspended in same volume, 200 uL of bacteria solution and 800 uL of a PGTphg enzyme solution (104 pg/mL) were taken to react at 37°C for 30 min, 100 uL of the mixture was coated to the LB plate, and three parallels were made for each sample. The plate was placed at 37°C to cultivate overnight, and the number of single colonies was calculated.

[72] The bactericidal effect of the lyase PGTphg on pathogenic bacteria is shown in Table 4:

Table 4 Validation on bactericidal activity of the lyase PGTphg to a series of pathogenic bacteria

Strain name Strain No. Bactericidal activity of the mr ee

[73] ++ represents that the number of the phages treated by the lyase decreases by two orders of magnitudes, and + represents that the number of the phages treated by the lyase decreases by one order of magnitudes.

[74] The result of FIG. 4 shows that the lyase has a very good bactericidal effect on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonellae,

Bacillus subtilis and shigellae, illustrating that the lyase PGTphg with the fluorescent maker purified has a good bactericidal activity.

[75] Test example 3

[76] The phage lytic enzyme PGTphg with the fluorescent maker purified can be adsorbed to the biofilm of Staphylococcus aureus and shows a good fluorescent activity.

[77] Analytical steps are as follows:

[78] (1) The Staphylococcus aureus ATCC6538 was prepared into a 1x10°

CFU/mL working bacteria solution with a TSB medium containing 5% glucose for later use;

[79] (2) 2 mL of the working bacteria solution was added into a 24-well plate for cultivation in a 37°C constant temperature incubator for 24 h, so that the biofilm of

Staphylococcus aureus was formed;

[80] (3) The culture solution was sucked out and washed with the PBS twice to remove the planktonic bacteria, 500 ul (the concentration of the lyase was 100 pg/ml) of lyase PGTphg was added, the PBS was added for control, the supernate was abandoned after reaction for 12 h, and the culture solution was washed with the PBS (pH 7.4) for 2-3 times to remove the planktonic bacteria; and

[81] (4) The culture solution was stained with PGTphg at 37°C for 30 min, was washed twice with the PBS and was immobilized with methanol for 15 min. The methanol solution was removed, the culture solution was washed with the PBS for 2-3 times, and the staining condition of the lyase PGTphg on the biofilm of

Staphylococcus aureus was observed by the fluorescence microscope.

[82] As shown in FIG. 5, we can observe that the lyase PGTphg is adsorbed to and acted on the biofilm of the Staphylococcus aureus ATCC6538 to successfully give off green fluorescence.

[83] Test example 4

[84] Inhibitory effect of the lyase PGTphg with the fluorescent marker on formation of the biofilm of Staphylococcus aureus

[85] Change of the viable count of the phages of the biofilm of Staphviococcus aureus after action of the lyase PGTphg is determined by means of an XTT reduction method, and the inhibitory effect of PGTphg on the process of forming the biofilm of

Staphylococcus aureus 1s studied. XTT as an acting substrate of mitochondria dehydrogenase can be reduced by living cells to a water soluble orange-yellow formazan product. When the XTT is applied in combination with an electron coupling agent (for example, PMS), the absorbancy of the water soluble formazan product generated by the XTT is in direct proportion to the number of the living cells. The steps are as follows:

[86] (1) 200 ul of an MH medium where Staphylococcus aureus (ATCC6538) cultivated overnight and multi-drug-resistant Staphylococcus aureus (1606BL 1486, preserved in the lab) were added was added into a 96-well plate, so that the concentration of the phages reached 10° CFU/mL; the lyase PGTphg (the final concentration was 50 ng/mL) was added, Kna® (the final concentration was 50 pg/mL) was added as a positive control, the PBS was added as a negative control, three parallels were made for each experiment, and the medium was cultivated at 37°C for 24 h, so that the number of the phages increased and the phages adhered to cell walls to form the biofilm.

[87] (2) 200 wl of the MH medium was added after the planktonic bacteria were cleaned with the PBS twice, and 20 ul of an XTT reagent was added, the medium was left still and cultivated at 37°C in a dark condition for 2 h and was taken out. Change of the OD value at 490 nm was determined with an enzyme-labeled instrument. The experimental group and the PBS control group were compared, and whether the result was of statistical meaning (significant difference if the P value was smaller than 0.05) was analyzed in a student t test.

[88] As shown in FIG. 6 and FIG. 7, compared with the control PBS, for the phage lytic enzyme PGTphg with the fluorescent marker, formation of the biofilms of the Staphylococcus aureus (ATCC6538) and the multi-drug-resistant Staphylococcus aureus (1606BL 1486, preserved in the lab) is significantly inhibited.

[89] A working bacteria solution (1x10° CFU/mL) of the Staphviococcus aureus

ATCC6538 was prepared with the TSB medium containing 5% glucose, 500 pl (the concentration of the lyase was 100 ug/ml) of the lyase PGTphg was added to react for 30 min and 60 min (0 min was set for control) at 37°C, respectively, and the experimental results with different action times were observed. Then the working solution was washed with PBA twice and immobilized with methanol for 15 min. The methanol solution was removed, the working solution was washed with the PBS for 2-3 times, and the result was shown in FIG. 8 through observation by the fluorescence microscope. As shown in FIG. 8, the process that the phage lytic enzyme PGTphg with the fluorescent marker acts on the biofilm of the Staphylococcus aureus can be directly observed by fluorescent staining. In conclusion, the above results demonstrate the application prospect of the phage lytic enzyme PGTphg with the fluorescent marker in preparing the preparation for preventing and treating formation of the biofilm of

Staphylococcus aureus.

[90] To sum up, the present invention provides a phage lytic enzyme with a fluorescent marker and an application thereof. Transgenic Escherichia coli which specifically produces the phage lytic enzyme PGTphg with the fluorescent marker is constructed by applying a genetic engineering technology, and combined expression of the fluorescent activity fused phage lytic enzyme is completed by using a single plasmid, so that there is no non-synchronous problem of stability and genetic expressions of multiple plasmids in engineering bacteria. The present invention features simple operation, low cost and high feasibility, and lays a foundation for industrial production application of lyase. By constructing a recombinant vector and expressing the phage lytic enzyme PGTphg with the fluorescent marker in a genetically engineered strain Escherichia coli BL21 (DE3), the phage lytic enzyme can serve as a fluorescence dye which has a long-term fluorescent activity and will not be quenched, so that a sample is not kept in a dark place, the cost is saved, the work efficiency can be greatly improved, and the research time can be shortened greatly. The phage lytic enzyme PGTphg with the fluorescent marker provided by the present invention can be used for preventing and treating a biofilm of Staphylococcus aureus, and has a good bioactivity for inhibiting formation of a biofilm of Staphylococcus aureus, so as to improve prevention and control of drug-resistant bacteria from a common bacterial level (state of planktonic bacteria) to a microbial community level (state of biofilm). The present invention is innovative and worthy of further promotion and application.

[91] The described embodiments are merely part of, rather than all of, the embodiments of the present invention. Detailed description on the embodiments of the present invention is not intended to limit the scope of the present invention claimed, and merely represents selected embodiments of the present invention. On a basis of the embodiments in the present invention, all other embodiments obtained by those skilled in the technical field without creative efforts fall into the scope of protection of the present invention.

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HKJP20221101442</ApplicantFileReference> <EarliestPriorityApplicationIdentification> <IPOfficeCode>CN</IPOfficeCode> <ApplicationNumberText>202210868273.2</ApplicationNumberT ext> <FilingDate>2022-07-22</FilingDate> </EarliestPriorityApplicationIdentification> <ApplicantName languageCode="en">Kunming University of

Science and Technology</ApplicantName> <InventionTitle languageCode="en">PHAGE LYTIC ENZYME WITH

FLUORESCENT MARKER AND APPLICATION THEREOF</InventionTitle> <SequenceTotalQuantity>4</SequenceTotalQuantity> <SequenceData sequenceIDNumber="1"> <INSDSeq> <INSDSeq length>501</INSDSeq length> <INSDSeq moltype>DNA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> u <INSDFeature> <INSDFeature key> source</INSDFeature key> <INSDFeature location>1..501</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name> mol type</INSDQualifier name> <INSDQualifier value> other DNA</INSDQualifier value> u </INSDQualifier> <INSDQualifier id="q6"> <INSDQualifier name> organism</INSDQualifier name> u <INSDQualifier value> synthetic construct</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> u </INSDSeq feature-table> <INSDSeg sequence» atgcgtctaccgactaagacttcccgctttggttatgtgcacggccagagaaaccacgaggg cattccccacccaggctatgacctgaataacggccctacgcctactagcgaccttggtcagc ctgtgtatgcccctgaggatggcgtggtggtctatgcceggactgggtcaggtacctggggt gggctggtggtggtcttgggcaaaagcggctttgcccatcggctaggccatgtgcgcaacat tcgggtcaaagagggacaggaggtgaaggaaggccagcaggtggccgagattggggagttcg tcaaggggcttccccacctgcactacgacatggtggagcccaaggttatccacaccatcagt atcctgatcaaggccccttatgttcggtgggacttctggcacgtaaactttcccaaactgtt 1 tgagcacatgtatgtggacccggccaggtttcaccctgagctggccaagttgctaggaggta aatga</INSDSeq sequence> </INSDSeq> </SequenceData> <SequenceData sequenceIDNumber="2"> <INSDSeq> <INSDSeq length>33</INSDSeq length> <INSDSeq moltype>DNA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key> source</INSDFeature key> <INSDFeature location>1..33</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name> mol type</INSDQualifier name> u <INSDQualifier value> other DNA</INSDQualifier value> </INSDQualifier> <INSDQualifier id="g7"> <INSDQualifier name> organism</INSDQualifier name> <INSDQualifier wvalue> synthetic construct</INSDQualifier value> u </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeg sequence> ggaattcatggcaatgcgtctaccgactaagac</INSDSeq sequence> </INSDSeq> </SequenceData> <SequenceData sequenceIDNumber="3"> <INSDSeq> <INSDSeq length>32</INSDSeq length> <INSDSeq moltype>DNA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key> source</INSDFeature key> <INSDFeature location>1l..32</INSDFeature location> u <INSDFeature quals> <INSDQualifier> <INSDQualifier name> mol type</INSDQualifier name> u u <INSDQualifier value> other DNA</INSDQualifier value> </INSDQualifier> <INSDQualifier id="g8"> <INSDQualifier name> organism</INSDQualifier name> 2

<INSDQualifier value> synthetic construct</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq sequence> atttgcggccgetttacctcctagcaacttgg</INSDSeq sequence> </INSDSeq> </SequenceData> <SequenceData sequenceIDNumber="4"> <INSDSeq> <INSDSeq length>166</INSDSeq length> <INSDSeq moltype>AA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key> source</INSDFeature key> u <INSDFeature location>1..166</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name> mol type</INSDQualifier name> <INSDQualifier wvalue> protein</INSDQualifier value> u </INSDQualifier> <INSDQualifier id="q9"> <INSDQualifier name> organism</INSDQualifier name> u <INSDQualifier value> synthetic construct</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq sequence>

MRLPTKTSRFGYVHGORNHEGIPHPGYDLNNGPTPTSDLGOPVYAPEDGVVVYARTGSGTWG

GLVVVLGKSGFAHRLGHVRNIRVKEGOEVKEGOOVAEIGEFVKGLPHLHYDMVEPKVIHTIS

ILIKAPYVRWDFWHVNFPKLFEHMYVDPARFHPELAKLLGGK</INSDSeq sequence> </INSDSeq> </SequenceData> </ST26SegquenceListing> 3

Claims

Conclusions l. Use of a phage lytic enzyme with a fluorescent marker in the preparation of a preparation for the prevention and treatment of Staphylococcus aureus biofilm formation.

Use according to claim 1, wherein the lytic enzyme of a phage with the fluorescent marker comprises a lyase obtained from a phage TSP4, Siphoviridae, Thermus and a green fluorescent GFP protein tag.

Use of the lytic enzyme of a phage with the fluorescent marker according to claim 2 in the preparation of an antibacterial agent for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonellae, Shigellae and/or Bacillus subtilis.

A preparation for preventing and treating biofilm formation of Staphylococcus aureus, comprising the lytic enzyme of a phage with the fluorescent marker according to claim 2.

The preparation according to claim 4, wherein the concentration of the lytic enzyme of a phage with the fluorescent marker is not less than 50 pg/ml.

6. Preparation according to claim 5, further containing 8-12 mg/ml NaCl, 0.15-0.25 mg/ml KCl, 1.5-2.0 mg/ml Na,HPO4 and 0.2-0.4 mg/ml KH>PO4.

7. Preparation according to claim 5, wherein the pH value of the preparation is 7.3-7.5.

A method of preparing a preparation for preventing and treating biofilm formation of Staphylococcus aureus according to any one of claims 4-7, comprising the following steps: sterilizing a buffer solution; and evenly mixing the buffer solution containing the lytic enzyme of phage with the fluorescent marker to obtain the preparation.