CN114736894B - Chimeric enzyme ClyQ for degrading staphylococcus biofilm and preparation method and application thereof - Google Patents

Abstract

The invention provides a chimeric enzyme ClyQ for degrading a staphylococcus biofilm, a preparation method and application thereof, and relates to the technical field of chimeric enzymes. The structure of the chimeric enzyme ClyQ comprises a catalytic domain CHAP of lyase LysGH15 and a cell wall binding domain SH3b of lyase PlyV12, can be used for killing various staphylococci, has good cleaning effect on a biological film of drug-resistant staphylococcus aureus, and provides a material source for industrially producing medicines for cleaning the staphylococcus biological film.

Description

A kind of chimeric enzyme ClyQ that degrades Staphylococcus biofilm and its preparation method and application

Technical field

The invention belongs to the technical field of chimeric enzymes, and specifically relates to a chimeric enzyme ClyQ that degrades Staphylococcus biofilm and its preparation method and application.

Background technique

Staphylococcus aureus is a common Gram-positive bacterium and an important zoonotic pathogen that is widely found in air, water, dust and human and animal excrement. In addition, Staphylococcus aureus can cause a variety of diseases in humans and animals, including wound infections, dairy mastitis, pseudomembranous colitis, sepsis, and sepsis, seriously threatening the lives of humans and animals. In recent years, due to the extensive use of antibiotics, drug-resistant "superbugs" have continued to emerge, such as Methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin has always been the drug of choice for the clinical treatment of Gram-positive bacterial infections. It was once called the "last bottom line" in clinical anti-infection. However, vancomycin-resistant Staphylococcus aureus (VRSA) )Already appeared. In addition, Staphylococcus aureus easily forms biofilms. The biofilm formed by Staphylococcus aureus increases the resistance of the bacteria inside to antibiotics, thereby accelerating the emergence of multi-drug-resistant bacteria. What's more serious is that biofilm-related infections caused by Staphylococcus aureus are increasing year by year. This creates an urgent need for the development of novel antibacterial infection treatments and antibiotic alternatives for clearing biofilms.

Phage lyase is a peptidoglycan hydrolase produced by double-stranded DNA phages in order to release the progeny of the phage after entering the host bacteria. It has strong peptidoglycan degradation activity and can destroy the bacteria from the "outside" after contact with the host bacteria. Its cell wall structure kills bacteria quickly. In nature, the size of phage lytic enzymes of Gram-positive bacteria is usually 25-40kDa, and its modular structure usually contains two domains: the N-terminal catalytic domain (CD) and the C-terminal cell wall binding structure. Domain (cell walls binding domains, CBD). The lytic enzyme catalytic domain mainly acts on most of the chemical bonds in the peptidoglycan network of the bacterial cell wall, while the binding domain is responsible for targeting the phage lytic enzyme to the host cell. The recombination strategy of obtaining chimeric lyases by randomly combining the catalytic domains and binding domains of different lyases has the advantages of simplicity and ease of operation, and can improve the bactericidal activity, expression level and stability of phage lyases.

As we all know, Staphylococcus aureus can easily form biofilms on surfaces such as medical equipment that are difficult to remove, causing great trouble to the medical system, especially ICU wards. At present, there are many studies on the use of natural phage lytic enzymes to clear biofilms, but the research on the use of chimeric enzymes to clear biofilms is still very limited. Therefore, it is important to develop a new chimeric enzyme for clearing biofilms formed by multidrug-resistant Staphylococcus aureus.

Contents of the invention

In view of this, the object of the present invention is to provide a chimeric enzyme ClyQ that degrades Staphylococcus biofilm and its preparation method and application. The chimeric enzyme ClyQ has a good removal effect on the biofilm formed by Staphylococcus aureus and can be used in vitro. Rapidly kill Staphylococcus aureus and provide a source of materials for the industrial production of drugs to remove Staphylococcus aureus biofilm.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The invention provides a chimeric enzyme ClyQ that degrades Staphylococcus biofilm. The structure of the chimeric enzyme ClyQ includes the catalytic domain CHAP of lytic enzyme LysGH15 and the cell wall binding domain SH3b of lytic enzyme PlyV12.

Preferably, the construction method of the catalytic domain CHAP of the lytic enzyme LysGH15 includes using the encoding gene of the lytic enzyme LysGH15 as a template, using LysGH15CHAP-F and LysGH15CHAP-R as primers, amplifying the catalytic domain CHAP of LysGH15; The nucleotide sequence of LysGH15CHAP-F is shown in SEQ ID NO.3, and the nucleotide sequence of LysGH15CHAP-R is shown in SEQ ID NO.4;

The construction method of the cell wall binding domain SH3b of the lytic enzyme PlyV12 includes using the PlyV12 gene as a template, using PlyV12SH3b-F and PlyV12SH3b-R as primers, amplifying the binding domain SH3b of PlyV12; the nucleotide of the PlyV12SH3b-F The sequence is shown in SEQ ID NO.5, and the nucleotide sequence of PlyV12SH3b-R is shown in SEQ ID NO.6.

Preferably, the nucleotide sequence encoding the chimeric enzyme ClyQ is shown in SEQ ID NO.1.

The invention also provides a method for constructing the above chimeric enzyme ClyQ, which includes the following steps: using the catalytic domain CHAP of the lytic enzyme LysGH15 and the cell wall binding domain SH3b of the lytic enzyme PlyV12 as templates, using LysGH15CHAP-F and PlyV12SH3b-R as primers, and using Overlap PCR is used to connect the catalytic domain CHAP of the lytic enzyme LysGH15 and the cell wall binding domain SH3b of the lytic enzyme PlyV12, and the amplified product is the encoding gene of the chimeric enzyme ClyQ.

Preferably, the overlapping PCR amplification program includes: pre-denaturation at 98°C for 10 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 57°C for 30 s, extension at 72°C for 25 s; and extension at 72°C for 5 min.

The invention also provides a method for preparing the above-mentioned chimeric enzyme ClyQ, which includes the following steps: (1) constructing a recombinant expression vector containing the encoding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector includes a prokaryotic expression vector ;

(2) Transform the recombinant expression vector into prokaryotic cells to obtain engineering bacteria expressing the chimeric enzyme ClyQ;

(3) Pick a single colony of the engineered bacteria for induction culture, centrifuge and break the bacteria, and collect the broken supernatant, which contains the chimeric enzyme ClyQ.

The present invention provides the application of the above chimeric enzyme ClyQ in preparing a reagent for removing Staphylococcus biofilm.

Preferably, the Staphylococcus aureus includes one or more of Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus wauerii, Staphylococcus coryi and Staphylococcus mimicus.

The present invention also provides a method for removing Staphylococcus biofilm, which includes the following steps: statically incubating the above chimeric enzyme ClyQ with the biofilm formed by Staphylococcus aureus.

Preferably, the static incubation temperature is 30-37°C and the time is 1 hour.

Beneficial effects: The present invention provides a chimeric enzyme ClyQ that degrades Staphylococcus biofilm. It combines the catalytic domain and the cell wall binding domain derived from two different lytic enzymes to obtain a chimeric enzyme that can degrade Staphylococcus biofilm. , and can quickly kill Staphylococcus aureus in vitro, especially for antibiotic-resistant "superbugs" with good killing effect. The chimeric enzyme ClyQ of the present invention can be solublely expressed through prokaryotic expression, and the performance of the enzyme is stable. The staphylococcal chimeric enzyme ClyQ can be prepared into an enzyme preparation to prevent and treat various infections caused by staphylococci, especially It has extremely high control effect on the biofilm formed by Staphylococcus aureus. In the embodiments of the present invention, elimination studies were conducted on biofilms formed by methicillin-resistant Staphylococcus aureus S3 and ATCC43300, confirming that the chimeric enzyme ClyQ can efficiently eliminate methicillin-resistant Staphylococcus aureus in a dose-dependent manner. Biofilm formation by Staphylococcus aureus.

Description of the drawings

Figure 1 is the SDS-PAGE picture of the chimeric enzyme ClyQ; M is EpiZyme WJ03 Marker, 1 is the unpurified pET28a-ClyQ protein, 2 represents the purified protein result, and the protein size is 45kDa;

Figure 2 shows the broad-spectrum results of the chimeric enzyme ClyQ in vitro lysing Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus varsii, Staphylococcus coryi and Staphylococcus mimicus;

Figure 3 shows the time change results of different concentrations of chimeric enzyme ClyQ cleavage of S. aureusATCC43300 in vitro;

Figure 4 shows the changes in cleavage efficiency of chimeric enzyme ClyQ under different pH buffers;

Figure 5 shows the changes in cleavage efficiency of chimeric enzyme ClyQ at different temperatures;

Figure 6 is a diagram showing the effect of the chimeric enzyme ClyQ in clearing biofilm of the methicillin-resistant Staphylococcus aureus standard strain ATCC43300;

Figure 7 is a diagram showing the effect of chimeric enzyme ClyQ in clearing methicillin-resistant Staphylococcus aureus S3 biofilm.

Detailed ways

The invention provides a chimeric enzyme ClyQ that degrades Staphylococcus biofilm. The structure of the chimeric enzyme ClyQ includes the catalytic domain CHAP of lytic enzyme LysGH15 and the cell wall binding domain SH3b of lytic enzyme PlyV12.

The method for constructing the catalytic domain CHAP of the lytic enzyme LysGH15 of the present invention preferably includes using the coding gene of the lytic enzyme LysGH15 (GenBank accession number: HM015284.1) as a template, using LysGH15CHAP-F and LysGH15CHAP-R as primers, and amplifying The catalytic domain CHAP of LysGH15 is obtained, wherein the 5' end of the LysGH15 CHAP-F is preferably provided with a BamHI restriction enzyme site, and the nucleotide sequence is preferably as shown in SEQ ID NO.3: 5'-GGATCCATGGCAAAAACACAAGCTGA-3', The nucleotide sequence of LysGH15CHAP-R is preferably shown in SEQ ID NO. 4: 5'-CGATCCAGACGAGCCTCCGGCTTTAACCGGGATC-3'. The amplification system of the present invention is calculated as 50 μL, and preferably includes: 2 μL of template, 2.5 μL of each 10 μM primer, 25 μL of Primerstar 2x Mix, and 50 μL of ddH ₂ O; and the amplification program preferably includes: pre-denaturation at 98°C for 10 min. ; Denaturation at 98°C for 10 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 15 seconds, 30 cycles; extension at 72°C for 5 minutes.

The method for constructing the cell wall binding domain SH3b of the lytic enzyme PlyV12 (GeneBank accession number: AY581208.1) of the present invention preferably includes using the PlyV12 gene as a template, using PlyV12SH3b-F and PlyV12SH3b-R as primers, and amplifying to obtain the binding of PlyV12 Domain SH3b; the nucleotide sequence of PlyV12SH3b-F is shown in SEQ ID NO.5: 5'-GGAGGCTCGTCTGGATCGCTAAATGGGGGATCAACA-3', the 5' end of PlyV12SH3b-R is set with a HindIII restriction enzyme site, and the nucleotide The sequence is shown in SEQ ID NO.6: 5'-AAGCTTTTATTTGAAAGTACCCCA-3'. The amplification system and procedure of the present invention are preferably the same as those mentioned above, and will not be described again here.

In the present invention, the nucleotide sequence encoding the chimeric enzyme ClyQ is shown in SEQ ID NO.1: ATGGCAAAAACACAAGCTGAAATAAATAAGCGCCTGGATGCGTACGCGAAAGGTACAGTGGACAGCCCGTATCGTATTAAAAAGGCTACCTCCTACGACCCGTCGTTCGGCGTGATGGAAGCGGGTGCAATTGACGCGGATGGCTACTACCATGCACAGTGCCAGGATCTGATCACCGATTATGTGCTGTGGCTGACCGATAACAAAGTTC GTACCTGGGGCAACGCGAAGGACCAAATCAAGCAAAGCTACGGCACTGGTTTTAAAATCCACGAAAACAAGCCAAGCACGGTGCCGAAAAAGGGCTGGATTGCTGTCTTCACGAGCGGTTCTTACCAGCAATGGGGTCATATTGGTATTGTTTATGACGGCGGTAACACCTCCACCTTCACCATCTTGGAGCAGAATTGGAATGGTTATGCCAATAAGAAGCCGACCAAACGTGTTGACAACTATTACGGCTTGACCACTTTATCG AGATCCCGGTTAAAGCCGGAGGCTCGTCTGGATCGCTAAATGGGGGATCAACACCACCTAAACCCAACACCAAGAAGGTGAAGGTGCTGAAGCACGCCACGAACTGGTCACCGTCTAGCAAAGGCGCAAAAATGGCTAGCTTTGTTAAGGGCGGCACCTTCGAGGTCAAGCAGCAGCGTCCGATTAGCTACTCCTACAGCAATCAAGAATATCTTATCGTGAATAAGGGTACGGTCTTGGGCTGGGTTTTGTCGCAA GACATCGAGGGTGGTTATGGTAGCGATCGTGTTGGTGGCTCCAAGCCGAAACTGCCCGGCAGGTTTTACCAAGGAGGAAGCGACCTTCATCAACGGTAACGCTCCGATTACCACCCGCAAAAACAAACCGTCCCTGTCTAGCCAGACCGCGACCCCGCTGTATCCCGGTCAAAGCGTTCGTTACCTGGGCTGGAAAAGCGCGGAAGGCTACATCTGGATTTATGCGACTGACGGCCGTTACATTCCGGTACGCCCAGTGGGTAAAGAGG CGTGGGGTACTTTCAAA; Its amino acid sequence is shown in SEQ ID NO.2: MAKTQAEINKRLDAYAKGTVDSPYRIKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYQQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVKAG GSSGSLNGGSTPPKPNTKKVKVLKHATNWSPSSKGAKMASFVKGGTFEVKQQRPISYSNQEYLIVNKGTVLGWVLSQDIEGGYGSDRVGGSKPKLPAGFTKEEATFINGNAPITTRKNKPSLSSQTATPLYPGQSVRYLGWKSAEGYIWIYATDGRYIPVRPVGKEAWGTFK.

The invention also provides a method for constructing the above chimeric enzyme ClyQ, which includes the following steps: using the catalytic domain CHAP of the lytic enzyme LysGH15 and the cell wall binding domain SH3b of the lytic enzyme PlyV12 as templates, using LysGH15CHAP-F and PlyV12SH3b-R as primers, and using Overlap PCR is used to connect the catalytic domain CHAP of the lytic enzyme LysGH15 and the cell wall binding domain SH3b of the lytic enzyme PlyV12, and the amplified product is the encoding gene of the chimeric enzyme ClyQ.

The present invention preferably uses the above method to amplify the catalytic domain CHAP of lytic enzyme LysGH15 and the cell wall binding domain SH3b of lytic enzyme PlyV12, and uses the amplified sequence as a template, and uses LysGH15CHAP-F and PlyV12SH3b-R as primers, The LysGH15CHAP fragment and the PlyV12SH3b fragment were connected together using overlapping PCR. The overlapping PCR amplification system of the present invention is calculated as 50 μL, and preferably includes: 1 μL of LysGH15CHAP fragment and PlyV12SH3b fragment as templates, 2.5 μL of 10 μM primers, 25 μL of Primerstar2x Mix, and 50 μL of ddH ₂ O. The overlapping PCR amplification program of the present invention preferably includes: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 10 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 25 seconds, 30 cycles; and extension at 72°C for 5 minutes.

The invention also provides a method for preparing the above-mentioned chimeric enzyme ClyQ, which includes the following steps: (1) constructing a recombinant expression vector containing the encoding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector includes a prokaryotic expression vector ;

(2) Transform the recombinant expression vector into prokaryotic cells to obtain engineering bacteria expressing the chimeric enzyme ClyQ;

(3) Pick a single colony of the engineered bacteria for induction culture, centrifuge and break the bacteria, and collect the broken supernatant, which contains the chimeric enzyme ClyQ.

The present invention constructs a recombinant expression vector containing the encoding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector includes a prokaryotic expression vector. The present invention preferably uses the above-mentioned overlapping PCR method to construct the product, uses BamHI restriction endonuclease and HindIII restriction endonuclease for double enzyme digestion, incubates it in a water bath at 37°C for 2 hours, and separates the target band through 1% agarose electrophoresis. The gel recovery kit purifies and recovers the fragments. The purified and recovered fragments are then used to construct a recombinant expression vector. The basic vector of the recombinant expression vector includes a prokaryotic expression vector. The present invention does not specifically limit the type of the prokaryotic expression vector. In the embodiment, the pET28a vector is used as an example. explanation, but it cannot be regarded as the entire protection scope of the present invention. In the present invention, the fragment is preferably inserted between the BamHI and HindIII restriction sites of the prokaryotic expression vector.

After obtaining the recombinant expression vector, the present invention uses the above recombinant expression vector to transform into the competent cell E. coli DH5α, extracts the plasmid and introduces it into E. coli BL21 (DE3) to obtain the engineering strain BL21/pET28a-ClyQ expressing the chimeric enzyme. In the present invention, it is preferred to apply the transformed bacterial liquid to an LB plate containing kanamycin (100 μg/ml), and culture it at 37°C overnight; select positive clones, conduct sequencing verification, and sequence the correct positive clones for subsequent research.

In the present invention, a single colony of the engineered bacteria is selected for induction culture, the bacteria are centrifugally broken, and the broken supernatant is collected, and the supernatant contains the chimeric enzyme ClyQ. In the present invention, IPTG is preferably used to induce the expression of the chimeric enzyme ClyQ. The final concentration of IPTG is preferably 0.8 mmol/L, and induction is carried out at 16°C for 16 to 18 hours under this condition.

In the present invention, it is preferred to collect the bacterial cells after the induction, use ultrasonic waves to disrupt the cells, and centrifuge at 4°C and 10,000 rpm/min for 20 minutes to collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, and analyze the lysed supernatant by SDS-PAGE. Protein expression in the supernatant; the filtered lysate supernatant was purified using His affinity chromatography nickel column (GE Healthcare, Sweden). In summary, after using the engineering bacteria of the present invention to induce expression with IPTG, there is a target protein band at about 45kD in the supernatant, which is consistent with the expected size. This shows that the engineering bacteria are constructed correctly and the expressed lytic enzyme protein The product ClyQ is a soluble protein.

The present invention provides the application of the above chimeric enzyme ClyQ in preparing a reagent for removing Staphylococcus biofilm.

The Staphylococcus aureus of the present invention preferably includes one or more of Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus wauerii, Staphylococcus coryi and Staphylococcus mimicus. In the examples, it is confirmed that the chimeric enzyme ClyQ has broad-spectrum lytic activity against Staphylococcus aureus, specifically as follows: it can efficiently lyse Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus varveneri, and Staphylococcus spp. and Staphylococcus mimicus. In the examples of the present invention, it is also confirmed that the chimeric enzyme ClyQ has good lytic activity against methicillin-resistant Staphylococcus aureus (MRSA), and the chimeric enzyme ClyQ is dose-dependent. Efficiently removes biofilm formed by MRSA.

The present invention also provides a method for removing Staphylococcus biofilm, which includes the following steps: statically incubating the above chimeric enzyme ClyQ with the biofilm formed by Staphylococcus aureus.

The temperature of static incubation in the present invention is preferably 30-37°C, more preferably 37°C; the time is preferably 1 hour.

The chimeric enzyme ClyQ provided by the present invention for degrading Staphylococcus biofilm and its preparation method and application will be described in detail below with reference to the examples. However, they should not be understood as limiting the scope of the present invention.

The reagents, bacterial strains and equipment used in the present invention are all commercially available unless otherwise specified.

The experimental methods used in the present invention are all conventional experimental methods unless otherwise specified; the primers and sequencing work used were all completed at Beijing Qingke Biotechnology Co., Ltd.

Example 1

Expression and purification of chimeric enzyme ClyQ

1.1 Construction of recombinant expression vector expressing chimeric enzyme ClyQ

1.1.1 Preparation of the catalytic domain CHAP of LysGH15 and the cell wall binding domain SH3b of PlyV12

The complete gene fragments of lytic enzymes LysGH15 and PlyV12 were synthesized (Beijing Qingke Biotechnology Co., Ltd.), and the synthesized sequences were connected to the pET28a vector respectively. Using the LysGH15 gene as a template and LysGH15CHAP-F (SEQ ID NO.3) and LysGH15CHAP-R (SEQ ID NO.4) as primers, the catalytic domain CHAP of LysGH15 was amplified. Using the PlyV12 gene as a template, PlyV12SH3b-F was amplified. (SEQ ID NO.5) and PlyV12SH3b-R (SEQ ID NO.6) were used as primers to amplify the binding domain SH3b of PlyV12.

The steps to amplify the above fragments are as follows:

Amplification system: 2 μL of template, 2.5 μL of each 10 μM primer, 25 μL of Primerstar 2x Mix, 50 μL of total system, and 50 μL of ddH ₂ O.

Amplification conditions: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 10 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 15 seconds, 30 cycles; extension at 72°C for 5 minutes.

After the reaction is completed, the PCR amplification product is detected by agarose gel electrophoresis to verify that the gene size is correct, and then the gene fragment is purified and recovered through a gel recovery kit.

1.1.2 Construction of recombinant expression vector expressing chimeric enzyme ClyQ

Use the LysGH15CHAP fragment and PlyV12SH3b fragment recovered in 1.1 as template, use LysGH15CHAP-F and PlyV12SH3b-R as primers, and connect the LysGH15CHAP fragment and PlyV12SH3b fragment together through overlap extension PCR technology. The specific steps are as follows:

Amplification system: 1 μL of LysGH15CHAP fragment and PlyV12SH3b fragment as templates, 2.5 μL of 10 μM primers, 25 μL of Primerstar 2x Mix, 50 μL of total system, and 50 μL of ddH ₂ O.

Amplification conditions: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 10 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 25 seconds, 30 cycles; extension at 72°C for 5 minutes.

After the reaction, the PCR amplification product was detected by agarose gel electrophoresis and the gene size was verified to be correct. Then, BamHI restriction endonuclease and HindIII restriction endonuclease were used for double enzyme digestion, and the product was incubated in a water bath at 37°C for 2 hours, and then dried for 1 hour. % agarose electrophoresis separates the target band, and the gel recovery kit purifies and recovers the fragment, and connects the fragment to the pET28a vector at 16°C overnight to obtain the recombinant expression vector pET28a-ClyQ, which is transformed into competent cells in the large intestine using the above recombinant expression vector. Bacillus DH5α, extract the plasmid and introduce it into E. coli BL21 (DE3) to obtain the engineering strain BL21/pET28a-ClyQ expressing the chimeric enzyme. The transformed bacterial solution was spread on an LB plate containing kanamycin (100 μg/ml) and cultured at 37°C overnight; positive clones were selected and sequenced for verification.

1.2 Inducible expression and purification of chimeric enzyme ClyQ

The recombinant strain BL21/pET28a-ClyQ was inoculated into LB culture medium containing kanapenicillin (100 μg/mL), and cultured overnight at 37°C with shaking; the next day, it was transferred to 1000 mL LB culture medium at a ratio of 1:100, and cultured at 37°C. Culture with shaking until the OD ₆₀₀ value is about 0.6, add IPTG to a final concentration of 0.8mmol/L, and induce at 16°C for 16 to 18 hours. Collect the bacterial cells, disrupt the cells by ultrasonic, centrifuge at 4°C and 10,000 rpm/min for 20 min, collect the supernatant, filter the supernatant through a 0.22 μm filter, and analyze the protein expression in the lysed supernatant by SDS-PAGE. The filtered lysate supernatant was purified using His affinity chromatography nickel column (GE Healthcare, Sweden).

The SDS-PAGE analysis results are shown in Figure 1. After the recombinant strain BL21/pET28a-ClyQ was induced and expressed by IPTG, there was a target protein band in the supernatant at about 45kD, which was consistent with the expected size. This showed that the recombinant strain BL21 /pET28a-ClyQ is constructed correctly, and the expressed lyase protein product ClyQ is a soluble protein.

Example 2

Broad-spectrum results of the chimeric enzyme ClyQ in in vitro lysis of Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus varsoni, Staphylococcus coryi and Staphylococcus mimicus.

A variety of different strains of Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus varsii, Staphylococcus coryi and Staphylococcus mimicus were cultured to the logarithmic phase (OD ₆₀₀ = 0.6), and centrifuged at low temperature ( 4°C, 6000rpm) to collect the precipitate, wash it twice with PBS and resuspend it in PBS to obtain bacterial liquids of different strains. Take 100 μL of the chimeric enzyme ClyQ in Example 1 and mix it with 100 μL of the above bacterial liquid, so that the final concentration of the chimeric enzyme ClyQ is 50 μg/ml, and the system OD ₆₀₀ = 0.6 to 0.8.

At the same time, a mixture of equal amounts of PBS and the above bacterial solution was used as a negative control. After incubating at 37°C for 30 minutes, the OD ₆₀₀ was measured. Repeat three times. The results obtained are shown in Figure 2.

As can be seen from the results in Figure 2, the chimeric enzyme ClyQ can efficiently lyse Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus squirrelii, Staphylococcus varvenerii, Staphylococcus coryi and Staphylococcus mimicus in vitro. This also indicates that the chimeric enzyme ClyQ has broad-spectrum lytic activity against Staphylococcus aureus.

Example 3

Time variation results of in vitro cleavage of MRSA standard strain ATCC43300 by chimeric enzyme ClyQ at different concentrations

Staphylococcus aureus MRSAATCC43300 was cultured to the logarithmic phase (OD ₆₀₀ =0.6), and the precipitate was collected by low-temperature centrifugation (4°C, 6000 rpm), washed twice with PBS and resuspended in PBS to obtain a strain liquid. Take 100 μL of the chimeric enzyme ClyQ in Example 1 and mix it with 100 μL of the above bacterial liquid, so that the final concentrations of the chimeric enzyme ClyQ are 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μg/ml respectively, and the system OD ₆₀₀ = 0.6 to 0.8 . At the same time, a mixture of equal amounts of PBS and the above bacterial liquid was used as a negative control, incubated at 37°C, and samples were taken at different time points to measure OD ₆₀₀ and repeated three times. The results are shown in Figure 3.

It can be seen from the results in Figure 3 that at the 5th minute of incubation, the absorbance of the experimental group with a chimeric enzyme ClyQ concentration of 50 μg/ml dropped significantly. At the 60th minute, the absorbance of the experimental group dropped from OD ₆₀₀ = 0.775 to OD ₆₀₀ =0.169. In addition, the chimeric enzyme ClyQ still has cleavage activity when the concentration is 0.78 μg/ml. The results show that the chimeric enzyme ClyQ has good cleavage activity against the MRSA standard strain ATCC43300.

Example 4

Changes in cleavage efficiency of chimeric enzyme ClyQ under different pH buffers

Staphylococcus aureus S. aureus S3 was cultured to the logarithmic phase (OD ₆₀₀ = 0.6), centrifuged at low temperature (4°C, 6000 rpm/min) to collect the precipitate, washed twice with PBS and resuspended in PBS to obtain a strain liquid. The chimeric enzyme ClyQ in Example 1 was diluted 2-fold to the usage concentration (25 μg/ml) with buffers of different pH values (5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0), and incubated at 37°C for 1 hour. After the incubation, mix 100 μL of bacterial solution and 100 μL of chimeric enzyme ClyQ so that the final concentration of chimeric enzyme ClyQ is 25 μg/ml. Incubate at 37°C for 30 minutes. The PBS group is used as a negative control. OD ₆₀₀ is measured and the turbidity reduction ratio is calculated. Repeat three times, and the results are shown in Figure 4.

As can be seen from the results in Figure 4, the chimeric enzyme ClyQ has efficient cleavage activity against S. aureus S3 at pH 7-11. The results show that the chimeric enzyme ClyQ maintains efficient and stable cleavage activity under neutral and weakly alkaline conditions.

Example 5

Changes in cleavage efficiency of chimeric enzyme ClyQ at different temperatures

Staphylococcus aureus S. aureus S3 was cultured to the logarithmic phase (OD ₆₀₀ = 0.6), centrifuged at low temperature (4°C, 6000 rpm/min) to collect the precipitate, washed twice with PBS and resuspended in PBS to obtain a strain liquid. The chimeric enzyme ClyQ in Example 1 was diluted with PBS to a final concentration of 25 μg/ml. The chimeric enzyme ClyQ with a final concentration of 25 μg/ml was placed at 37, 40, 42.5 and 45°C and incubated for 1 hour respectively. The incubation was completed. Afterwards, take 100 μL of the above-mentioned chimeric enzyme ClyQ and 100 μL of the above-mentioned bacterial liquid and incubate it at 37°C for 30 minutes. The PBS group is used as a negative control. OD ₆₀₀ is measured and the turbidity reduction ratio is calculated. Repeat three times, and the results are shown in Figure 5.

As can be seen from the results in Figure 5, the chimeric enzyme ClyQ can efficiently cleave S. aureus S3 at temperatures below 42.5°C. It can be seen from the results that the chimeric enzyme ClyQ can maintain stable bactericidal activity at temperatures below 42.5°C.

Example 6

Effect of chimeric enzyme ClyQ in eliminating methicillin-resistant Staphylococcus aureus standard strain ATCC43300

The methicillin-resistant Staphylococcus aureus standard strain ATCC43300 was cultured to the logarithmic phase (OD ₆₀₀ =0.6), and the bacterial liquid was adjusted to 1×10 ⁷ CFU/ml using LB medium. Add 200 μL of the bacterial solution of the above concentration to the 96-well plate, incubate at 30°C for 24 hours or 48 hours, and then wash three times with sterile PBS. After drying, add 200 μL of the chimeric enzyme ClyQ in Example 1 with PBS at a concentration of 5 μg/ml or 40 μg/ml to each well as a negative control. Incubate at 37°C for 1 hour and then wash three times with sterile PBS. After washing, use 200 μL of sterile PBS to resuspend the biofilm in each well. The resuspension was diluted and spread on an LB plate containing 1.5% agar to count the bacteria in the biofilm. The above experiment was repeated three times. The results are shown in Figure 6.

It can be seen from the results in Figure 6 that when the chimeric enzyme ClyQ concentration is 40 μg/ml, the bacterial concentration in the 48-h biofilm formed by ATCC43300 can be reduced from 7.43 logs to 5.90 logs. The 24-h biofilm formed by ATCC43300 The bacterial concentration in was reduced from 7.56 log to 5.61 log. Moreover, the biofilm can still be cleared even when the chimeric enzyme ClyQ concentration is 5 μg/ml. The results showed that the chimeric enzyme ClyQ efficiently eliminated the biofilm formed by the methicillin-resistant Staphylococcus aureus standard strain ATCC43300 in a dose-dependent manner.

Example 7

Effect of chimeric enzyme ClyQ in eliminating methicillin-resistant Staphylococcus aureus S3

Methicillin-resistant Staphylococcus aureus S3 was cultured to the logarithmic phase (OD ₆₀₀ =0.6), and the bacterial liquid was adjusted to 1×10 ⁷ CFU/ml using LB medium. Add 200 μL of the bacterial solution of the above concentration to the 96-well plate, incubate at 30°C for 24 hours or 48 hours, and then wash three times with sterile PBS. After drying, add 200 μL of the chimeric enzyme ClyQ in Example 1 with PBS at a concentration of 5 μg/ml or 40 μg/ml to each well as a negative control. Incubate at 37°C for 1 hour and then wash three times with sterile PBS. After washing, use 200 μL of sterile PBS to resuspend the biofilm in each well. The resuspension was diluted and spread on an LB plate containing 1.5% agar to count the bacteria in the biofilm. The above experiment was repeated three times. The results are shown in Figure 7.

It can be seen from the results in Figure 7 that when the chimeric enzyme ClyQ concentration is 40 μg/ml, the bacterial concentration in the biofilm formed by S3 for 48 hours can be reduced from 7.54 logs to 5.46 logs, and the bacterial concentration in the biofilm formed by S3 for 24 hours can be reduced. The bacterial concentration in the film decreased from 7.23 log to 6.28 log. Moreover, the biofilm can still be cleared even when the chimeric enzyme ClyQ concentration is 5 μg/ml. The results showed that the chimeric enzyme ClyQ efficiently cleared the biofilm formed by methicillin-resistant Staphylococcus aureus S3 in a dose-dependent manner.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

sequence list

<110> Huazhong Agricultural University

<120> A chimeric enzyme ClyQ that degrades Staphylococcus biofilm and its preparation method and application

<160> 6

<170> SIPOSequenceListing 1.0

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<211> 1020

<212> DNA

<213> Artificial Sequence

<400> 1

atggcaaaaa cacaagctga aataaataag cgcctggatg cgtacgcgaa aggtacagtg 60

gacagcccgt atcgtattaa aaaggctacc tcctacgacc cgtcgttcgg cgtgatggaa 120

gcgggtgcaa ttgacgcgga tggctactac catgcacagt gccaggatct gatcaccgat 180

tatgtgctgt ggctgaccga taacaaagtt cgtacctggg gcaacgcgaa ggaccaaatc 240

aagcaaagct acggcactgg ttttaaaatc cacgaaaaca agccaagcac ggtgccgaaa 300

aagggctgga ttgctgtctt cacgagcggt tctttaccagc aatggggtca tattggtatt 360

gtttatgacg gcggtaacac ctccaccttc accatcttgg agcagaattg gaatggttat 420

gccaataaga agccgaccaa acgtgttgac aactattacg gcttgaccca ctttatcgag 480

atcccggtta aagccggagg ctcgtctgga tcgctaaatg ggggatcaac accacctaaa 540

cccaacacca agaaggtgaa ggtgctgaag cacgccacga actggtcacc gtctagcaaa 600

ggcgcaaaaa tggctagctt tgttaagggc ggcaccttcg aggtcaagca gcagcgtccg 660

attagctact cctacagcaa tcaagaatat cttatcgtga ataagggtac ggtcttgggc 720

tgggttttgt cgcaagacat cgagggtggt tatggtagcg atcgtgttgg tggctccaag 780

ccgaaactgc cggcaggttt taccaaggag gaagcgacct tcatcaacgg taacgctccg 840

attaccaccc gcaaaaacaa accgtccctg tctagccaga ccgcgacccc gctgtatccc 900

ggtcaaagcg ttcgttacct gggctggaaa agcgcggaag gctacatctg gatttatgcg 960

actgacggcc gttacattcc ggtacgccca gtgggtaaag aggcgtgggg tactttcaaa 1020

<210> 2

<211> 340

<212> PRT

<213> Artificial Sequence

<400> 2

Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala

1 5 10 15

Lys Gly Thr Val Asp Ser Pro Tyr Arg Ile Lys Lys Ala Thr Ser Tyr

20 25 30

Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly

35 40 45

Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp

50 55 60

Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile

65 70 75 80

Lys Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser

85 90 95

Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr

100 105 110

Gln Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser

115 120 125

Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys

130 135 140

Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu

145 150 155 160

Ile Pro Val Lys Ala Gly Gly Ser Ser Ser Gly Ser Leu Asn Gly Gly Ser

165 170 175

Thr Pro Pro Lys Pro Asn Thr Lys Lys Val Lys Val Leu Lys His Ala

180 185 190

Thr Asn Trp Ser Pro Ser Ser Lys Gly Ala Lys Met Ala Ser Phe Val

195 200 205

Lys Gly Gly Thr Phe Glu Val Lys Gln Gln Arg Pro Ile Ser Tyr Ser

210 215 220

Tyr Ser Asn Gln Glu Tyr Leu Ile Val Asn Lys Gly Thr Val Leu Gly

225 230 235 240

Trp Val Leu Ser Gln Asp Ile Glu Gly Gly Tyr Gly Ser Asp Arg Val

245 250 255

Gly Gly Ser Lys Pro Lys Leu Pro Ala Gly Phe Thr Lys Glu Glu Ala

260 265 270

Thr Phe Ile Asn Gly Asn Ala Pro Ile Thr Thr Arg Lys Asn Lys Pro

275 280 285

Ser Leu Ser Ser Gln Thr Ala Thr Pro Leu Tyr Pro Gly Gln Ser Val

290 295 300

Arg Tyr Leu Gly Trp Lys Ser Ala Glu Gly Tyr Ile Trp Ile Tyr Ala

305 310 315 320

Thr Asp Gly Arg Tyr Ile Pro Val Arg Pro Val Gly Lys Glu Ala Trp

325 330 335

Gly Thr Phe Lys

340

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 3

ggatccatgg caaaaacaca agctga 26

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 4

cgatccagac gagcctccgg ctttaaccgg gatc 34

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 5

ggaggctcgt ctggatcgct aaatggggga tcaaca 36

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 6

aagcttttatttgaaagtacccca 24

Claims (9)

1. A chimeric enzyme ClyQ that degrades a staphylococcal biofilm, wherein the structure of the chimeric enzyme ClyQ comprises a catalytic domain CHAP of a lyase LysGH15 and a cell wall binding domain SH3b of a lyase PlyV 12; the nucleotide sequence of the coded chimeric enzyme ClyQ is shown as SEQ ID NO. 1.

2. The chimeric enzyme ClyQ according to claim 1, characterized in that the construction method of the catalytic domain CHAP of the lyase LysGH15 comprises the steps of using the coding gene of the lyase LysGH15 as a template and LysGH15CHAP-F and LysGH15CHAP-R as primers to amplify and obtain the catalytic domain CHAP of LysGH 15; the nucleotide sequence of LysGH15CHAP-F is shown as SEQ ID NO.3, and the nucleotide sequence of LysGH15CHAP-R is shown as SEQ ID NO. 4;

the construction method of the cell wall binding domain SH3b of the lyase PlyV12 comprises the steps of taking a PlyV12 gene as a template, taking PlyV12SH3b-F and PlyV12SH3b-R as primers, and amplifying to obtain the binding domain SH3b of the PlyV 12; the nucleotide sequence of the PlyV12SH3b-F is shown as SEQ ID NO.5, and the nucleotide sequence of the PlyV12SH3b-R is shown as SEQ ID NO. 6.

3. The method for constructing chimeric enzyme ClyQ according to claim 1 or 2, comprising the steps of: connecting the catalytic domain CHAP of the lysGH15 and the cell wall binding domain SH3b of the lyase PlyV12 by using the catalytic domain CHAP of the lyase LysGH15 and the cell wall binding domain SH3b of the lyase PlyV12 as templates, using LysGH15CHAP-F and PlyV12SH3b-R as primers and using an overlap PCR method, wherein an amplification product is a coding gene of the chimeric enzyme ClyQ; the nucleotide sequence of LysGH15CHAP-F is shown as SEQ ID NO.3, and the nucleotide sequence of LysGH15CHAP-R is shown as SEQ ID NO. 4.

4. The method of claim 3, wherein the overlapping PCR amplification procedure comprises: pre-denaturation at 98℃for 10min; denaturation at 98℃for 10s, annealing at 57℃for 30s, elongation at 72℃for 25s,30 cycles; extending at 72℃for 5min.

5. The method for producing the chimeric enzyme ClyQ according to claim 1 or 2, comprising the steps of: (1) Constructing a recombinant expression vector containing a coding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector comprises a prokaryotic expression vector;

(2) Converting the recombinant expression vector into prokaryotic cells to obtain engineering bacteria expressing the chimeric enzyme ClyQ;

(3) And (3) picking a single colony of the engineering bacteria for induction culture, centrifugally crushing the bacterial body, and collecting the supernatant after crushing, wherein the supernatant contains the chimeric enzyme ClyQ.

6. Use of the chimeric enzyme ClyQ of claim 1 or 2 for the preparation of a reagent for removing a biofilm from staphylococci.

7. The use according to claim 6, wherein the staphylococcus comprises one or more of staphylococcus aureus, staphylococcus hemolyticus, staphylococcus epidermidis, staphylococcus squirrel, staphylococcus warsiensis, staphylococcus colestis and staphylococcus mimicus.

8. A method of removing a biofilm from staphylococci comprising the steps of: incubating the chimeric enzyme ClyQ of claim 1 or 2 with a biofilm formed by staphylococci at rest.

9. The method of claim 8, wherein the incubation at rest is at a temperature of 30-37 ℃ for a period of 1h.