A streptococcal prophage lytic enzyme lys1459 and its application

Abstract

The invention discloses a streptococcal prophage lytic enzyme Lys1459 and application thereof, belonging to the technical field of streptococcal related disease prevention and treatment. The amino acid sequence of the lytic enzyme Lys1459 is shown in SEQ ID NO.1, and the coding gene of the lytic enzyme can be prepared into a recombinant vector or an engineered bacterium, which provides a basis for further application of the lytic enzyme Lys1459; the lytic enzyme has a broad-spectrum antibacterial activity against streptococci related to cow mastitis, which provides a theoretical basis for the prevention and treatment of cow mastitis.

Description

Technical Field

[0001] The invention relates to the technical field of streptococcal related disease prevention and treatment, and more specifically to a streptococcal prophage lytic enzyme lys1459 and an application thereof. Background Art

[0002] Streptococcus bacteria belong to Gram-positive bacteria, which are widely distributed in nature, human and animal feces, and the nasopharynx of healthy people. They include a wide variety of pathogenic bacteria. Human pathogens include Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus faecalis. These streptococci can cause clinical diseases such as sepsis, arthritis, meningitis, endocarditis, encephalitis, rheumatic fever, and scarlet fever; Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis infecting cattle can cause symptoms such as mastitis in dairy cows; Streptococcus suis can cause meningitis, sepsis, and pneumonia in pigs and humans; Streptococcus equi infection can cause equine glanders in horses, wound infections, and bacteremia in humans. Streptococcal disease has caused great harm to human health and caused huge losses to the animal husbandry industry. The emergence of antibiotics has brought streptococcal infections under certain control. However, with the irregular use of antibiotics in human medicine and animal husbandry industry, their drug resistance has gradually strengthened. The resulting multidrug-resistant bacteria have no available antibiotics to treat. Therefore, there is an urgent need to develop a new class of antimicrobial drugs or antimicrobial agents for the prevention and control of streptococcal infections.

[0003] Bacteriophages are viruses that can infect prokaryotic microorganisms such as bacteria and archaea. They can quickly lyse bacteria through the "perforin-lyase" binary system. Therefore, bacteriophages and their derived lyases can be used as alternative antibacterial agents to antibiotics. The lysis spectrum of bacteriophages is generally narrow, and their application is limited because bacteria can quickly evolve resistance to surface receptors. However, lyases derived from bacteriophages have a relatively wide lysis spectrum and can be used as an efficient and safe natural antibacterial agent for the prevention and control of drug-resistant pathogenic bacteria.

[0004] Phage lytic enzymes can be used as antimicrobial agents to specifically and rapidly kill host bacteria. Lytic enzymes act on the peptidoglycan on the surface of bacteria. Since peptidoglycan is a key component of the cell wall and is highly conserved within the species, lytic enzymes exhibit a broader spectrum of lytic activity, which also determines that it is more difficult for bacteria to develop resistance to lytic enzymes. At the same time, when lytic enzymes are used to treat animals infected with pathogenic bacteria, the host animals will produce antibodies against them, but the active center of the lytic enzyme is generally not an antigenic epitope exposed on the surface, so antibodies against the lytic enzyme will not weaken its lytic and bactericidal effect. Moreover, lytic enzymes have a synergistic antibacterial effect when used in combination with other antimicrobial agents such as antibiotics, which can not only broaden the lytic spectrum, but also more effectively exert antibacterial activity and reduce the probability of bacteria developing resistance.

[0005] At present, there is no broad-spectrum effective streptococcal phage lytic enzyme developed to prevent and control streptococcal infections related to bovine mastitis and diseases caused by other streptococci such as Streptococcus suis, Streptococcus equi, Streptococcus pneumoniae and Streptococcus pyogenes.

[0006] Therefore, how to provide a streptococcal phage lytic enzyme targeting a spectrum of streptococci is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0007] In view of this, the present invention provides a streptococcal prophage lytic enzyme lys1459, which has a wide spectrum of antibacterial activity against streptococci associated with bovine mastitis and bacteria causing other bacterial infectious diseases, providing a theoretical basis for the prevention and treatment of bovine mastitis and bacterial infections.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A streptococcal prophage lytic enzyme lys1459, the amino acid sequence of the lytic enzyme lys1459 is shown as SEQ ID NO.1.

[0010] Preferably, the domain of the streptococcal prophage lytic enzyme lys1459 includes an N-terminal CHAP cleavage domain and a C-terminal Amidase-2 cleavage domain.

[0011] Preferably, the N-terminal CHAP cleavage domain is from amino acid 1 to amino acid 151, and the C-terminal Amidase-2 cleavage domain is from amino acid 181 to amino acid 325.

[0012] As an inventive concept identical to the above-mentioned technical scheme, the present invention also requests protection for the use of the streptococcal prophage lytic enzyme lys1459 in the preparation of drugs related to Streptococcus lyticus, Enterococcus, Aerococcus, and Staphylococcus aureus; wherein the streptococci include: Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus infantis, Streptococcus suis, Streptococcus equi, human Streptococcus pneumoniae, and Streptococcus pyogenes.

[0013] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of Streptococcus prophage lytic enzyme lys1459 in the preparation of an antibacterial agent for treating bovine mastitis.

[0014] A drug comprises streptococcal prophage lytic enzyme lys1459, wherein the amino acid sequence of the lytic enzyme lys1459 is shown as SEQ ID NO.1, and the use concentration of the streptococcal prophage lytic enzyme lys1459 is 50 μg / mL.

[0015] As an inventive concept identical to the above-mentioned technical scheme, the present invention also requests protection for the use of the streptococcal prophage lytic enzyme lys1459 in the preparation of biological materials for lytic streptococci, staphylococci, enterococci faecalis, aerobic bacteria, listeria monocytogenes or pseudomonas aeruginosa, wherein the biological materials include a recombinant expression vector and a recombinant engineered bacterium; the recombinant expression vector is formed by recombination of an expression vector and a coding gene as shown in SEQ ID NO.2; the recombinant engineered bacterium is formed by transforming the recombinant expression vector into a host Escherichia coli.

[0016] The present invention also provides a method for preparing streptococcal prophage lytic enzyme lys1459, the specific steps of which are as follows:

[0017] (1) Using the genome of Streptococcus SX5-2 as a template, amplify the nucleotide sequence of prophage lytic enzyme lys1459;

[0018] (2) cloning the nucleotide sequence obtained in step (1) into an expression vector to obtain a recombinant expression vector;

[0019] (3) transforming the recombinant expression vector obtained in step (2) into competent Escherichia coli cells to obtain recombinant engineered bacteria;

[0020] (4) Using recombinant engineered bacteria to induce expression of lysase lys1459;

[0021] (5) Extract and purify the lysate lys1459 obtained in step (4).

[0022] Preferably, the enzyme digestion method used in the cloning process of the lysine lys1459 nucleotide sequence is double digestion with Bam HI and Kpn I.

[0023] Preferably, the expression vector is pEC, with a 6xHis tag at the C-terminus; a T7lac promoter, and a Ni + Affinity chromatography purification. The vector can also be selected from other vectors suitable for expressing foreign genes in E. coli, such as pET series, pGEX series, pMAL series or pBAD series.

[0024] Preferably, the engineering bacteria is Escherichia coli BL21 (DE3), and may also be expression host bacteria such as Escherichia coli Rosseta.

[0025] Preferably, the inducing agent used to induce expression is IPTG, the induction concentration is 0.5 mmol / L, and the induction time is 16 h.

[0026] Preferably, the culture temperature of the engineered bacteria is 37°C and the induction expression temperature is 25°C.

[0027] It can be known from the above technical scheme that compared with the prior art, the present invention clones the prophage lytic enzyme lys1459 from the Streptococcus uberis SX5-2 genome, and adopts prokaryotic expression to induce the expression of soluble lytic enzyme. The lytic enzyme lys1459 has a broad-spectrum antibacterial effect on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus infantis, Streptococcus suis, Streptococcus equi, Streptococcus pneumoniae, Streptococcus pyogenes and other bacteria such as enterococci, aerobic bacteria, Staphylococcus aureus, etc. in the genus Streptococcus, laying the foundation for the prevention and treatment of diseases infected by the above bacteria. The present invention can obtain a large number of products with clear enzyme activity and physicochemical properties, and the product production cost is low, the product activity is strong, and the production demand for promotion and application can be met. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The attached figure is a SDS-PAGE result diagram of prokaryotic expression and purification of lysase protein lys1459; wherein M is a standard protein molecular weight marker; 1 is the whole protein of the engineering strain transformed with an empty vector; 2 is the whole protein of the recombinant engineering strain transformed with a recombinant expression plasmid; 3 and 4 are the purification results of lysase protein lys1459;

[0030] Figure 2 The attached figure is a schematic diagram of the protein sequence structure of the lysase lys1459; wherein CHAP is the first cleavage domain and Amidase-2 is the second cleavage domain;

[0031] Figure 3 The attached figure is a diagram of the three-dimensional structure of Streptococcus prophage lytic enzyme lys1459 predicted by AlphaFold;

[0032] Figure 4 The accompanying drawings are diagrams showing the in vitro antibacterial effects of different concentrations of lytic enzyme lys1459 on Streptococcus dysgalactiae;

[0033] Figure 5 The accompanying drawing is a diagram showing the pH stability test results of the lysase lys1459;

[0034] Figure 6 The attached figure is a graph showing the temperature stability of the lytic enzyme lys1459. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1 Genomic sequence amplification and recovery.

[0037] The whole genome of Streptococcus uberis SX5-2 isolated from bovine mastitis milk samples was sequenced, the prophage genome in the genome was located by bioinformatics analysis, and the full-length nucleotide sequence of the lytic enzyme lys1459 was retrieved in the prophage. The primer pair lys1459-F / R was used to amplify the nucleotide sequence shown in Seq ID NO.2.

[0038] Upstream primer lys1459-F: 5'- aggagatataccatgggatcc atggtctcatacatagattt-3′, as shown in SEQ ID NO.3.

[0039] Downstream primer lys1459-R: 5' aaaatacaggttttcggtacc cttgtctaaattgacaaggt-3′, as shown in SEQ ID NO.4.

[0040] The primer sequence contains the homology arm fragment at the expression vector connection site, and the underlined base sequence is the homology arm fragment.

[0041] The reaction system is shown in Table 1;

[0042] Table 1: PCR reaction system

[0043] <![CDATA[2× Phanta ·Mix·Master·Buffer]]> 25μL Template DNA 1μL lys1459-F 2μL lys1459-R 2μL <![CDATA[ddH2O·]]> 20μL

[0044] Reaction conditions:

[0045]

[0046] After the amplification was completed, the PCR amplification product was checked by 1% agarose gel electrophoresis, and the target fragment amplified by PCR was recovered using the Novagen gel recovery kit.

[0047] Example 2: Connection of recombinant expression plasmid and transformation of competent cells.

[0048] 1) The expression vector was double-digested with enzymes, and the enzyme digestion system is shown in Table 2.

[0049] Table 2: Enzyme digestion system

[0050] <![CDATA[ pEC Plasmids ]]> 10μL |Bam HI 1μL <![CDATA[ Kpn ·I·]]> 1μL 10×M Buffer 2μL <![CDATA[ddH2O·]]> 6μL

[0051] The enzyme digestion process was as follows: 37°C, water bath for 4 hours, the enzyme digestion system was verified by 1% agarose gel electrophoresis, and the gel was recovered to obtain a vector with sticky ends.

[0052] According to the target gene PCR gel recovery product and the plasmid concentration after enzyme digestion, the 20 μL one-step cloning and ligation system was determined, as shown in Table 3.

[0053] Table 3: Connection system

[0054] Target gene 2μL Plasmid digestion products 2μL 5×CE·II·Buffer 4μL <![CDATA[ddH2O]]> 12μL

[0055] 2) Mix the ligation system in Table 3 and ligate for 30 minutes at 37°C. Add 10 μL of the ligation product to 100 μL of E. coli DH5α competent cells, mix gently, and place on ice for 30 minutes. Then heat shock at 42°C for 90 seconds and quickly place on ice for 2 minutes. Add 890 μL of LB medium and culture at 37°C with shaking for 1 hour. After centrifugation at 5000 rpm for 5 minutes, discard 900 μL of supernatant, use the remaining 100 μL of resuspended bacterial solution, and evenly spread it on solid LB medium containing Kan (final concentration 50 μg / mL), and culture it inverted in a 37°C incubator overnight.

[0056] 3) Pick a single colony on the Kan plate and inoculate it into LB liquid medium containing Kan for cultivation. After obtaining a bacterial suspension, identify the positive bacterial suspension by PCR, and send the PCR identification system to Shanghai Sangon Biotech Co., Ltd. for sequencing, and compare the sequencing results with the target gene sequence. After the recombinant DH5α strain with correct sequencing is cultured overnight, the recombinant plasmid is extracted using the Novozymes plasmid extraction kit.

[0057] 4) Take 10 μL of recombinant plasmid and add it to 100 μL of E. coli BL21 competent cells, mix well and place on ice for 30 minutes. Then heat shock at 42℃ for 90 seconds and quickly place on ice for 2 minutes. Add 890 μL of LB medium and culture at 37℃ with shaking for 1 hour. Take 100 μL of bacterial solution and evenly spread it on solid LB medium containing Kan (final concentration 50 μg / mL), and culture it upside down in a 37℃ incubator overnight.

[0058] Example 3 Prokaryotic expression and purification of lysase protein lys1459

[0059] Pick a single colony on the Kan plate and inoculate it into LB liquid medium containing Kan for cultivation. After obtaining a bacterial suspension, identify the positive bacterial suspension by PCR. Transfer the positive bacterial suspension to LB liquid medium and culture at 37℃ with shaking until OD 600After about 0.6-0.8, add IPTG with a final concentration of 0.5mmol / L and shake at 25℃ overnight to induce expression. Use the expression engineering bacteria transferred with empty plasmid as negative control. After the recombinant engineering bacteria are expanded and induced to express, use ultrasonic disruption to lyse the bacteria.

[0060] After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was taken for SDS-PAGE electrophoresis analysis. Figure 1 As shown, compared with the empty plasmid engineering bacteria (lane 1), the recombinant engineering bacteria showed thicker bands at 50 KDa and 38 KDa (lane 2).

[0061] The cells were resuspended in 50 mM imidazole solution, lysed by ultrasonication, and the supernatant was taken and purified by Ni + The affinity chromatography column is used to purify the lytic enzyme protein. The column is equilibrated with the equilibrium solution for 2-3 times, and then the supernatant is loaded onto the column multiple times to allow the target protein to interact with the affinity chromatography Ni. + The filler was fully combined and washed 6 times with 50mM imidazole to remove the impurities. Then 200mM imidazole was used to elute the target protein. The results of protein purification are shown in Figure 1 Lanes 3 and 4. The amino acid sequence of the purified lysate protein lys1459 is shown in SEQ ID NO.1, and the encoding gene is shown in SEQ ID NO.2.

[0062] MVSYIDFKSRVLNHGYDHDGGAYGWQCWDGFAEWCVANGIPIINTDPVNHTGYAMDLWELRQSNGILTYFDEVEIMEGGDVVVFKEVAGVTPSSHVAIFDSDIDGT YGWFLGQNQGSTITHPNGGSAFNLVMLPYSATYPTAFRIKKKVEVQTSMGTKNVNGDIYSDLITSSRPELFGNWGVRDMKSIKYIVIHGTLGLSVQSAVNAWTGGR EASANYIVHDNQIVGCVGENYTAWHCGGQGAITNQNSIGIEHVNATLNGANSTFSAQTVDTGAKLVAEICKRLGIIPSRKTIVGHREVFATACPQSLNLDDYVNKV LSYYNGKTSSQTKQETLKTASSQPNKQPSARIIVFDEDIDNFKKNQNWLFNFDRGTYNYIANTEELKFIKKQYPDIQTEHVSKKYPAHVRYIEGFNLVNLDK, as in SEQ Shown as ID NO.1.

[0063] atggtctcat acatagattt taaaagtcgt gtattaaatc acggctacga tcatgacggtggagcatacg gttggcaatg ctgggacggg ttcgcagaat ggtgtgtagc taatggaatt ccaattattaacacggatccagtaaatcat actggctatg ctatggacct atgggaactt agacaatcga atggaatactgacatatttt gatgaggttg aaataatgga aggtggtgat gtcgtagtct ttaaagaggt tgccggcgtcacaccatcta gtcatgtagc tatttttgat agtgacattg acggcactta cggatggttc cttgggcaaaatcagggctc gacgattacg catcctaatg gaggctcagc atttaacctt gtcatgttac cgtactctgcaacatatcca actgcgtttagaataaaaaa gaaagttgag gtgcaaacat ctatgggaac taaaaatgtaaatggagaca tttactctgatttaatcaca agctcacgac ctgagctatt tggaaactgg ggtgttcgtgacatgaaatc tattaaatac attgttattc atggcacgct cggcctgtca gtgcaaagcg cagtaaatgcatggactggt ggccgtgaag caagcgcaaa ctatattgtt catgataacc agattgttgg ttgtgtaggcgaaaattaca cggcatggca ttgtggtggt caaggtgcaa ttactaacca aaattcaatt gggatagaacatgttaatgc aacattaaat ggtgctaattcaacattttc tgcgcagact gtcgatactg gtgctaaattggtggcagaa atttgtaaac gccttggaat tataccgagc agaaaaacaa ttgttggcca cagagaggtgtttgccacag catgccctcagagcttaaat ttggacgatt atgtcaataa agttttatct tattacaacggcaaaacaag ctctcaaacg aagcaagaga cactaaaaac ggcatctagc caaccaaaca aacaaccaagtgctagaatt atcgtatttg atgaagatat tgacaatttt aaaaagaatc aaaattggct ttttaattttgatcgtggca catataacta tattgctaat actgaagaat taaaattcat caaaaagcaa tatccagatattcagacaga acatgtatct aaaaaatatc cagctcatgt tcgatatatc gaaggtttca accttgtcaatttagacaag, as shown in SEQ ID NO. 2.

[0064] Example 4 Structural identification of lysase lys1459.

[0065] The structure of the purified lysase lys1459 was identified. Figure 2 shown by Figure 2 It can be seen that CHAP is the first cleavage domain and Amidase-2 is the second cleavage domain; CHAP starts at the numerical value 1 and ends at the numerical value 151 in the protein sequence; Amidase-2 starts at the numerical value 181 and ends at the numerical value 325 in the protein sequence.

[0066] The three-dimensional structure of Streptococcus prophage lytic enzyme lys1459 was predicted using the AlphaFold Colab website (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb#scrollTo=AzIKiDiCaHAn), and its amino acid sequence (SEQ ID NO.1) was submitted. After downloading the prediction results, the domain was drawn using pymol software. The results are shown in the figure. Figure 3 shown.

[0067] Example 5 Analysis of the physicochemical properties of the lysase LYS1459.

[0068] Number of amino acids: 420; relative molecular mass: 46639.24; theoretical pI: 5.95; number and proportion of amino acids: Ala(A)26 6.2%; Arg(R)12 2.9%; Asn(N)32 7.6%; Asp(D)24 5.7%; Cys(C)6 1.4%; Gln(Q)18 4.3%; Glu(E)19 4.5%; Gly(G)38 9.0%; His(H)12 2.9%; Ile(I)31 7.4%; Leu(L)22 5.2%; Lys(K)24 5.7%; Met(M)6 1.4%; Phe(F)17 4.0%; Pro(P)13 3.1%; Ser(S)29 6.9%;Thr(T)29 6.9%;Trp(W)9 2.1%;Tyr(Y)20 4.8%;Val(V)33 7.9%;Pyl(O)0 0.0%;Sec(U)0 0.0%;(B)0 0.0%;(Z)0 0.0%;(X)0 0.0%;Total number of negatively charged residues (Asp+Glu): 43;Total number of positively charged residues (Arg+Lys): 36;Atomic composition: Carbon C 2082;Hydrogen H3177;Nitrogen N 563;Oxygen O 635;Sulfur S 12; molecular formula: C2082H3177N563O635S12; total number of atoms: 6469; extinction coefficient: 79675; extinction coefficient is measured in M-1cm-1 in water at 280nm; absorbance 0.1% (=1g / l) 1.708.

[0069] Estimated half-life:

[0070] The N-terminal of the protein sequence is methionine (Met).

[0071] Estimated half-life: 30 hours (mammalian reticulocytes, in vitro).

[0072] >20 hours (yeast, in vivo).

[0073] >10 hours(Escherichia coli,invivo).

[0074] Instability Index:

[0075] The instability index (II) was calculated to be 23.97, which indicated that the protein was classified as stable.

[0076] The aliphatic index is 78.19 and the overall average hydrophilicity (GRAVY) is -0.546.

[0077] Example 6 Determination of in vitro antibacterial activity and antibacterial spectrum of the lytic enzyme Lys1459.

[0078] Streptococci were cultured in BHI medium until the logarithmic phase. After centrifugation, the precipitate was washed twice with Tris-HCl (pH 7.5). The bacteria were then resuspended with Tris-HCl and the OD was adjusted. 600 100 μL of concentrated bacterial solution was mixed with 100 μL of lysase lys1459 at different concentrations (0, 50, 100, 150, 200 μg / mL) in a 96-well plate to adjust the OD of the mixture to 0.6. 600 The value was about 0.6, and the final concentrations of the lytic enzyme were 0, 25, 50, 75, and 100 μg / mL. 100 μL of concentrated bacterial solution and 100 μL of Tris-HCl mixed solution were used as controls. Three parallels were placed in a multifunctional microplate reader and cultured at 37°C. The OD was measured every 10 minutes. 600 Numerical value, plotting OD 600 A curve that changes over time.

[0079] The results are as follows Figure 4 As shown, the OD of the groups treated with different concentrations of lysase lys1459 600 The value showed a gradual downward trend, the bacterial solution gradually became clear, and the OD without adding lysing enzyme 600 The values ​​were basically unchanged. Among them, the downward trend of the 25μg / mL lyase group was slightly worse; when the lyase concentration was greater than 50μg / mL, OD 600 The decrease in values ​​tends to be consistent, indicating that the application concentration of 50μg / mL can achieve the best antibacterial effect.

[0080] Commercially available Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus infantum, Streptococcus suis, Streptococcus equi, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Aerococcus equi, Aerococcus viride, etc. were used for the cleavage spectrum determination of the lytic enzyme lys1459. The strain to be tested was cultured at 37°C to the mid-logarithmic growth period, and the bacterial solution was centrifuged and the precipitate was washed twice with Tris-HCl (pH 7.5), and then the bacterial cells were resuspended with Tris-HCl and the OD was adjusted. 600 100 μL of concentrated bacterial solution was mixed with 100 μL of lysase lys1459 at 100 μg / mL in a 96-well plate to adjust the OD of the mixture to 0.6. 600The value was about 0.6, the final concentration of the lytic enzyme was 50 μg / mL, 100 μL of concentrated bacterial solution and 100 μL of Tris-HCl mixed solution were used as controls, 3 parallels per group were placed in a multifunctional microplate reader and cultured at 37°C. OD was measured at 0h and 1h 600 Numerical value, calculate OD 600 The value decreases in proportion to OD 600 >20% indicated that the cleavage was possible, so as to determine the cleavage spectrum of the cleavage enzyme Lys1644.

[0081] The lysis spectrum results are shown in Table 4. The lytic enzyme LYS1459 has a lysis effect on the tested Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus infantis, Streptococcus suis, Streptococcus equi, Streptococcus pneumoniae, and Streptococcus pyogenes. It also has a lysis effect on other tested Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Aeromonas. The results show that the lytic enzyme LYS1459 has a broad-spectrum antibacterial ability.

[0082] Table 4: Results of cleavage spectrum determination of lysase lys1459

[0083] Strain name Cracking capacity Streptococcus uberis SX5-2 + Streptococcus uberis HB4 + Streptococcus uberis HB19-2 + Streptococcus uberis SD2-1 + Streptococcus uberis SD22 + Streptococcus uberis SD25 + Streptococcus uberis SD7 + Streptococcus dysgalactiae SD24 + Streptococcus dysgalactiae SD5-1 + Streptococcus dysgalactiae 4-4 + Streptococcus agalactiae H-11-1 + Streptococcus infantis HB·10-1 + <![CDATA[Streptococcus suis ss > + Streptococcus equi MF / 1 / 2 + Streptococcus pneumoniae CCUG·6798 + Streptococcus pneumoniae CCUG·1407 + Streptococcus pneumoniae ATCC 49619 + Streptococcus pyogenes ATCC·12344 + Staphylococcus aureus H4 + Enterococcus faecalis V583 +. Enterococcus faecium SX4-1 + Aeromonas cyprinifolia SX16-1 + Green balloon fungus HB11-4 +

[0084] Example 7 Analysis of pH stability and temperature stability of lysase lys1459.

[0085] pH stability analysis of lysase lys1459: Streptococcus was cultured in BHI medium to the logarithmic phase, and the bacterial suspension was divided into 8 groups. After centrifugation, the precipitate was washed twice with different pH buffers (50mM NaCl, 50mM Na2HPO4, pH adjusted to 4, 5, 6, 7, 8, 9, 10, 11, respectively). Then, the bacteria were resuspended in the corresponding pH buffer in each group and the OD was adjusted. 600 In a 96-well plate, 100 μL of concentrated bacterial solution at different pH conditions was mixed with 100 μL of lysase lys1459 (lysase was prepared by diluting Tris-HCl at different pH) at 100 μg / mL at the same pH, and the OD of the mixed solution was adjusted to 0.6. 600 The value is about 0.6, and the final concentration of the lyase is 50 μg / mL. After mixing, quickly place it in an ELISA reader to measure the initial A 630 Then, the mixture of bacteria and lytic enzymes under different pH conditions was incubated at 37°C, and the final A was measured after 1 hour. 630 Value, and calculate △A 630 100 μL of concentrated bacterial solution and 100 μL of Tris-HCl mixed solution were used as controls, with 3 parallels in each group.

[0086] The results of the test are as follows Figure 5 As shown, the lysase lys1459 has a △A value of 6 under the condition of pH value. 630 The highest value has the highest lytic activity, and the lytic enzyme activity decreases to varying degrees with the increase or decrease of pH value.

[0087] Temperature stability analysis of lysase lys1459: Streptococci were cultured in BHI medium until the logarithmic phase. After centrifugation, the precipitate was washed twice with Tris-HCl (pH 7.5), and then the cells were resuspended with Tris-HCl and the OD was adjusted. 600 The concentration of the bacterial solution was about 0.6, and then concentrated to 1 / 2 of the original volume. The concentrated bacterial solution was placed at 4°C, 16°C, 25°C, 37°C, 42°C, and 56°C for 30 minutes. At the same time, the lysase Lys1459 to be added was treated in the same way at the corresponding temperature. 100 μL of the concentrated bacterial solution was mixed with 100 μL of lysase Lys1459 at 100 μg / mL in a 96-well plate to make the OD of the mixed solution 600 The value is about 0.6, and the final concentration of the lyase is 50 μg / mL. After mixing, quickly place it in an ELISA reader to measure the initial A 630 Then the mixture of bacteria and lytic enzyme was placed under different temperature conditions for incubation, and A was measured after 1 hour. 630 Final value, and calculate △A 630 100 μL of concentrated bacterial solution and 100 μL of Tris-HCl mixed solution were used as controls, with 3 parallels in each group.

[0088] The results of the test are as follows Figure 6 As shown, the lysase lys1459 at 37°C △A 630 The value is the highest, with the highest lytic activity, and with the increase or decrease of temperature, the lytic enzyme activity decreases to varying degrees, and the lys1459 enzyme activity is lost at 56℃.

[0089] In summary, the optimal enzyme activity conditions for the lysase lys1459 are pH = 6, 37°C.

[0090] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0091] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A streptococcal prophage lytic enzyme lys1459, characterized in that The amino acid sequence of the lysase lys1459 is shown in SEQ ID NO.

1.

2. A Streptococcus prophage lytic enzyme lys1459 according to claim 1, characterized in that The structural domain of the streptococcal prophage lytic enzyme lys1459 includes an N-terminal CHAP cleavage domain and a C-terminal Amidase-2 cleavage domain.

3. A Streptococcus prophage lytic enzyme Lys1459 according to claim 2, characterized in that The N-terminal CHAP cleavage domain is from amino acids 1 to 151, and the C-terminal Amidase-2 cleavage domain is from amino acids 181 to 325.

4. Use of the streptococcal prophage lytic enzyme lys1459 according to any one of claims 1 to 3 in the preparation of drugs related to lytic streptococci, enterococci, aerobic bacteria, and Staphylococcus aureus; wherein, Streptococci include: Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus infantis, Streptococcus suis, Streptococcus equi, human Streptococcus pneumoniae and Streptococcus pyogenes.

5. Use of the streptococcal prophage lytic enzyme Lys1459 according to any one of claims 1 to 3 in the preparation of an antibacterial agent for treating bovine mastitis.

6. A drug, characterized in that The invention comprises streptococcal prophage lytic enzyme lys1459, the amino acid sequence of the lytic enzyme lys1459 is shown as SEQ ID NO.1, and the use concentration of the streptococcal prophage lytic enzyme lys1459 is 50 μg / mL.

7. Use of the streptococcal prophage lytic enzyme lys1459 according to any one of claims 1 to 3 in the preparation of biomaterials of Streptococcus lysate, Enterococcus, Aerococcus, and Staphylococcus aureus, wherein the biomaterial comprises a recombinant expression vector and a recombinant engineered bacterium; the recombinant expression vector is formed by recombining the expression vector and the SEQ ID NO.2 sequence; the recombinant engineered bacterium is formed by transforming the recombinant expression vector into a host Escherichia coli.

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