A hybrid antibiotic enzyme for killing animal-derived pathogens and its application

Abstract

The present invention discloses a technical field involving antibiotic enzymes, and in particular, relates to a hybrid antibiotic enzyme for killing animal-derived pathogens and its application. The hybrid antibiotic enzyme includes a hybrid protein E1C1, and the hybrid protein E1C1 includes a catalytic domain E1 as shown in SEQ ID NO:2 and a binding domain C1 as shown in SEQ ID NO:3. The present invention also provides an application of a hybrid antibiotic enzyme in the preparation of a pharmaceutical composition for controlling microbial infection, and a pharmaceutical composition comprising the hybrid antibiotic enzyme as an effective ingredient. The hybrid antibiotic enzyme of the present application can quickly lyse common animal-derived pathogens Escherichia coli, Salmonella, Staphylococcus aureus and Helicobacter pylori, and can also efficiently kill common pathogens such as clinically isolated Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention relates to the technical field of antibiotic enzymes, in particular to a hybrid antibiotic enzyme for killing animal-derived pathogens and application thereof. Background Art

[0002] With the extensive use of antibiotics, the problem of bacterial resistance to antibiotics has become increasingly serious, causing difficulties in clinical treatment and increased mortality worldwide, seriously threatening human health. Some drug-resistant bacteria / resistance genes with important public health significance have begun to appear and spread in livestock and poultry in my country, posing a threat to food safety and human health. It has been found that many animal-derived bacteria in my country, such as Staphylococcus aureus and Enterococcus aureus, carry cfr and optrA genes. This gene can mediate resistance to five types of antimicrobial drugs. If the carrying bacteria infect humans, it may cause people to be infected with clinically relevant pathogens such as MRSA and VRE without drug treatment. The ermB gene that can mediate high-level drug resistance was found in Campylobacter from pigs and chickens in my country. This gene has been integrated into the resistance gene island, which has the possibility of causing the carrying bacteria to develop into "super bacteria". In addition, carbapenem-resistant Enterobacteriaceae carrying NDM-1 and MCR-1 resistance genes have been found in chickens and pigs in my country. Will these bacteria become widespread and endanger public safety? Recently, researchers from South China Agricultural University discovered a "super super bacteria" in a sick pet cat. A strain of Escherichia coli carried a hybrid plasmid that made the strain resistant to both carbapenems and colistin (the relevant research results were published online in Nature Microbiology). Due to its strongest antibacterial ability, polymyxin is generally regarded as the "last line of defense" among antibiotics. The emergence of polymyxin resistance has put humanity's last line of defense at risk.

[0003] "Super bacteria" have already appeared, but the pace of human development of new antibiotics has stagnated for many years. Humans have not discovered new types of antibiotics for nearly 30 years, but the speed at which bacteria develop drug resistance has not slowed down. According to the latest research results of Harvard Medical School, it only takes 12 days for ordinary E. coli to evolve into super-resistant bacteria under experimental conditions! The drug development cycle of human beings for new antibiotics takes at least 10 years. After a new innovative drug was launched, drug-resistant bacteria appeared in just 2 years. According to market rules, innovative drugs need to be sold for at least 5 years after they are launched to be profitable, which has led to companies withdrawing from antibiotic research and development and production.

[0004] Undoubtedly, screening or synthesizing new antibacterial drugs with different mechanisms of action from antibiotics and less prone to drug resistance is an effective way to solve this problem. Peptidoglycan is the main component of bacterial cell walls. It maintains the mechanical strength of the cell wall and protects bacteria from external factors. Enzybiotics is a hybrid of enzymes and antibiotics. It was introduced in 2001 by Dr. Vincent Fischetti's team at Rockefeller University in New York. It refers to a special class of antibacterial proteins with the function of cleaving peptidoglycan, also known as peptidoglycan hydrolases (PGH). It was first defined as a bacteriophage lytic enzyme that has antibacterial ability by degrading bacterial cell walls. Now it generally includes bacteriocin, endolysin and lysozyme. Enzymes can quickly and specifically cleave cell wall peptidoglycan, thereby releasing cell contents and killing bacteria.

[0005] The structure of antibiotic enzymes often differs depending on whether they target Gram-positive or Gram-negative bacteria. For Gram-negative antibiotic enzymes, the catalytic function is usually located within a single domain. In contrast, antibiotic enzymes from Gram-positive bacteria have evolved modular structures, typically containing two common functional domains: a cell wall binding domain (CBD) and an enzyme catalytic domain (EAD), which are connected by a short link. The EAD is responsible for cleaving specific bonds in the PG, while the CBD is responsible for targeting the protein to the peptidoglycan substrate and tightly binding to cell wall fragments after cell lysis, thereby preventing spread and subsequent infection of surrounding intact cells that have not yet been infected. Of course, this common modular structure is not absolute, and more complex architectures exist, such as multiple EADs and / or CBDs in different locations. Most of the lytic enzymes derived from staphylococcal phages, such as LysK, have a unique three-domain structure with two EADs (one at the N-terminus and the other in the center) and a C-terminal SH3b-type CBD. In addition, antibiotic enzymes from streptococcal phages, such as B30 and λSA2, have a dual EAD structure with a glycosidase activity domain and an endopeptidase activity domain in addition to one or several CBDs. Peptidoglycan hydrolases generally consist of two domains, namely, a catalytic domain responsible for hydrolyzing peptidoglycan (catalytic domain) and a domain responsible for specific targeting to the bacterial cell wall (binding domain).

[0006] The antibacterial activity of antibiotics in vitro and in vivo has been confirmed by a large number of studies. Its bactericidal mechanism is different from that of traditional antibiotics. It can be used repeatedly for a long time and is not easy to induce drug resistance. Antibiotics can directly lyse bacteria in each growth cycle. Antibiotics have almost no effect on normal flora.

[0007] However, direct application of natural antibiotic enzymes has some problems, such as low antibacterial activity, too narrow antibacterial spectrum, low expression, poor solubility, the catalytic domain and the binding domain are not necessarily the best combination, or other unnecessary domains are sandwiched between the two binding domains. Therefore, the construction of highly active hybrid antibiotic enzymes is of great significance for promoting the development of new antibacterial drugs, and can provide an effective solution to the problem of bacterial resistance. Summary of the invention

[0008] The present application provides a hybrid antibiotic enzyme for killing animal-derived pathogens and its application, so as to improve the defects of existing antibiotic enzymes with low antibacterial activity or limited application capacity, and meet people's needs.

[0009] In a first aspect, the present application provides a hybrid antibiotic enzyme, using the following technical solution:

[0010] A hybrid antibiotic enzyme includes a hybrid protein E1C1. In a specific embodiment, the hybrid protein E1C1 includes a catalytic domain E1 as shown in SEQ ID NO:2 and a binding domain C1 as shown in SEQ ID NO:3, connected by a Linker1 sequence as shown in SEQ ID NO:4.

[0011] In a specific embodiment, the hybrid protein E1C1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0012] The catalytic domain E1 (1-184 aa) sequence described in this application is the phage lytic enzyme PVP-SE1gp146 (from Salmonella enterica serovar Enteritidis phage PVP-SE1) has peptidoglycan cleavage activity. The activity of the native protein PVP-SE1gp146 of the present application is described in the literature Walmagh M, Briers Y, Santos SBd (2012) Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201φ2-1 and PVP-SE1. PLoS ONE 7(5): e36991. doi:10.1371 / journal.pone.0036991. The binding domain C1 (1-56 aa) sequence is Pseudomonas The amino acid sequence from No. 8 to No. 63 in phage 201phi2-1 has peptidoglycan targeting and binding function. PseudomonasPhage 201phi2-1 is from Thomas, JA, Rolando, MR, Carroll, CA et al (2008). Characterization of Pseudomonas chlororaphis myovirus 201varphi2-1 via genomic sequencing, mass spectrometry, and electron microscopy. Virology 376 (2), 330-338, which has a detailed report. Linker1 sequence is used to maintain the stable spatial structure of the hybrid protein.

[0013] Therefore, the hybrid protein E1C1 of the present application has a completely new protein amino acid sequence and physicochemical properties different from PVP-SE1gp146. The hybrid protein E1C1 of the present application contains 256 amino acids, a molecular weight of 27.7kD, a theoretical isoelectric point of 8.35, has high activity, and can effectively kill common animal pathogens Escherichia coli, Salmonella, Staphylococcus aureus and Helicobacter pylori, and has the same effect on clinically isolated Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0014] The inventors found that the native protein PVP-SE1gp146 had a log value drop of about 0.31 after 30 minutes of action on Escherichia coli, and a log value drop of about 0.21 after 30 minutes of action on Salmonella. The E1C1 modified by the present application had a log value drop of more than 4.24 after 30 minutes of action on Escherichia coli, and a log value drop of more than 4.54 after 30 minutes of action on Salmonella. It can be seen that the bactericidal activity of E1C1 of the present application, especially the bactericidal activity against common animal pathogens, has been greatly improved. At the same time, after 30 minutes of action on Staphylococcus aureus, Helicobacter pylori, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, the log value drop was greater than 4.0.

[0015] Moreover, the hybrid protein E1C1 of the present application has a high temperature tolerance. The hybrid protein E1C1 remains fully active after being incubated at 55°C for 48 hours. At a higher temperature, after being incubated at 80°C for 1 hour, it can maintain more than 90% of its activity. It can tolerate up to 90°C without inactivation within 30 minutes. Therefore, compared with the native protein PVP-SE1gp146, the hybrid protein E1C1 of the present application can exert a bactericidal effect at a higher temperature.

[0016] In addition, the inventors found that the hybrid protein E1C1 of the present application also has high activity under complex conditions such as certain ionic strength, culture medium, serum and body fluids. The activity of the hybrid protein E1C1 is maintained at more than 90% under 0-150 mM NaCl conditions, and the activity of the hybrid protein E1C1 is basically unaffected in the presence of 10% and 20% BSA at different concentrations. Therefore, the hybrid protein E1C1 of the present application can adapt to more complex environments.

[0017] In a specific embodiment, the hybrid antibiotic enzyme further comprises a hybrid protein composed of a sequence similar to the catalytic domain E1, wherein the sequence similar to the catalytic domain E1 has at least 75% homology with the catalytic domain E1.

[0018] The hybrid antibiotic enzyme composed of a sequence similar to the catalytic domain E1 also has high activity, as shown in SEQ ID NO: 5, and is named hybrid protein E2C1.

[0019] In a specific embodiment, the hybrid antibiotic enzyme further comprises a hybrid protein composed of a sequence similar to the binding domain C1, wherein the sequence similar to the binding domain C1 has at least 75% homology with the binding domain C1.

[0020] A hybrid antibiotic enzyme composed of a sequence similar to the binding domain C1 also has high activity, as shown in SEQ ID NO: 6, and is named hybrid protein E1C2.

[0021] In a specific embodiment, the hybrid antibiotic enzyme further comprises a hybrid protein composed of the catalytic domain E1 and the binding domain C1 in an interchanged position.

[0022] The hybrid antibiotic enzyme composed of the catalytic domain E1 and the binding domain C1 with their positions interchanged also has high activity, as shown in SEQ ID NO: 7, and is named hybrid protein C1E1.

[0023] Compared with the hybrid protein E1C1, the hybrid protein E2C1, hybrid protein E1C2 and hybrid protein C1E1 of the present application have similar high activity and high temperature tolerance, and have efficient killing effects on common animal pathogens such as Escherichia coli, Salmonella, Staphylococcus aureus and Helicobacter pylori.

[0024] In a second aspect, the present application provides an application of an antibiotic enzyme in the preparation of a drug for controlling microbial infection, wherein the microorganisms are bacteria and fungi.

[0025] In a third aspect, the present application provides a pharmaceutical composition, which comprises an effective amount of the above-mentioned hybrid antibiotic enzyme, and a pharmaceutically acceptable carrier, excipient or diluent.

[0026] In a specific embodiment, the dosage form of the pharmaceutical composition is an injection, an oral agent or an external agent, the content of the hybrid antibiotic enzyme in the injection is 0.01-500 mg / vial, the content of the hybrid antibiotic enzyme in the oral agent is 0.01-500 mg / vial, and the content of the hybrid antibiotic enzyme in the external agent is 1 / 10000-10% / vial.

[0027] The hybrid antibiotic enzyme of the present application can be obtained by expression using Escherichia coli or yeast.

[0028] The pharmaceutical composition containing the hybrid antibiotic enzyme can be used for preventing and treating bacterial infections, as well as sterilizing and disinfecting equipment and physical places, etc. The content of the hybrid antibiotic enzyme is controlled within the above range, so that the pharmaceutical composition can be suitable for most users.

[0029] The hybrid antibiotic enzyme of the present application not only maintains the high temperature resistance of the native protein, but also further improves the high temperature resistance, and is very suitable for developing products with special processing technology, such as pharmaceutical compositions, feeds or feed additives, etc. The above-mentioned pharmaceutical compositions, feeds or feed additives can effectively kill common animal pathogens such as bacteria and fungi in animals.

[0030] In summary, the beneficial technical effects of this application are:

[0031] Based on a large number of studies, this application successfully constructed a new hybrid antibacterial protein, which was expressed by yeast or Escherichia coli, and a high-purity hybrid antibacterial protein was obtained after purification; the protein can quickly lyse common animal pathogens, such as Escherichia coli, Salmonella, Staphylococcus aureus and Helicobacter pylori, and can also efficiently kill common pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae isolated from clinical sites. A new hybrid antibacterial protein constructed in this application not only maintains the high temperature resistance of the native protein, but also further improves the high temperature resistance on its basis, which is very suitable for the development of products with special processing technology, such as feed or feed additives. The antibacterial preparation made from the hybrid antibacterial protein can be used to prevent and treat bacterial infections. It is a new, efficient antibacterial substance that can directly lyse bacteria. The hybrid antibacterial protein can be made into antibacterial preparations for the prevention and treatment of infections caused by various pathogens, as well as sterilization and disinfection of equipment and physical places. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison of the antibacterial activities of the hybrid protein E1C1 and the native protein PVP-SE1gp146.

[0033] Figure 2This is a comparison of the antibacterial activities of the hybrid protein E1C1 and the native protein PVP-SE1gp146 under 0-150 mM NaCl conditions.

[0034] Figure 3 This is a comparison of the antibacterial activities of the hybrid protein E1C1 and the native protein PVP-SE1gp146 under different BSA concentrations (10% and 20%).

[0035] Figure 4 It is a comparison of the effects of different incubation times at 55°C on the antibacterial activities of the hybrid protein E1C1 and the native protein PVP-SE1gp146.

[0036] Figure 5 This is a comparison of the high temperature tolerance of the hybrid protein E1C1 and the native protein PVP-SE1gp146. DETAILED DESCRIPTION

[0037] The following is combined with Figure 1-5 The present application is further described in detail with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0038] The present application artificially designs a hybrid protein with high antibacterial activity by analyzing the domain of antibacterial proteins, and the amino acid sequence of the hybrid protein is SEQ ID NO: 1. The present application adopts molecular biological methods to achieve secretory expression of the hybrid protein in Pichia pastoris or Escherichia coli, and obtains high-purity protein after purification for activity detection.

[0039] The native protein PVP-SE1gp146 in the present application is reported in detail in the literature Walmagh M, Briers Y, Santos SBd (2012) Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201φ2-1 and PVP-SE1. PLoS ONE 7(5): e36991. doi:10.1371 / journal.pone.0036991.

[0040] The term "approximately" is used herein to mean "about or around..." in this application. When the term "approximately" is used in conjunction with a numerical value or a numerical range, it defines the range by extending the boundaries above and below the stated numerical values. In general, the term "approximately" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the specified value. When the term "approximately" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., no "about") is preferred. Example

[0041] Example 1

[0042] Construction, expression and purification of E1C1:

[0043] 1. Construction of E1C1 engineered bacteria

[0044] According to the codon preference of Pichia pastoris, the nucleic acid sequence encoding E1C1 was artificially designed and synthesized. The restriction sites Xho Ⅰ (CTCGAG) and Xba Ⅰ (TCTAGA) were introduced at both ends, TAA and TAG were stop codons, and CG was added between the restriction site and the stop codon to ensure the correctness of the code frame, and then connected between the Xho Ⅰ and Xba Ⅰ sites of the pGAPZαA vector to obtain the recombinant plasmid pGAPZαA-E1C1. Yeast SMD1168 was made into competent bacteria according to the method in the manual of Invitrogen Company. According to the manual, 10 μg of the digested product was used to transform the competent yeast SMD1168 at 25 μF and 1500 V using a Bio-Rad electroporator. The transformation products were spread on YPDS plates with Zeocin resistance at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, respectively, and cultured in a 30°C incubator for 2 to 4 days until a single colony grew. The highest concentration of Zeocin resistance bacteria growing colonies was picked with a toothpick, shaken and cultured overnight, and the bacteria were collected by centrifugation for PCR identification, and the engineered bacteria with the target gene integrated were screened to obtain positive yeast.

[0045] 2. Expression and purification of E1C1

[0046] The positive yeasts screened were inoculated into 100 mL of BMGY medium and cultured at 30 °C and 250 r / min until OD 600 When the number of cells reached 2-6, the cells were collected by centrifugation and resuspended in 500 mL of BmMY medium for induction expression. The cells were cultured at 28°C and 250 r / min for 96 h, during which methanol with a final concentration of 0.5% (V / V) was added every 24 h. After 96 h of induction expression, the cells were centrifuged at 10,000 r / min for 5 min and the culture supernatant was collected.

[0047] The fermentation supernatant was purified by cation exchange and gel filtration. The purified sample was frozen and stored at -20°C for future use. The hybrid protein E1C1 was obtained.

[0048] Example 2

[0049] Construction, expression and purification of E1C1:

[0050] In order to avoid the degradation of the hybrid protein E1C1 by proteases and the toxic effect on the host Escherichia coli, this embodiment adopts a fusion expression strategy to express the target protein in Escherichia coli. The main carrier proteins of fusion expression include but are not limited to the following four categories: fusion tags that enhance solubility, fusion tags that promote inclusion body formation, fusion tags that can be self-cleaved, and fusion tags of signal peptides. The main carrier protein of fusion expression used in this embodiment is the prokaryotic expression vector pGEX-4T-1 with a glutathione transferase (GST) tag.

[0051] According to the codon preference of Escherichia coli, a nucleic acid sequence encoding E1C1 was artificially designed and synthesized. A protein that can be expressed in Escherichia coli was inserted into the N-terminus for fusion expression. In this example, the fusion gene containing protein E1C1 was cloned into the prokaryotic expression vector pGEX-4T-1 with a glutathione transferase (GST) tag, transformed into Escherichia coli BL21 (DE3), induced by lactose or IPTG, centrifuged at 10000 r / min for 5 minutes, and the culture supernatant was collected. The GST-antimicrobial peptide fusion protein was obtained by separation and purification by a chromatographic column. The fusion protein was removed by a site-specific protease such as thrombin or Xa-factor to obtain the target protein. The purified sample was frozen at -20°C for standby use. The hybrid protein E1C1 was obtained.

[0052] Example 3

[0053] Comparison of the antibacterial activities of the hybrid protein E1C1 of Example 1 and the native protein PVP-SE1gp146.

[0054] The overnight culture of Micrococcus lysodeikticus was transferred and grown to the mid-logarithmic phase, that is, to OD 600 About 0.5, then centrifuged at 5,000 g for 5 minutes, and then collected the bacteria. Wash the bacteria twice with 20mM PBS at pH 7.2, and then resuspend in PBS buffer. Add hybrid protein E1C1 (final concentration 2μg / mL), native protein PVP-SE1gp146 (final concentration 2μg / mL) or PBS control to a 96-well plate, add 10μl of 200 mM EDTA (final concentration 0.5 mM) and 50μl of bacterial suspension, add PBS to the total reaction system 200μl, the reaction temperature is 37°C, and read 5 times at 600 nm wavelength every 1 minute. The results are as follows Figure 1 shown.

[0055] Example 4

[0056] Comparison of the antibacterial activities of the hybrid protein E1C1 of Example 1 and the native protein PVP-SE1gp146 under 0-150 mm NaCl conditions.

[0057] The overnight culture of Micrococcus lysodeikticus was transferred and grown to the mid-logarithmic phase, that is, to OD 600 About 0.5, and then centrifuged at 5,000 g for 5 minutes to collect the bacteria. The bacteria were washed twice with 20mM PBS at pH 7.2, and then resuspended in PBS buffer containing 0, 75mM and 150mM NaCl, respectively. Hybrid protein E1C1 (final concentration 2μg / mL), native protein PVP-SE1gp146 (final concentration 2μg / mL) or PBS control was added to a 96-well plate, and 10 μl of 200 mM EDTA (final concentration 0.5 mM) and 50 μl of bacterial suspension were added, and PBS was added to the total reaction system of 200 μl. The reaction temperature was 37°C and the reading was 5 times at a wavelength of 600 nm with an interval of 1 minute. The results are as follows Figure 2 shown.

[0058] Example 5

[0059] The antibacterial activities of the hybrid protein E1C1 of Example 1 and the native protein PVP-SE1gp146 were compared under the conditions of 10% and 20% BSA volume concentrations.

[0060] The overnight culture of Micrococcus lysodeikticus was transferred and grown to the mid-logarithmic phase, that is, to OD 600 About 0.5, and then centrifuged at 5,000 g for 5 minutes to collect the bacteria. The bacteria were washed twice with 20mM PBS at pH 7.2, and then resuspended in buffer containing 10% and 20% BSA, respectively. Hybrid protein E1C1 (final concentration 2μg / mL), native protein PVP-SE1gp146 (final concentration 2μg / mL) or PBS control was added to a 96-well plate, and 10 μl of 200mM EDTA (final concentration 0.5 mM) and 50 μl of bacterial suspension were added, and PBS was added to the total reaction system of 200 μl. The reaction temperature was 37°C, and the reading was taken 5 times at a wavelength of 600 nm with an interval of 1 minute. The results are as follows Figure 3 shown.

[0061] Example 6

[0062] The hybrid protein E1C1 of Example 1 has the effect of killing Escherichia coli, Salmonella, Staphylococcus aureus, Helicobacter pylori, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae in vitro in a timely and quantitative manner.

[0063] The overnight cultured Escherichia coli, Salmonella, Staphylococcus aureus, Helicobacter pylori, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were transferred and grown to the mid-logarithmic phase, that is, to OD 600The cells were centrifuged at 5,000 g for 5 minutes to collect the cells. The cells were diluted to about 1×10 8 cfu / mL.

[0064] Add 1 mL of hybrid antibiotic sample, native protease sample or PBS sample to 250 μl of bacterial solution. Hybrid antibiotic sample contains E1C1 (final concentration of 10 μg / mL or 50 μg / mL), NaCl (final concentration of 150 mM) and EDTA (final concentration of 0.5 mM). Native protease sample contains native protein PVP-SE1gp146 (final concentration of 10 μg / mL or 50 μg / mL) and EDTA (final concentration of 0.5 mM). PBS sample contains PBS and EDTA (final concentration of 0.5 mM).

[0065] After the hybrid antibiotic enzyme sample acted on the bacterial solution at room temperature for 30 minutes, 0.5 mL of the sample solution was taken for serial dilution, and then 0.5 mL of the sample solution was taken for live bacteria counting and culture, which was expressed as logarithmic Log value. The greater the decrease in Log value, the better the bactericidal effect.

[0066] After the native protease sample and PBS sample were treated with the bacterial solution at room temperature for 2 h, 0.5 mL of the sample solution was taken for serial dilution, and then 0.5 mL of the sample solution was taken for viable bacteria counting and culture, which was expressed as logarithmic Log value. The greater the decrease in Log value, the better the bactericidal effect. The results are shown in Tables 1 and 2.

[0067] Table 1. Bactericidal activity of E1C1 (10 µg / mL) / EDTA against different pathogens

[0068]

[0069] Table 2. Bactericidal activity of E1C1 (50 µg / mL) / EDTA against different pathogens

[0070]

[0071] Example 7

[0072] Comparison of temperature tolerance between the hybrid protein E1C1 of Example 1 and the native protein PVP-SE1gp146.

[0073] Protein sample treatment: The hybrid protein E1C1 sample or native protein PVP-SE1gp146 sample of the same concentration was dissolved in a buffer solution, incubated at 55°C for 2h, 6h, 12h, 24h, and 48h, and then naturally cooled to room temperature, and the antibacterial activity was determined. The antibacterial activity at 4°C was used as the initial value to calculate the activity retention rate. Alternatively, the hybrid protein E1C1 sample or native protein PVP-SE1gp146 sample of the same concentration was dissolved in a buffer solution, incubated at 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 0min, 30min, and 60min, and then naturally cooled to room temperature, and the antibacterial activity was determined. The antibacterial activity at 4°C was used as the initial value to calculate the activity retention rate.

[0074] Antimicrobial activity assay: The overnight cultured Micrococcus was transferred and grown to the mid-logarithmic phase, i.e., to OD 600 About 0.5, and then centrifuged at 5,000 g for 5 minutes, the bacteria were collected. The bacteria were washed twice with 20mM PBS at pH 7.2, and then resuspended in PBS buffer. Hybrid protein E1C1 samples (final concentration 2 μg / mL), native protein PVP-SE1gp146 samples (final concentration 2 μg / mL) or PBS control after incubation at different temperatures for different times were added to a 96-well plate, and 10 μl 200 mM EDTA (final concentration 0.5 mM) and 50 μl of bacterial suspension were added, and PBS was added to the total reaction system of 200μl. The reaction temperature was 37°C, and the reading was taken 5 times at a wavelength of 600 nm with an interval of 1 minute. The results are as follows Figure 4 and Figure 5 shown.

[0075] Example 8

[0076] Construction, expression and purification of E2C1:

[0077] In this example, a nucleic acid sequence encoding E2C1 was artificially designed and synthesized based on the codon preference of Pichia pastoris. The sequence was ligated between the Xho Ⅰ and Xba Ⅰ sites of the pGAPZαA vector to obtain the recombinant plasmid pGAPZαA-E2C1. The yeast SMD1168 was transformed to obtain positive yeast, which was fermented, expressed and purified to obtain the hybrid protein E2C1.

[0078] Example 9

[0079] Construction, expression and purification of E1C2:

[0080] In this example, a nucleic acid sequence encoding E1C2 was artificially designed and synthesized based on the codon preference of Pichia pastoris. The sequence was ligated between the Xho Ⅰ and Xba Ⅰ sites of the pGAPZαA vector to obtain the recombinant plasmid pGAPZαA-E1C2. The yeast SMD1168 was transformed to obtain positive yeast, which was fermented, expressed and purified to obtain the hybrid protein E1C2.

[0081] Example 10

[0082] Construction, expression and purification of C1E1:

[0083] This Example This Example is different from Example 2 in that, according to the codon preference of Pichia pastoris, a nucleic acid sequence encoding C1E1 was artificially designed and synthesized. It was connected between the Xho Ⅰ and Xba Ⅰ sites of the pGAPZαA vector to obtain the recombinant plasmid pGAPZαA-C1E1. The yeast SMD1168 was transformed to obtain positive yeast, which was fermented, expressed and purified to obtain the hybrid protein C1E1.

[0084] Embodiment 11

[0085] In this example, 100 ml of a biological antibacterial preparation containing E1C1 was prepared.

[0086] prescription:

[0087] E1C1 of Example 2 0.02 g

[0088] Anhydrous potassium dihydrogen phosphate 0.16 g

[0089] Anhydrous disodium hydrogen phosphate 0.12 g

[0090] Disodium ethylenediaminetetraacetate 0.037 g

[0091] Glycerol 3 ml

[0092] NaCl 0.6 g

[0093] Make up to 100 ml water

[0094] The preparation method of the biological antibacterial preparation of this embodiment is as follows:

[0095] a) Calculate the required amount of excipients according to the prescription and total preparation liquid volume, and accurately weigh them into a clean container.

[0096] b) Add 70% of the total preparation solution into the mixing vessel with water. Dissolve NaCl, disodium EDTA, disodium hydrogen phosphate and potassium dihydrogen phosphate first. Add glycerol after they are fully dissolved. Mix well and add E1C1. Finally, make up to volume with water and mix well.

[0097] c) Sterile filtration: Pass the prepared solution through a sterilizing filter and transfer the outflowing solution into a sterile container.

[0098] d) Filling: Fill the sterile solution into plastic or glass bottles.

[0099] The antibacterial preparation can be used for sterilization and disinfection of wounds or wound surfaces, 1-2 times a day.

[0100] Example 12

[0101] In this example, 100 ml of a biological antibacterial preparation containing E2C1 was prepared.

[0102] prescription:

[0103] E2C1 of Example 8 0.02 g

[0104] Anhydrous potassium dihydrogen phosphate 0.16 g

[0105] Anhydrous disodium hydrogen phosphate 0.12 g

[0106] Disodium ethylenediaminetetraacetate 0.037 g

[0107] Glycerol 3 ml

[0108] NaCl 0.6 g

[0109] Make up to 100 ml water

[0110] The preparation method of the biological antibacterial preparation of this embodiment is as follows:

[0111] a) Calculate the required amount of excipients according to the prescription and total preparation liquid volume, and accurately weigh them into a clean container.

[0112] b) Add 70% of the total preparation solution into the mixing vessel with water. Dissolve NaCl, disodium EDTA, disodium hydrogen phosphate and potassium dihydrogen phosphate first. Add glycerol after they are fully dissolved. Mix well and add E2C1. Finally, make up to volume with water and mix well.

[0113] c) Sterile filtration: Pass the prepared solution through a sterilizing filter and transfer the outflowing solution into a sterile container.

[0114] d) Filling: Fill the sterile solution into plastic or glass bottles.

[0115] The antibacterial preparation can be used for sterilization and disinfection of wounds or wound surfaces, 1-2 times a day.

[0116] Embodiment 13

[0117] In this example, 100 ml of a biological antibacterial preparation containing E1C2 was prepared.

[0118] prescription:

[0119] E1C2 of Example 9 0.02 g

[0120] Anhydrous potassium dihydrogen phosphate 0.16 g

[0121] Anhydrous disodium hydrogen phosphate 0.12 g

[0122] Disodium ethylenediaminetetraacetate 0.037 g

[0123] Glycerol 3 ml

[0124] NaCl 0.6 g

[0125] Make up to 100 ml water

[0126] The preparation method of the biological antibacterial preparation of this embodiment is as follows:

[0127] a) Calculate the required amount of excipients according to the prescription and total preparation liquid volume, and accurately weigh them into a clean container.

[0128] b) Add 70% of the total preparation solution into the mixing vessel, dissolve NaCl, disodium EDTA, disodium hydrogen phosphate and potassium dihydrogen phosphate first, add glycerol after they are fully dissolved, add E1C2 after mixing, and finally make up to volume with water and mix thoroughly.

[0129] c) Sterile filtration: Pass the prepared solution through a sterilizing filter and transfer the outflowing solution into a sterile container.

[0130] d) Filling: Fill the sterile solution into plastic or glass bottles.

[0131] The antibacterial preparation can be used for sterilization and disinfection of wounds or wound surfaces, 1-2 times a day.

[0132] Embodiment 14

[0133] In this example, 100 ml of a biological antibacterial preparation containing C1E1 was prepared.

[0134] prescription:

[0135] C1E1 of Example 10 0.02 g

[0136] Anhydrous potassium dihydrogen phosphate 0.16 g

[0137] Anhydrous disodium hydrogen phosphate 0.12 g

[0138] Disodium ethylenediaminetetraacetate 0.037 g

[0139] Glycerol 3 ml

[0140] NaCl 0.6 g

[0141] Make up to 100 ml water

[0142] The preparation method of the biological antibacterial preparation of this embodiment is as follows:

[0143] a) Calculate the required amount of excipients according to the prescription and total preparation liquid volume, and accurately weigh them into a clean container.

[0144] b) Add 70% of the total preparation solution into the mixing vessel, dissolve NaCl, disodium EDTA, disodium hydrogen phosphate and potassium dihydrogen phosphate first, add glycerol after they are fully dissolved, mix well and add C1E1, finally make up to volume with water and mix well.

[0145] c) Sterile filtration: Pass the prepared solution through a sterilizing filter and transfer the outflowing solution into a sterile container.

[0146] d) Filling: Fill the sterile solution into plastic or glass bottles.

[0147] The antibacterial preparation can be used for sterilization and disinfection of wounds or wound surfaces, 1-2 times a day.

[0148] Combined with Example 1 and Example 3 Figure 1 It can be seen that the hybrid protein E1C1 of Example 1 and the native protein PVP-SE1gp146 can both cause the OD value of the bacterial solution to decrease by lysing the pathogens. The faster the OD value of the pathogenic bacteria solution decreases, the greater the decrease, indicating that the antibacterial activity of the protein is higher. The results show that the hybrid protein E1C1 has significantly higher antibacterial activity than the native protein PVP-SE1gp146. Comparing the decrease in OD value at the end point of the reaction for 5 minutes, the hybrid E1C1 of the present invention is 2.3 times that of the native protein.

[0149] Combined with Example 1 and Example 4 Figure 2 It can be seen that the hybrid protein E1C1 of Example 1 still maintains a high bactericidal activity in a high salt environment. As the salt concentration increases, the activity decreases, but remains above 90%. The activity of the native protein decreases by 50% and 80% in the presence of 75mM and 150mM NaCl, respectively.

[0150] Combined with Example 1 and Example 5 Figure 3 It can be seen that the hybrid protein E1C1 of Example 1 still maintains a high bactericidal activity in a high concentration of organic matter environment, and 10% and 20% BSA have little effect on the bactericidal activity of E1C1. However, the activity of the native protein PVP-SE1gp146 decreased by 70% in the presence of 10% BSA, and almost completely lost in the presence of 20% BSA.

[0151] Combining Example 1 and Example 6 with Tables 1-2, it can be seen that the in vitro bactericidal experimental results in Table 1 show that 10 μg / mL of antimicrobial protein E1C1 can effectively kill Escherichia coli, Salmonella, Helicobacter pylori, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae after 30 minutes of action, and the decrease values ​​are all greater than 3 Log values.

[0152] The results in Table 2 show that 50 μg / mL of the antimicrobial protein E1C1 can effectively kill Escherichia coli, Salmonella, Helicobacter pylori, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae after 30 min of action, with the decrease values ​​being greater than 4 Log values.

[0153] The above results show that the hybrid E1C1 protein has a very strong bactericidal effect on Escherichia coli, Salmonella, Helicobacter pylori, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, which is significantly improved compared with the native protein before hybridization.

[0154] Combined with Example 1 and Example 7 Figure 4-5 It can be seen that the hybrid protein E1C1 of Example 1 has a high temperature tolerance. The hybrid protein E1C1 of Example 1 remains fully active after being incubated at 55°C for 48 hours, while the activity of the native protein can only be maintained for 24 hours at 55°C, and the activity decreases significantly after 48 hours. At a higher temperature, the hybrid protein E1C1 of Example 1 maintains more than 90% of its activity after being incubated at 80°C for 1 hour, and can tolerate up to 90°C without inactivation within 30 minutes.

[0155] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A hybrid antibiotic enzyme, characterized in that It comprises a hybrid protein E1C1, wherein the hybrid protein E1C1 comprises a catalytic domain E1 as shown in SEQ ID NO:2 and a binding domain C1 as shown in SEQ ID NO:

3.

2. A hybrid antibiotic enzyme according to claim 1, characterized in that: The amino acid sequence of the hybrid protein E1C1 is shown in SEQ ID NO:

1.

3. A hybrid antibiotic enzyme according to claim 1, characterized in that: The hybrid antibiotic enzyme also includes a hybrid protein C1E1, which is a hybrid protein composed of the catalytic domain E1 and the binding domain C1 in an interchanged position.

4. A hybrid antibiotic enzyme according to claim 3, characterized in that: The amino acid sequence of C1E1 is shown in SEQ ID NO:

7.

5. Use of the hybrid antibiotic enzyme according to any one of claims 1 to 4 in the preparation of a drug for controlling microbial infection, characterized in that: The microorganisms are Escherichia coli, Salmonella, Staphylococcus aureus, Helicobacter pylori, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

6. A pharmaceutical composition, characterized in that: The active ingredient of the pharmaceutical composition comprises the hybrid antibiotic enzyme according to any one of claims 1 to 4.

7. A pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. A pharmaceutical composition according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is injection, oral preparation or external preparation.

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