CN115851690A - Hybrid antibiotic enzyme for killing animal-derived pathogenic bacteria and application thereof - Google Patents

Abstract

The invention discloses the technical field of antibiotic enzymes, and particularly relates to a heterozygous antibiotic enzyme for killing pathogenic bacteria of animal origin and application thereof. The hybrid antibiotic enzyme comprises a hybrid protein E1C1, wherein the hybrid protein E1C1 comprises a catalytic domain E1 shown as SEQ ID NO. 2 and a binding domain C1 shown as SEQ ID NO. 3. The invention also provides the use of a hybrid antibiotic enzyme in the manufacture of a pharmaceutical composition for controlling microbial infections, and a pharmaceutical composition comprising as an active ingredient said hybrid antibiotic enzyme. The heterozygosis antibiotic enzyme can rapidly crack common animal-derived pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus and helicobacter pylori, and can efficiently kill clinically separated common pathogenic bacteria such as acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae and the like.

Description

Hybrid antibiotic enzyme for killing animal-derived pathogenic bacteria and application thereof

Technical Field

The invention relates to the technical field of antibiotic enzymes, in particular to a heterozygous antibiotic enzyme for killing animal-derived pathogenic bacteria and application thereof.

Background

With the use of a large amount of antibiotics, the problem of drug resistance of bacteria to the antibiotics becomes more and more serious, which causes difficulties in clinical treatment and higher fatality rate worldwide, and seriously threatens human health. Some drug-resistant bacteria/drug-resistant genes with important public health significance begin to appear and spread in livestock breeding in China, and threaten food safety and human health, and it has been found that various animal-derived bacteria such as staphylococcus and enterococcus in China all carry cfr and optrA genes. The gene can mediate drug resistance of five antibacterial drugs, and if people are infected with epidemic diseases, the infection of the people with clinically relevant pathogenic bacteria such as MRSA, VRE and the like can not be cured by the drug. The ermB gene which can mediate high-level drug resistance is found in domestic swine and chicken campylobacter. The gene is integrated into a drug-resistant gene island, and the possibility that the carrying bacteria can be developed into 'super bacteria' exists. In addition, carbapenemase-resistant bacteria carrying NDM-1 and MCR-1 resistance genes have been found in chicken and pig bodies in China. Whether the bacteria are widely epidemic or not endangers public safety, recently, researchers at southern China agriculture university find a super-super bacterium on a diseased pet cat, an escherichia coli carries a hybrid plasmid, and the strain can simultaneously resist two medicines of carbapenems and colistin (related research results are published on line in Nature Microbiology), because of the strongest antibacterial ability, the colistin is generally regarded as the 'last line of defense' in the antibiotics, and the medicine resistance of the colistin enables the last line of defense of humans to be dangerous.

"Superbacterias" have appeared and the human step of developing new antibiotics has been arrested for many years. In recent thirty years, no new antibiotic species have been discovered in humans, but the rate of bacterial resistance has not been slowed. According to the latest research results of Harvard medical college, the common Escherichia coli evolves into super-strong drug-resistant bacteria under the experimental environment, and only 12 days are needed! However, the period of developing new antibiotic drugs by human needs at least 10 years, and after a new innovative drug is put into the market, drug-resistant bacteria appear in only 2 years. According to the market law, innovative drugs are profitable only after being sold for at least 5 years after being put into the market, which leads enterprises to quit antibiotic research and development and production in a dispute.

Undoubtedly, screening or synthesizing novel antibacterial drugs with different action mechanisms from antibiotics and difficult generation of drug resistance is an effective way to solve the problem. Peptidoglycan is a major component of bacterial cell walls, which maintains the mechanical strength of the cell wall and protects the bacteria from external elements. Antibiotic enzymes (enzymes), a mixed word of enzymes and antibiotics, introduced in 2001 by the team of knent Fischetti, university of rocky, new york, in scientific language, refer to a specific class of antibacterial proteins with the function of cleaving peptidoglycans, also known as Peptidoglycan hydrolases (PGH), which were first defined as phage lyases with antibacterial ability by degrading the bacterial cell wall, and now commonly include bacteriocins (bacteriociins), autolysins (endolysins) and lysozymes (lysozymes). Antibiotic enzymes are capable of rapidly specifically cleaving cell wall peptidoglycans to allow release of cellular contents to kill bacteria.

The structure of a biocide enzyme generally varies depending on whether it targets gram-positive or gram-negative bacteria. The catalytic function of gram-negative antibiotic enzymes is usually within a single domain. In contrast, antibiotic enzymes from gram-positive bacteria have evolved into modular structures, typically comprising two common functional domains: a cell wall binding domain (CBD) and an enzyme catalytic domain (EAD), the two domains being linked by a short chain. The EAD is responsible for cleaving specific bonds in PG and the CBD is responsible for targeting proteins to peptidoglycan substrates and tightly binding to cell wall fragments after cell lysis, thereby preventing diffusion and subsequent infection of surrounding intact cells that have not been infected. Of course this common modular structure is not absolute and more complex architectures exist, such as having multiple EADs and/or CBDs in different locations. Most are derived from staphylococcal phage lytic enzymes, such as LysK, with a unique 3-domain structure, two EADs (one at the N-terminal and the other centrally located) and one C-terminal SH3 b-type CBD. Furthermore, the antibiotic enzymes from streptococcal bacteriophages, such as B30 and λ SA2, have, in addition to one or several CBDs, a dual EAD-structure peptidoglycan hydrolase with a glycosidase active domain and an endopeptidase active domain, generally consisting of two domains, a catalytic domain (catalytic domain) responsible for the hydrolysis of peptidoglycan and a domain (binding domain) responsible for the specific targeting of the bacterial cell wall.

The in vivo and in vitro antibacterial activity of the antibiotic enzyme is proved by a great deal of research, and the antibiotic enzyme has a different sterilization mechanism from that of the traditional antibiotic, can be repeatedly used for a long time and is not easy to induce the generation of drug resistance. In each growth cycle of the bacteria, the antibiotic enzyme can directly crack the bacteria. Antibiotic enzymes hardly affect the normal flora.

However, direct use of natural antibiotic enzymes has problems such as low antibacterial activity, too narrow antibacterial spectrum, low expression level, poor solubility, not necessarily optimal combination of catalytic domain and binding domain, or other non-essential domains sandwiched between two binding domains. Therefore, the construction of the high-activity hybrid antibiotic enzyme has very important significance for promoting the research and development of novel antibacterial drugs, and can provide an effective solution for the problem of bacterial drug resistance.

Disclosure of Invention

The application provides a heterozygous antibiotic enzyme for killing pathogenic bacteria of animal origin and application thereof, so as to overcome the defects of low antibacterial activity or limited application capability of the existing antibiotic enzyme and meet the requirements of people.

In a first aspect, the present application provides a hybrid antibiotic enzyme, which adopts the following technical scheme:

a hybrid antibiotic enzyme comprising hybrid protein E1C1. In a specific embodiment, 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, connected by a Linker1 sequence as shown in SEQ ID NO. 4.

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

The catalytic domain E1 (1-184 aa) sequence of bacteriophage lytic enzyme PVP-SE1gp146 (fromSalmonella entericaAn amino acid sequence of serovar Enteritidis phase PVP-SE 1) having peptidoglycan cleaving activity. The activity of the protamine PVP-SE1gp146 of the present application is described in Walmagh M, briers Y, santos SBd (2012) Characterisation of Modular Bacteriophage Endolysis fromMyoviridaePhages OBP,201 φ 2-1 and PVP-SE1 PLoS ONE 7 (5): e36991 doi 10.1371/journal. Bean.0036991 are reported in detail. The sequence of the binding domain C1 (1-56 aa) isPseudomonasNo. 8 to No. 63 amino acid sequences in the phage 201phi2-1 have a target binding function of peptidoglycan. In the present applicationPseudomonasphase 201phi2-1 is from Thomas, J.A., rolando, M.R., carroll, C.A. et al (2008) Spectrification of Pseudomonas chlororaphis myovirus 201 varchi 2-1 via genetic sequencing, mass spectrometry, and electron microscopy. Virology 376 (2), 330-338, all of which are reported in detail. Linker1 sequences were used to maintain a stable spatial structure of the hybrid proteins.

Therefore, the hybrid protein E1C1 of the present application has a completely new protein amino acid sequence and physicochemical properties different from PVP-SE1gp 146. The hybrid protein E1C1 contains 256 amino acids, has the molecular weight of 27.7kD, has the theoretical isoelectric point of 8.35, has high activity, can effectively kill common animal-derived pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus and helicobacter pylori, and has the same effect on clinically separated acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae.

The inventor finds that the log value of the protogenic protein PVP-SE1gp146 is reduced by about 0.31 after the protogenic protein PVP-SE1gp146 acts on escherichia coli for 30min, and is reduced by about 0.21 after the protogenic protein PVP-SE1gp146 acts on salmonella for 30 min. The log value of E1C1 modified by the method is more than 4.24 after the E1C1 acts on escherichia coli for 30min, and the log value of the E1C1 modified by the method is more than 4.54 after the E1C acts on salmonella for 30 min. Therefore, the bactericidal activity of the E1C1 is greatly improved, particularly the bactericidal activity to common animal-derived pathogenic bacteria. Meanwhile, the log value of E1C1 of the application is more than 4.0 after the E1C1 acts on staphylococcus aureus, helicobacter pylori, acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae for 30 min.

Moreover, the hybrid protein E1C1 of the present application has high temperature tolerance, and the hybrid protein E1C1 maintains complete activity after being incubated at 55 ℃ for 48 hours; after the culture is carried out for 1 h at the temperature of 80 ℃ at a higher temperature, the activity can be kept more than 90 percent; can endure 90 ℃ at most and does not lose activity within 30 min. Therefore, the hybrid protein E1C1 of the present application may exert bactericidal effect at higher temperature than the primary protein PVP-SE1gp 146.

In addition, the inventors found that the hybrid protein E1C1 of the present application has high activity under complex conditions such as a certain ionic strength, a medium, serum, and a body fluid. The activity of the hybrid protein E1C1 is kept above 90% under the condition of 0-150 mM NaCl, and the activity of the hybrid protein E1C1 is basically not influenced under the condition that BSA with different concentrations of 10% and 20% exists. Thus, the hybrid protein E1C1 of the present application can accommodate more complex environments.

In a specific possible embodiment, the hybrid antibiotic enzyme further comprises a hybrid protein consisting of a sequence similar to the catalytic domain E1, said sequence similar to the catalytic domain E1 having at least 75% homology with the catalytic domain E1.

The hybrid antibiotic enzyme consisting of a sequence similar to the catalytic domain E1 also has high activity, as shown in SEQ ID NO:5, designated the hybrid protein E2C1.

In a specific embodiment, said hybrid antibiotic enzyme further comprises a hybrid protein consisting of a sequence similar to said binding domain C1, said binding domain C1 similar sequence having at least 75% homology to said binding domain C1.

The hybrid antibiotic enzyme consisting of a sequence similar to the binding domain C1 also has high activity, as shown in SEQ ID NO 6, designated hybrid protein E1C2.

In a particular embodiment, the hybrid antibiotic enzyme further comprises a hybrid protein consisting of the catalytic domain E1 and the binding domain C1 exchanged at positions.

The hybrid antibiotic enzyme composed of the catalytic domain E1 and the binding domain C1 with the mutual exchange position also has high activity, as shown in SEQ ID NO:7, and is named as hybrid protein C1E1.

Compared with the hybrid protein E1C1, the hybrid protein E2C1, the hybrid protein E1C2 and the hybrid protein C1E1 have similar high activity and high temperature tolerance, and have high-efficiency killing effect on common animal-derived pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus, helicobacter pylori and the like.

In a second aspect, the present application provides the use of an antibiotic enzyme in the manufacture of a medicament for the control of microbial infections, said microorganisms being bacteria and fungi.

In a third aspect, the present application provides a pharmaceutical composition comprising an effective amount of the above hybrid antibiotic enzyme, together with a pharmaceutically acceptable carrier, excipient or diluent.

In a specific embodiment, the pharmaceutical composition is in the form of injection, oral preparation or external preparation, wherein the injection contains 0.01-500mg of hybrid antibiotic enzyme per unit, the oral preparation contains 0.01-500mg of hybrid antibiotic enzyme per unit, and the external preparation contains 1/10000-10% of hybrid antibiotic enzyme per unit.

The hybrid antibiotic enzyme of the present application can be obtained by expression using escherichia coli or yeast, etc.

The medicine composition containing the hybrid antibiotic enzyme can be used for preventing and treating bacterial infection, sterilizing instruments and physical places, and the like. Controlling the content of the hybrid antibiotic enzyme within the above range enables the pharmaceutical composition to be suitable for most users.

The hybrid antibiotic enzyme not only maintains the high temperature resistance of the original protein, but also further improves the high temperature resistance on the basis of the original protein, and is very suitable for developing products with special processing technologies, such as pharmaceutical compositions, feeds or feed additives. The pharmaceutical composition, the feed or the feed additive can effectively kill common animal pathogenic bacteria such as bacteria and fungi in the animal body.

To sum up, the beneficial technical effect of this application is:

on the basis of a large amount of researches, a new hybrid antibacterial protein is successfully constructed, expressed by saccharomycetes or escherichia coli and the like, and purified to obtain high-purity hybrid antibacterial protein; the protein can rapidly crack common animal-derived pathogenic bacteria, such as: escherichia coli, salmonella, staphylococcus aureus and helicobacter pylori, and can effectively kill common pathogenic bacteria such as clinically separated acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae and the like. The new hybrid antibacterial protein constructed by the application not only maintains the high temperature resistance of the original protein, but also further improves the high temperature resistance on the basis of the new hybrid antibacterial protein, and is very suitable for developing products with special processing technology, such as feed or feed additives. The antibacterial agent prepared from the hybrid antibacterial protein can be used for preventing and treating bacterial infection. It is a new and efficient antibacterial substance capable of directly cracking bacteria. The hybrid antibacterial protein can be made into antibacterial preparations for preventing and treating infection caused by various pathogenic bacteria, and sterilizing instruments and physical places.

Drawings

FIG. 1 is a graph comparing the antimicrobial activity of hybrid protein E1C1 with that of the protist PVP-SE1gp 146.

FIG. 2 is a graph comparing the antibacterial activity of the hybrid protein E1C1 with that of the protogenic protein PVP-SE1gp146 under 0-150 mM NaCl.

FIG. 3 is a graph comparing the antimicrobial activity of the hybrid protein E1C1 with the native protein PVP-SE1gp146 at different concentrations of BSA (10% and 20%).

FIG. 4 is a graph comparing the effect of incubation at 55 ℃ for various times on the antimicrobial activity of the hybrid protein E1C1 and the native protein PVP-SE1gp 146.

FIG. 5 is a graph comparing the tolerance of hybrid protein E1C1 to the native protein PVP-SE1gp146 to high temperatures.

Detailed Description

The present application will be described in further detail below with reference to fig. 1-5 and examples, but the scope of the present invention is not limited thereto.

The application artificially designs a hybrid protein with high antibacterial activity by analyzing the antibacterial protein structural domain, wherein the amino acid sequence of the hybrid protein is SEQ ID NO. 1. The application adopts a molecular biology method to realize secretory expression of the hybrid protein in pichia pastoris or escherichia coli, and high-purity protein is obtained after purification for activity detection.

The protist PVP-SE1gp146 in this application is reported in detail in Walmagh M, briers Y, santos SBd (2012) Characterisation of modulation bacterial enzymes from Myoviridae Phages OBP,201 φ 2-1 and PVP-SE1.PLoS ONE 7 (5): e36991. Doi:10.1371/journal. Pole.0036991.

The term "about" is used herein to mean "about or at \8230; left or right of the 8230;. When the term "about" is used in connection with a numerical value or range of values, it defines the range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to limit the numerical values to variations of 20%, preferably 10% up or down (higher or lower) above and below the stated values. When the term "about" is used in the context of the present invention (e.g., in combination with temperature or molecular weight values), precise values (i.e., without "about") are preferred.

Examples

Example 1

E1C1 engineering bacteria construction, expression and purification:

1. construction of E1C1 engineering bacteria

According to the codon preference of pichia pastoris, a nucleic acid sequence capable of coding E1C1 is artificially designed and synthesized. Enzyme cutting sites Xho I (CTCGAG) and Xba I (TCTAGA) are respectively introduced into two ends, TAA and TAG are termination codons, CG is added between the enzyme cutting sites and the termination codons to ensure the correctness of a code frame, and the CG is connected between the Xho I and Xba I sites of a pGAPZ alpha A vector to obtain a recombinant plasmid pGAPZ alpha A-E1C1. The yeast SMD1168 was made into competent yeast SMD1168 by the method described in the Invitrogen corporation, and the competent yeast SMD1168 was transformed with 10. Mu.g of the enzyme-digested product under the conditions of 25. Mu.F and 1500V using a Bio-Rad electric transfer instrument according to the specification, and the transformed products were spread on Zeocin-resistant YPDS plates having concentrations of 100. Mu.g/mL, 200. Mu.g/mL, 300. Mu.g/mL, 400. Mu.g/mL and 500. Mu.g/mL, respectively, and cultured in an incubator at 30 ℃ for 2 to 4 days until a single colony grew. And (3) selecting the thalli with highest concentration Zeocin resistance, which grow out bacterial colonies, by using a toothpick, carrying out shaking culture overnight, centrifugally collecting the thalli, carrying out PCR identification, and screening out engineering bacteria integrated with target genes to obtain the positive saccharomycetes.

2. Expression and purification of E1C1

Inoculating the screened positive yeast on 100mL BMGY medium, culturing at 30 deg.C and 250r/min to OD ₆₀₀ And when the concentration reaches 2-6, centrifugally collecting the thalli, suspending the thalli on 500mL BmMY culture medium, carrying out induced expression, culturing for 96 hours at the temperature of 28 ℃ and at the speed of 250r/min, and supplementing methanol with the final concentration of 0.5% (V/V) every 24 hours. After the induction expression for 96h, centrifuging for 5min at 10000 r/min, and collecting the culture supernatant.

The fermentation supernatant was purified by two steps of cation exchange and gel filtration. And (4) freezing and storing the purified sample at-20 ℃ for later use. Obtaining the hybrid protein E1C1.

Example 2

E1C1 engineering bacteria construction, expression and purification:

in order to avoid the degradation of the hybrid protein E1C1 by proteases and the toxic effect on the host E.coli, this example adopted the strategy of fusion expression to express the target protein in E.coli. The major carrier proteins for fusion expression include, but are not limited to, the following four major classes: enhancing soluble fusion tag, promoting inclusion body formation fusion tag, self-cutting fusion tag, and signal peptide fusion tag. The main carrier protein for fusion expression adopted in the embodiment is a prokaryotic expression vector pGEX-4T-1 with a glutathione transferase (GST) label.

The nucleic acid sequence encoding E1C1 was designed and synthesized artificially according to the codon preference of E.coli. And a protein capable of being expressed in Escherichia coli is inserted into the N-terminus for fusion expression. In the embodiment, the fusion gene containing the protein E1C1 is cloned to a prokaryotic expression vector pGEX-4T-1 with a glutathione transferase (GST) label, escherichia coli BL21 (DE 3) is transformed, lactose or IPTG is induced and expressed, centrifugation is carried out for 5min at 10000 r/min, culture solution supernatant is collected, GST-antibacterial peptide fusion protein is obtained by chromatographic column separation and purification, and GST part is cut off by site-specific protease such as thrombin or Xa-factor to obtain the target protein. The purified sample is frozen and stored at-20 ℃ for later use. Obtaining the hybrid protein E1C1.

Example 3

The antibacterial activity of the hybrid protein E1C1 of example 1 was compared with that of the native protein PVP-SE1gp 146.

After the overnight cultured Micrococcus muralis was transferred, it grew to mid-log phase, i.e., to OD ₆₀₀ About 0.5, and then centrifuged at 5,000 g for 5 minutes, and the cells were collected. The mycelia were washed 2 times with 20mM PBS pH 7.2 and then resuspended in PBS buffer. Hybrid protein E1C1 (final concentration 2. Mu.g/mL), the native protein PVP-SE1gp146 (final concentration 2. Mu.g/mL) or PBS control were added to 96-well plates, 10. Mu.l of 200mM EDTA (final concentration 0.5 mM) and 50. Mu.l of bacterial suspension were added, PBS was added to the total reaction volume of 200. Mu.l, the reaction temperature was 37 ℃ and readings were performed 5 times at 600 nm wavelength for 1 minute intervals. The results are shown in FIG. 1.

Example 4

The antibacterial activity of the hybrid protein E1C1 of example 1 was compared with that of the native protein PVP-SE1gp146 under 0-150 mm NaCl conditions.

After the overnight cultured Micrococcus muralis was transferred, it grew to mid-log phase, i.e., to OD ₆₀₀ About 0.5, and then centrifuged at 5,000 g for 5 minutes, and the cells were collected. The cells were washed 2 times with 20mM PBS pH 7.2 and then resuspended in PBS buffer containing 0, 75mM and 150 mM NaCl, respectively. The hybrid protein E1C1 (final concentration 2. Mu.g/mL), the primary protein PVP-SE1gp146 (final concentration 2. Mu.g/mL) or PBS control were added to 96-well plates along with 10. Mu.l of 200mM EDTA (final concentration 0.5 mM) and 50. Mu.l of bacterial suspension, and PBS was added to the total reaction volume of 200. Mu.l, the reaction temperature was 37 ℃ and readings were performed 5 times at 600 nm wavelength for 1 minute intervals. The results are shown in FIG. 2.

Example 5

The antibacterial activity of the hybrid protein E1C1 of example 1 was compared with that of the native protein PVP-SE1gp146 under BSA conditions at volume concentrations of 10% and 20%.

After the overnight cultured Micrococcus muralis was transferred, it grew to mid-log phase, i.e., to OD ₆₀₀ About 0.5, howeverAfter centrifugation at 5,000 g for 5 minutes, the cells were collected. The cells were washed 2 times with 20mM PBS pH 7.2 and then resuspended in 10% and 20% BSA in buffer, respectively. The hybrid protein E1C1 (final concentration 2. Mu.g/mL), the primary protein PVP-SE1gp146 (final concentration 2. Mu.g/mL) or PBS control were added to 96-well plates along with 10. Mu.l of 200mM EDTA (final concentration 0.5 mM) and 50. Mu.l of bacterial suspension, and PBS was added to the total reaction volume of 200. Mu.l, the reaction temperature was 37 ℃ and readings were performed 5 times at 600 nm wavelength for 1 minute intervals. The results are shown in FIG. 3.

Example 6

The hybrid protein E1C1 of example 1 can kill Escherichia coli, salmonella, staphylococcus aureus, helicobacter pylori, acinetobacter baumannii, pseudomonas aeruginosa and Klebsiella pneumoniae in vitro at regular time and quantity.

Respectively inoculating overnight cultured Escherichia coli, salmonella, staphylococcus aureus, helicobacter pylori, acinetobacter baumannii, pseudomonas aeruginosa, and Klebsiella pneumoniae, and growing to mid-log phase, i.e. OD ₆₀₀ About 0.5, and then centrifuged at 5,000 g for 5 minutes, and the cells were collected. The mycelia were diluted to about 1X 10 with PBS pH 7.2 ⁸ cfu/mL。

1 mL of either the hybrid antibiotic enzyme sample, the primary protease sample or the PBS sample was added to 250. Mu.l of the cell suspension. The hybrid antibiotic enzyme sample contained E1C1 (final concentration of 10. Mu.g/mL or 50. Mu.g/mL), naCl (final concentration of 150 mM), and EDTA (final concentration of 0.5 mM). The protain sample contained the protain PVP-SE1gp146 (final concentration 10. Mu.g/mL or 50. Mu.g/mL) and EDTA (final concentration 0.5 mM). PBS samples contained PBS and EDTA (0.5 mM final concentration).

After the heterozygosis antibiotic enzyme sample acts on the bacterial liquid for 30min at room temperature, 0.5 mL of sample liquid is taken for serial dilution, then 0.5 mL of sample liquid is taken for viable count culture, and the higher the Log value is, the better the sterilization effect is.

After the protase sample and the PBS sample act on the bacterial liquid for 2 hours at room temperature respectively, 0.5 mL of sample liquid is taken for serial dilution, then 0.5 mL of sample liquid is taken for viable count culture, and the Log value shows that the larger the Log value is reduced, the better the sterilization effect is. The results are shown in tables 1 and 2.

TABLE 1, E1C1 (10. Mu.g/mL)/EDTA fungicidal Activity against different pathogenic bacteria

TABLE 2 fungicidal Activity of E1C1 (50. Mu.g/mL)/EDTA against different pathogenic bacteria

Example 7

Temperature tolerance of the hybrid protein E1C1 of example 1 was compared to the native protein PVP-SE1gp 146.

Protein sample treatment: dissolving a hybrid protein E1C1 sample or a protogenic protein PVP-SE1gp146 sample with the same concentration in a buffer solution, respectively culturing at 55 ℃ for 2h, 6h, 12h, 24h and 48h, naturally cooling to room temperature, measuring the antibacterial activity, and calculating the activity retention rate by taking the antibacterial activity at 4 ℃ as an initial value. Or dissolving the hybrid protein E1C1 sample or the protogenic protein PVP-SE1gp146 sample with the same concentration in a buffer solution, respectively culturing at 50 ℃, 60 ℃, 70 ℃,80 ℃, 90 ℃ and 100 ℃ for 0min, 30min and 60min, naturally cooling to room temperature, respectively determining the antibacterial activity, and calculating the activity retention rate by taking the antibacterial activity at 4 ℃ as an initial value.

And (3) determination of antibacterial activity: after the overnight culture of Micrococcus muralis was transferred, it grew to mid-log phase, i.e., to OD ₆₀₀ About 0.5, and then centrifuged at 5,000 g for 5 minutes to collect the cells. The mycelia were washed 2 times with 20mM PBS, pH 7.2, and then resuspended in PBS buffer. The hybrid protein E1C1 sample (final concentration 2. Mu.g/mL), the primary protein PVP-SE1gp146 sample (final concentration 2. Mu.g/mL) or the PBS control after incubation at different temperatures for different periods of time were added to a 96-well plate, 10. Mu.l of 200mM EDTA (final concentration 0.5 mM) and 50. Mu.l of bacterial suspension were added, PBS was added to 200. Mu.l of the total reaction system, the reaction temperature was 37 ℃, and the reading was 5 at 600 nm wavelength for 1 minute at intervalsNext, the process is carried out. The results are shown in FIGS. 4 and 5.

Example 8

E2C1 engineering bacteria construction, expression and purification:

this example artificially designed and synthesized a nucleic acid sequence encoding E2C1 according to pichia codon preferences. Is connected between Xho I and Xba I sites of pGAPZ alpha A vector to obtain the recombinant plasmid pGAPZ alpha A-E2C1. And (3) converting the yeast SMD1168 to obtain positive yeast, fermenting, expressing and purifying to obtain the hybrid protein E2C1.

Example 9

E1C2 engineering bacteria construction, expression and purification:

this example artificially designed and synthesized a nucleic acid sequence encoding E1C2 according to Pichia codon preference. Is connected between Xho I and Xba I sites of pGAPZ alpha A vector to obtain the recombinant plasmid pGAPZ alpha A-E1C2. And (3) converting the yeast SMD1168 to obtain positive yeast, fermenting, expressing and purifying to obtain the hybrid protein E1C2.

Example 10

C1E1 engineering bacteria construction, expression and purification:

this example differs from example 2 in that the nucleic acid sequence encoding C1E1 was artificially designed and synthesized according to Pichia codon preferences. Ligated between Xho I and Xba I sites of pGAPZ alpha A vector to obtain recombinant plasmid pGAPZ alpha A-C1E1. And (3) converting the yeast SMD1168 to obtain positive yeast, fermenting, expressing and purifying to obtain the hybrid protein C1E1.

Example 11

This example produces 100ml of a bio-antimicrobial formulation containing E1C1.

Prescription:

E1C 1.02 g of example 2

Anhydrous potassium dihydrogen phosphate 0.16 g

Anhydrous disodium hydrogen phosphate 0.12 g

Ethylene diamine tetraacetic acid disodium 0.037 g

Glycerol 3 ml

NaCl 0.6 g

Water to make up 100ml

The preparation method of the biological antibacterial agent of the embodiment is as follows:

a) And calculating the required quantity of the auxiliary materials according to the prescription and the total preparation liquid volume, and accurately weighing the auxiliary materials into a clean container.

b) Adding 70% of water into a dosing vessel, dissolving NaCl, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate and potassium dihydrogen phosphate, adding glycerol after full dissolution, adding E1C1 after uniform mixing, and finally, fixing the volume by using water and fully mixing.

c) And (3) degerming and filtering: the prepared solution passes through a sterilizing filter, and the effluent solution is connected into a sterile container.

d) Filling: the sterile solution is filled into plastic or glass bottles.

The antibacterial preparation can be used for sterilizing wound or wound surface 1-2 times per day.

Example 12

This example produces 100ml of a bio-antimicrobial formulation containing E2C1.

Prescription:

E2C 1.02 g from example 8

Anhydrous potassium dihydrogen phosphate 0.16 g

Anhydrous disodium hydrogen phosphate 0.12 g

Ethylene diamine tetraacetic acid 0.037 g

Glycerol 3 ml

NaCl 0.6 g

Water to make up 100ml

The preparation method of the biological antibacterial agent of the embodiment is as follows:

a) And calculating the required quantity of the auxiliary materials according to the prescription and the total preparation liquid volume, and accurately weighing the auxiliary materials into a clean container.

b) Adding 70% of water into a batching vessel, dissolving NaCl, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate and potassium dihydrogen phosphate, adding glycerol after fully dissolving, adding E2C1 after uniformly mixing, and finally adding water to a constant volume and fully mixing.

c) And (3) degerming and filtering: the prepared solution is passed through a sterilizing filter, and the effluent solution is connected into a sterile container.

d) Filling: the sterile solution is filled into plastic or glass bottles.

The antibacterial preparation can be used for sterilizing wound or wound surface 1-2 times per day.

Example 13

This example produces 100ml of a bio-antimicrobial formulation containing E1C2.

Prescription:

E1C 2.02 g of example 9

Anhydrous potassium dihydrogen phosphate 0.16 g

Anhydrous disodium hydrogen phosphate 0.12 g

Ethylene diamine tetraacetic acid disodium 0.037 g

Glycerol 3 ml

NaCl 0.6 g

Water to make up 100ml

The preparation method of the biological antibacterial agent of the embodiment is as follows:

a) And calculating the required quantity of the auxiliary materials according to the prescription and the total preparation liquid volume, and accurately weighing the auxiliary materials into a clean container.

b) Adding 70% of water into a batching vessel, dissolving NaCl, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate and potassium dihydrogen phosphate, adding glycerol after fully dissolving, adding E1C2 after uniformly mixing, and finally adding water to a constant volume and fully mixing.

c) And (3) degerming and filtering: the prepared solution passes through a sterilizing filter, and the effluent solution is connected into a sterile container.

d) Filling: the sterile solution is filled into plastic or glass bottles.

The antibacterial preparation can be used for sterilizing wound or wound surface 1-2 times per day.

Example 14

This example produces 100ml of a C1E 1-containing biocide formulation.

Prescription:

C1E 1.02 g of example 10

Anhydrous potassium dihydrogen phosphate 0.16 g

Anhydrous disodium hydrogen phosphate 0.12 g

Ethylene diamine tetraacetic acid disodium 0.037 g

Glycerol 3 ml

NaCl 0.6 g

Water make up to 100ml

The preparation method of the biological antibacterial agent of the embodiment is as follows:

a) And calculating the required quantity of the auxiliary materials according to the prescription and the total preparation liquid volume, and accurately weighing the auxiliary materials into a clean container.

b) Adding 70% of water into a dosing vessel, dissolving NaCl, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate and potassium dihydrogen phosphate, adding glycerol after full dissolution, adding C1E1 after uniform mixing, and finally, fixing the volume by using water and fully mixing.

c) And (3) degerming and filtering: the prepared solution is passed through a sterilizing filter, and the effluent solution is connected into a sterile container.

d) Filling: the sterile solution is filled into plastic or glass bottles.

The antibacterial preparation can be used for sterilizing wound or wound surface 1-2 times per day.

As can be seen by combining example 1 and example 3 with FIG. 1, the hybrid protein E1C1 and the protist PVP-SE1gp146 of example 1 both can cause the OD value of the bacterial liquid to be reduced by cracking pathogenic bacteria, and the faster the OD value of the bacterial liquid of the pathogenic bacteria is reduced, the more the reduction shows that the antibacterial activity of the protein is higher. The results show that the hybrid protein E1C1 has higher antibacterial activity than the primary protein PVP-SE1gp 146. Compared with the degree of decrease in OD value at the end of the reaction time of 5min, the hybrid E1C1 of the present invention is 2.3 times that of the native protein.

Combining example 1 and example 4 and combining fig. 2, it can be seen that the hybrid protein E1C1 of example 1 still maintains high bactericidal activity in high salt environment, and as the salt concentration increases, the activity decreases, but remains above 90%. While the activity of the native protein was reduced by 50% and 80% in the presence of 75mM and 150 mM NaCl, respectively.

As can be seen by combining example 1 with example 5 and combining FIG. 3, the hybrid protein E1C1 of example 1 still maintains higher bactericidal activity in a high-concentration organic environment, and 10% and 20% BSA have no substantial effect on the bactericidal activity of E1C1. While the protamine PVP-SE1gp146 showed a 70% reduction in activity in the presence of 10% BSA, and a substantial total loss of activity in the presence of 20% BSA.

As can be seen by combining the results of the in vitro sterilization experiments in the embodiment 1 and the embodiment 6 and the tables 1-2, the in vitro sterilization experiment results in the table 1 show that 10 mug/mL of the antimicrobial protein E1C1 can effectively kill Escherichia coli, salmonella, helicobacter pylori, staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa and Klebsiella pneumoniae for 30min, and the reduction values are all more than 3 Log values.

The results in Table 2 show that 50 mug/mL of the antimicrobial protein E1C1 can effectively kill Escherichia coli, salmonella, helicobacter pylori, staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae for 30min, and the reduction values are all more than 4 Log values.

The results show that the E1C1 protein after heterozygosis has very strong bactericidal effect on escherichia coli, salmonella, helicobacter pylori, staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae, and has obvious improvement compared with the original protein before heterozygosis.

As can be seen by combining example 1 and example 7 with FIGS. 4-5, the hybrid protein E1C1 of example 1 has a higher temperature tolerance. The hybrid protein E1C1 of example 1 maintained full activity after 48 hours of incubation at 55 ℃ and the native protein only maintained activity at 55 ℃ for 24h, with a significant decrease in activity after 48h. At higher temperatures, the hybrid protein E1C1 of example 1 maintained more than 90% activity after incubation at 80 ℃ for 1 h, tolerating up to 90 ℃ and not being inactivated within 30 min.

The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.

Claims (11)

1. A hybrid antibiotic enzyme, comprising a hybrid protein E1C1, said hybrid protein E1C1 comprising a catalytic domain E1 as set forth in SEQ ID NO. 2 and a binding domain C1 as set forth in SEQ ID NO. 3.

2. A hybrid antibiotic enzyme according to claim 1 wherein: 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 wherein: the hybrid antibiotic enzymes further include a hybrid protein E2C1, the catalytic domain of the hybrid protein E2C1 having at least 75% homology with the catalytic domain E1.

4. A hybrid antibiotic enzyme according to claim 3 wherein: the amino acid sequence of the E2C1 is shown in SEQ ID NO. 5.

5. A hybrid antibiotic enzyme according to claim 1 wherein: the hybrid antibiotic enzymes further include a hybrid protein E1C2, the binding domain of the hybrid protein E1C2 having at least 75% homology to the binding domain C1.

6. A hybrid antibiotic enzyme according to claim 5 wherein: the amino acid sequence of the E1C2 is shown in SEQ ID NO. 6.

7. A hybrid antibiotic enzyme according to claim 1 wherein: the hybrid antibiotic enzymes also include hybrid protein C1E1, wherein the hybrid protein C1E1 is a hybrid protein composed of the catalytic domain E1 and the binding domain C1 by position exchange.

8. A hybrid antibiotic enzyme according to claim 7 wherein: the amino acid sequence of the C1E1 is shown as SEQ ID NO. 7.

9. Use of a hybrid antibiotic enzyme of any one of claims 1 to 8 in the manufacture of a medicament for controlling a microbial infection.

10. A pharmaceutical composition characterized by: the active ingredient of the pharmaceutical composition comprises the hybrid antibiotic enzyme according to any one of claims 1 to 8.

11. A pharmaceutical composition according to claim 10, wherein: the pharmaceutical composition also comprises a pharmaceutically acceptable carrier, and the dosage form of the pharmaceutical composition is injection, oral administration or external application.

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