Patent Description:
Antibiotics have been widely used in the fields of medical and health services and agricultural breeding, and have played an important role in treatment of infectious diseases and protection of public health security. However, with extensive use of the antibiotics, bacteria have become increasingly resistant to the antibiotics. It resulted clinical treatment difficulties and increased mortality in the world and therefor seriously threatened human health.

Multi-drug resistant bacteria mainly include Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Enterococcus and Staphylococcus aureus, in which Gram-negative bacteria (G- bacteria) have a largest proportion, and particularly drug resistance of the Acinetobacter baumannii has become more and more serious in recent years (<NPL>. Among <NUM> deadliest drug-resistant bacteria ranked by the World Health Organization (WHO), the Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae which resistant to carbapenem antibiotics are the most serious. It is urgent to develop new antimicrobial drugs, especially antimicrobial drugs with novel action modes different from the traditional antibiotics.

The traditional antibiotics have broad-spectrum antimicrobial properties and take the same effect on normal microbial flora. During extensive use of the antibiotics, the normal microbial flora also envolved drug resistance. As drug-resistant genes are transmitted between the normal microbial flora and pathogenic bacteria, the probability of drug-resistant bacteria is significantly increased. As a main component of a bacterial cell wall, peptidoglycan is a three-dimensional network structure formed by cross-linking a sugar chain, a peptide chain and a peptide bond bridge. The peptidoglycan is used for maintaining the mechanical strength of the cell wall and preventing bacteria from being affected by external factors. As a class of proteins that can specifically lyse the peptidoglycan of the bacterial cell wall, peptidoglycan hydrolases mainly include bacteriophage lysins, bacteriocins and bacterial autolysins (<NPL>. For example, a Staphylococcus aureus bacteriophage lysin LysK, a Staphylococcus simulans bacteriocin lysostaphin and a Streptococcus pneumoniae autolysin LytA are included. A peptidoglycan hydrolase generally consists of a catalytic structural domain (catalytic domain) used for hydrolyzing the peptidoglycan and a targeting structural domain (binding domain) used for specifically targeting a bacterial cell wall.

When being added into a bacterial solution, the peptidoglycan hydrolase can specifically bind to sensitive bacteria and rapidly lyse cell walls. It has been extensively proved that the peptidoglycan hydrolase has antimicrobial activity in vivo and in vitro (<NPL>. The peptidoglycan hydrolase can quickly kill specific bacteria by directly lysing the peptidoglycan of the bacteria, has a narrow antimicrobial spectrum and a bactericidal mechanism different from the traditional antibiotics, and can be used repeatedly for a long time without easy induction drug resistance. The peptidoglycan hydrolase can directly lyse the bacteria in every growth cycle. By using the narrow-spectrum peptidoglycan hydrolase, the normal microbial flora is hardly affected.

However, there are some problems need to be improved when natural peptidoglycan hydrolases are directly used, for example, low antimicrobial activity, very narrow antimicrobial spectrum, low expression amount, poor solubility. In addition, the combination of the catalytic domain and the binding domain are not in a best condition, or there are other non-essential domains between them. Wild-type LysK includes two catalytic domains (CHAP and Amidase structural domains) while the Amidase structural domain has very low catalytic activity. When the Amidase structural domain is removed from the LysK (LysKΔ221-<NUM>), the activity of a protein is not changed (<NPL>. An isolated CHAP catalytic domain of the LysK also has no antimicrobial activity in a TSB culture medium. In a complex environment, the catalytic domain needs to be combined with a binding domain to achieve more effect. However, the binding domain of the LysK has no great effect, and is leading to low affinity efficiency and narrow identification range. Therefore, construction of a highly active hybrid antimicrobial protein has great significance for promoting research and development of novel antimicrobial drugs. It will provide an effective solution for solving the problem of the bacteria of drug resistance.

In <NUM>, Gladskin, as the first bacteriophage lysin based antimicrobial protein product on the market in the world, is used for treatment of inflammatory skin diseases, such as eczema, rosacea and acne, caused by methicillin-resistant Staphylococcus aureus (MRSA) in intact skin. At present, clinical researches have already been conducted on many antimicrobial proteins, such as Lysostaphin (China, used for treatment of a wound infection with the Staphylococcus aureus), P128 (India, used for removal of the Staphylococcus aureus fixed to the nasal mucosa), CF301 (the United States of America, used for treatment of septicemia caused by the Staphylococcus aureus) and SAL200 (South Korea, used for treatment of the septicemia caused by the Staphylococcus aureus). In addition, clinical researches have also been conducted on some antimicrobial proteins, such as Cpl-<NUM>, ClyS, ClyH and PlyV12, which are used for treatment of infections caused by Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus, respectively. All these antimicrobial proteins with clinical researches are focused on treatment of infections with G+ bacteria, and at present, there are no clinical researches on antimicrobial proteins used for treatment of G- bacteria. Based on the severity of the drug-resistant G-bacteria and the imbalance in development of antimicrobial protein drugs, it is urgent to develop new antimicrobial drugs against the G-bacteria.

<CIT> discloses polypeptides comprising an amino acid sequence of a globular Gram-negative endolysin and a cell wall-binding domain. These polypeptides can be used for treating humans or animals through surgery or therapy, as well as for diagnostic purposes. This document further describes corresponding nucleic acids, vectors, bacteriophages, host cells, and compositions, which can also be used as antimicrobial agents in food, cosmetics, or as disinfecting agents.

<NPL>) describes a phage endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1, exhibiting antibacterial activity against Gram-negative pathogens, including multidrug-resistant Acinetobacter baumannii. ABgp46 functions as an N-acetylmuramidase, active across a broad pH range and temperatures up to <NUM>. It reduces multidrug-resistant A. baumannii strains by up to <NUM> logs within <NUM> hours. In combination with citric and malic acid, ABgp46 shows enhanced antibacterial effects against A. baumannii, Pseudomonas aeruginosa, and Salmonella typhimurium, suggesting potential use in human and veterinary medicine.

According to <NPL>), endolysins and their derivatives have emerged as a novel class of antibacterials. Their rapid action and proteinaceous nature distinguish them from other antibiotics. A key feature is their modularity, enabling customization of specificity, activity, stability, and solubility. Protein engineering has expanded their activity against drug-resistant Gram-negative bacteria and improved efficacy against Gram-positive pathogens. Additionally, specific cell wall binding domains from endolysins are used for diagnostic development.

An objective of the present invention is to provide a hybrid antimicrobial protein having a strong bactericidal effect and an application thereof, so as to overcome the defects of the prior art and meet human needs.

The hybrid antimicrobial protein of the present invention, having a strong bactericidal effect against Gram-negative bacteria, is a hybrid protein with the amino acid sequence of SEQ ID NO: <NUM> which named AB469, a hybrid protein named AB46M9 with an amino acid sequence shown in SEQ ID NO <NUM>, a hybrid protein named AB469M with an amino acid sequence shown in SEQ ID NO <NUM>, or a hybrid protein named B946 with an amino acid sequence shown in SEQ ID NO <NUM>.

The AB469 includes <NUM> amino acids and has a molecular weight of <NUM> kD and a theoretical isoelectric point of <NUM>. After purification in <NUM> steps, the purity can reach <NUM>% or above. According to SDS-PAGE electrophoresis, it is shown that the AB469 has a molecular weight of about <NUM> kD.

A nucleic acid sequence encoding the AB469 is shown in SEQ ID NO: <NUM> and named ab469.

According to a test, it is proven that the AB469 has high solubility and stability.

According to an in vitro test, it is proven that the AB469 has high activity, a broad spectrum and a strong bactericidal effect on Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi and Escherichia coli. After adding <NUM>µg/mL of the AB469 into bacterial fluid and acts for <NUM>, the Acinetobacter baumannii (ATCC <NUM>), the Pseudomonas aeruginosa (ATCC <NUM>) and the Salmonella typhi (CMCC <NUM>) can be decreased by more than <NUM> log values, and the Klebsiella pneumoniae (ATCC <NUM>) and the Escherichia coli (ATCC <NUM>) are decreased by more than <NUM> log values. After adding <NUM>µg/mL of the AB469 acts for <NUM>, the Klebsiella pneumoniae (ATCC <NUM>) and the Escherichia coli (ATCC <NUM>) can be decreased by more than <NUM> log values, and Enterobacter cloacae (ATCC <NUM>) and Staphylococcus aureus (ATCC <NUM>) are decreased by about <NUM> log values.

The antimicrobial protein AB469 can effectively kill the clinically isolated Acinetobacter baumannii, the Pseudomonas aeruginosa and the Klebsiella pneumoniae. In addition, the AB469 has the same effect on the Acinetobacter baumannii, the Pseudomonas aeruginosa and the Klebsiella pneumoniae with multi-drug resistance (to ceftazidime, ceftriaxone, meropenem, imipenem, tobramycin and sulbactam).

The AB469 also has high activity under complex conditions such as serum and body fluid.

A nucleic acid sequence encoding the AB46M9 is shown in SEQ ID NO: <NUM> and named ab46m9.

A nucleic acid sequence encoding the AB469M is shown in SEQ ID NO: <NUM> and named ab469m.

A nucleic acid sequence encoding the B946 is shown in SEQ ID NO: <NUM> and named b946.

Compared with wild-type ABgp46, the AB469 has one more binding domain, the antimicrobial activity in a complex environment can be significantly improved, and the complex environment includes certain ionic strength, a culture medium, serum and a body fluid.

According to a test, it is shown that under certain ionic strength (<NUM> NaCl), the bactericidal activity of the ABgp46 is significantly reduced (Table <NUM>), and a log decrease value is decreased from <NUM> to <NUM>. The results are consistent with those of many proteins with only one catalytic domain (<NPL>. From Table <NUM>, it can be seen that the effect of the ionic strength on the ABgp46 is significantly higher than that on the AB469. A bactericidal effect of Oliverira-expressed ABgp46 (<NUM>, ~<NUM>µg/mL) on the Pseudomonas aeruginosa (decreased by <NUM> log values) is lower than that on the Acinetobacter baumannii (decreased by more than <NUM> log values). The AB469 of the present invention with a lower protein concentration (<NUM>, <NUM>µg/mL), has a strong bactericidal effect on the Acinetobacter baumannii and the Pseudomonas aeruginosa under <NUM> NaCl (decreased by more than <NUM> log values).

The ABgp46 is a bacteriophage lysin only including one catalytic domain and has been reported in detail in the following literature: <NPL>.

The hybrid antimicrobial protein having a strong bactericidal effect can be used for preparing an antimicrobial preparation. The antimicrobial preparation includes an antimicrobial effective amount of the hybrid antimicrobial protein having a strong bactericidal effect on Gram-negative bacteria and a drug carrier. The drug carrier includes an excipient, such as water and hydroxypropyl methyl cellulose.

Preferably, in the antimicrobial preparation, the hybrid antimicrobial protein having a strong bactericidal effect on Gram-negative bacteria has a weight percentage of <NUM>-<NUM>%.

The antimicrobial preparation can be applied to a surface of a human body or an object in need by spraying.

The present invention has the following beneficial effects.

In the present invention, on the basis of a large number of researches, a new hybrid antimicrobial protein is successfully constructed. The Escherichia coli or yeast is used for expression, and after purification, the hybrid antimicrobial protein with high purity is obtained. The protein can rapidly lyse the Gram-negative bacteria, such as the Acinetobacter baumannii, the Pseudomonas aeruginosa, the Klebsiella pneumoniae, the Salmonella typhi and the Escherichia coli. The antimicrobial preparation prepared from the antimicrobial protein can be used for prevention and treatment of bacterial infection. The antimicrobial protein is a new and efficient antimicrobial substance that can directly lyse bacteria. The antimicrobial protein can be prepared into an antimicrobial preparation used for prevention and treatment of bacterial infection and sterilization of medical apparatus and instruments and medical places.

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto;
In the present invention, through analysis of a structural domain of an antimicrobial protein, a hybrid protein having high antimicrobial activity against Gram-negative bacteria is artificially designed, and an amino acid sequence is shown in SEQ ID NO: <NUM>. Secretion expression of the protein in Escherichia coli is achieved by using a molecular biology method. After purification, a protein with high purity is obtained for activity detection.

According to the preference of a codon of Escherichia coli, a nucleic acid sequence ab469 (SEQ ID NO: <NUM>) encoding the AB469 was artificially designed and synthesized. A Ncol restriction endonuclease site (italic part) was added into a <NUM>' end, and a nucleic acid sequence ct was introduced with g at the end of the restriction site to form a codon encoding an alanine. A HindIII restriction endonuclease site was added into a <NUM>' end (italic part). The sequence was as follows:
<IMG>.

In order to facilitate expression in the Escherichia coli and to form a soluble target protein, Cys in an SP10 binding domain was mutated into Ser. An artificially synthesized ab469 gene inserted to a vector pET28a(+) through being connected between the Ncol and HindIII sites to obtain a recombinant plasmid pET28a-ab469. The recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3) to obtain an engineering bacterium expressing the AB469.

A monoclonal engineering bacterium was inoculated into an LB culture solution (containing <NUM>/L of kanamycin) and subjected to shaking culture overnight at <NUM>, then inoculated into the same LB culture solution with a ratio of <NUM>%, and subjected to shaking culture at <NUM> until an optical density (wavelength <NUM>) was about <NUM>. An IPTG with a final concentration of <NUM> was added to induce protein expression and continuously subjected to shaking culture at <NUM>, induction was conducted for about <NUM>, and a fermentation supernatant was collected by centrifugation.

The fermentation supernatant was purified by two steps of cation exchange and gel filtration. A purified sample was cryopreserved at -<NUM> for later use. The weight and purity of a recombinant protein sample were determined by using <NUM>% SDS-PAGE. According to results, it could be seen that a main electrophoresis band of a recombinant hybrid antimicrobial protein AB469 was about <NUM> kD (see <FIG>).

The mutant AB46M9 (SEQ ID NO: <NUM>) consisted of a catalytic domain AB46M (SEQ ID NO: <NUM>) having <NUM>% similarity with an amino acid sequence of AB46 (<NUM>-185aa) (SEQ ID NO: <NUM>), a flexible linker, and a binding domain SP10 (<NUM>-236aa) (SEQ ID NO: <NUM>), in sequence.

The mutant AB469M (SEQ ID NO: <NUM>) consisted of a catalytic domain AB46 (<NUM>-185aa), a flexible linker, and a binding domain B9M (SEQ ID NO: <NUM>) having <NUM>% similarity with an amino acid sequence of SP10 (<NUM>-236aa), in sequence.

The B946 (SEQ ID NO: <NUM>) consisted of a binding domain SP10 (<NUM>-236aa), a flexible linker in the middle, and a catalytic domain AB46 (<NUM>-185aa), in sequence.

According to the preference of a codon of Escherichia coli, nucleic acid sequences ab46m9 (SEQ ID NO: <NUM>), ab469m (SEQ ID NO: <NUM>) and b946 (SEQ ID NO: <NUM>) that can encode AB46M9, AB469M and B946 were artificially designed and synthesized.

Construction of expression vectorsand engineering bacteria of the ab46m9, the ab469m and the b946, and specific expression and purification methods of the ab46m9, the ab469m and the b946 were the same as those of the ab469. Purified AB46M9, AB469M and B946 samples were cryopreserved at -<NUM> for later use. The weight and purity of recombinant protein samples were determined by using <NUM>% SDS-PAGE (see <FIG>).

The Acinetobacter baumannii (ATCC <NUM>), the Pseudomonas aeruginosa (ATCC <NUM>) and the Klebsiella pneumoniae (ATCC <NUM>) cultured overnight were transferred and allowed to grow to a mid-log phase (OD<NUM> was about <NUM>). Bacterial bodies were collected by centrifugation (<NUM>,<NUM>, <NUM>). The bacteria bodies were washed twice with <NUM> PBS (pH <NUM>) and then resuspended in PBS buffers with different concentrations of NaCl (<NUM>-<NUM>). A sample (to a final concentration of <NUM>µg/mL) or a PBS control, <NUM>µl of a <NUM> EDTA (to a final concentration of <NUM>) and <NUM>µl of a bacterial suspension were added into a <NUM>-well plate, and the PBS was supplemented until a total reaction system was <NUM>µl. A reaction temperature was <NUM>, and reading results was conducted at a wavelength of <NUM> (for <NUM> times at an interval of <NUM>).

Turbidimetric detection results were shown in <FIG>. The AB469 could lyse pathogenic bacteria, thereby causing the OD value of a bacterial solution decreased. When the OD value of the bacterial solution of the pathogenic bacteria was rapidly decreased, it was indicated that the AB469 had a rapid bactericidal effect on the Acinetobacter baumannii (ATCC <NUM>), the Pseudomonas aeruginosa (ATCC <NUM>) and the Klebsiella pneumoniae (ATCC <NUM>). With increase of the salt concentration, the AB469 still had high bactericidal activity. The bactericidal activity of the AB469 could be significantly improved by using some non-ionic surfactants (such as BriJ98, Tween <NUM> and Peregal).

The turbidimetric method was the same as that in Embodiment <NUM> for detection, and turbidimetric detection results were shown in <FIG>. Compared with the AB469, the bactericidal activity of the mutants AB46M9, AB469M and B946 against the Acinetobacter baumannii was not significantly reduced.

Bacteria cultured overnight were transferred and allowed to grow to a mid-log phase (OD<NUM> was about <NUM>). Bacterial bodies were collected by centrifugation (<NUM>,<NUM>, <NUM>). The bacterial bodies were diluted with PBS (pH <NUM>) to about <NUM>*<NUM><NUM> cfu/mL. <NUM> of a sample containing the AB469 (to a final concentration of <NUM>-<NUM>µg/mL); NaCl (to a final concentration of <NUM>); and an EDTA (to a final concentration of <NUM>) or sodium citrate (to a final concentration of <NUM>) was added into <NUM>µl of a bacterial solution. After reaction at room temperature for <NUM>, <NUM> of a sample solution was taken for serial dilution, and then <NUM> of a sample solution was taken and subjected to viable count culture. <NUM>µh/mL of AB46 and an EDTA (to a final concentration of <NUM>) were used as a control (after reaction at room temperature for <NUM>). A preparation method of the AB46 was the same as that of the AB469.

According to results of an in vitro bactericidal experiment, it was shown that compared with the isolated AB46, the hybrid antimicrobial protein AB469 had stronger activity in killing Acinetobacter baumannii (ATCC <NUM>) (Table <NUM>) and was more resistant to ionic strength.

According to results of an in vitro bactericidal experiment (Table <NUM>), it was shown that after <NUM>µg/mL of the AB469 acted for <NUM>, the Acinetobacter baumannii (ATCC <NUM>), Pseudomonas aeruginosa (ATCC <NUM>) and Salmonella typhi (CMCC <NUM>) could be effectively killed and decreased by more than <NUM> log values; Klebsiella pneumoniae (ATCC <NUM>) and Escherichia coli (ATCC <NUM>) decreased by more than <NUM> log values; Enterobacter cloacae (ATCC <NUM>) and Staphylococcus aureus (ATCC <NUM>) decreased by about <NUM> log value.

According to results in Table <NUM>, it was shown that after <NUM>µg/mL of the AB469 acted for <NUM>, the Klebsiella pneumoniae (ATCC <NUM>) and the Escherichia coli (ATCC <NUM>) could be effectively killed; the Enterobacter cloacae (ATCC <NUM>) and the Staphylococcus aureus (ATCC <NUM>) decreased by about <NUM> log values.

After <NUM>µg/mL of the antimicrobial protein AB469 acted for <NUM>, the clinically isolated Acinetobacter baumannii and Pseudomonas aeruginosa could be effectively killed (Table <NUM>). The AB469 had the same effect on multi-drug (comprising ceftazidime, ceftriaxone, meropenem, imipenem, tobramycin and sulbactam, etc.) resistant strains. After <NUM>µg/mL of the antimicrobial protein AB469 acted for <NUM>, the clinically isolated Klebsiella pneumoniae including multi-drug resistant bacteria could be effectively killed.

According to the results above, the hybrid AB469 had a very strong bactericidal effect on Gram-negative bacteria and the same effect on drug-resistant bacteria. The AB469 also had a strong bactericidal effect on the Staphylococcus aureus (Gram-positive bacteria).

<NUM> of a biological antimicrobial preparation containing AB469 was prepared.

Claim 1:
A hybrid antimicrobial protein having a strong bactericidal effect, wherein the hybrid antimicrobial protein is:
<NUM>) a hybrid protein named AB469 with the amino acid sequence shown in SEQ ID NO <NUM>;
<NUM>) a hybrid protein named AB46M9 with the amino acid sequence shown in SEQ ID NO <NUM>;
<NUM>) a hybrid protein named AB469M with the amino acid sequence shown in SEQ ID NO <NUM>; or
<NUM>) a hybrid protein named B946 with the amino acid sequence shown in SEQ ID NO <NUM>.