CN116478966B - Novel acinetobacter baumannii phage endolysin protein, preparation and application thereof - Google Patents

Abstract

The application provides a novel acinetobacter baumannii phage endolysin protein and preparation and application thereof, and in particular provides a recombinant solvent protein derived from multi-drug resistant acinetobacter baumannii phage, and preparation and application thereof. The protein can be used for treating diseases or symptoms caused by Acinetobacter baumannii infection.

Description

Novel acinetobacter baumannii phage endolysin protein, preparation and application thereof

Technical Field

The application relates to the field of recombinant proteins, and in particular provides a recombinant menstruum protein derived from multi-drug-resistant Acinetobacter baumannii phage, and preparation and application thereof.

Background

Acinetobacter baumannii is widely found in nature and is a non-fermented, oxidase-negative, unpowered, aerobic, gram-negative bacillus. As a globally widespread conditional pathogen, it often infects people with low immunity, usually elderly patients, ICU patients, and it can cause wound infection, hospital-acquired pneumonia, bacteremia, meningitis, etc. In recent years, clinical treatment is more and more difficult due to the wide drug resistance of Acinetobacter baumannii, particularly the appearance of Acinetobacter baumannii with 'full drug resistance', and high vigilance is caused in the medical community. In the seventies of the 19 th century, acinetobacter baumannii was sensitive to general, traditional antibiotics, however, today, most clinical strains developed resistance to first-line antibiotics, so multi-drug resistant acinetobacter baumannii (MDR-AB) is very common today. MDR-AB refers to Acinetobacter baumannii strains that develop resistance to at least three of the following five classes of antibacterial agents: cephalosporins, carbapenems, beta-lactamase inhibitors (including piperacillin/tazobactam, cefoperazone/sulbactam and ampicillin/sulbactam), quinolones and aminoglycosides. The treatment of MDR-AB is a challenge in the world today and this trend has forced the clinical use of new antibiotics, including tigecycline, colistin, polymyxin antibiotics in the new generation of minocycline class. Unfortunately, reports of tigecycline and polymyxin strains still appear, indicating that acinetobacter baumannii resistance is obtained very rapidly.

Phage is a bacterial virus that is widely found in nature, in amounts that are more than ten times greater than bacteria, and which lyses and kills bacteria. Phage were used to treat infectious diseases at the beginning of their discovery, and prior to the second war, a great deal of research was done on phage therapy and positive therapeutic effects were achieved, however, when antibiotics appeared, research on phage became increasingly cooler. Until today, the abuse of antibiotics has led to a pace of embarrassment in the treatment of bacterial infections, and the study of phage therapy has again attracted a great deal of attention from researchers.

Many reports have been made on the successful treatment of diseases with phage preparations, but the stringent host specificity of phage makes the phage cleavage spectrum narrow, which is an obstacle in the phage preparation development process. With the continuous emergence of multi-drug resistant strains worldwide, the development of novel antibiotics is more and more difficult, and the search for new strategies for treating bacterial infections is urgent.

In view of the above, there is a lack in the art of non-antibiotic therapeutic agents directed against bacterial infections, particularly against multi-drug resistant bacterial infections.

Disclosure of Invention

The application aims to provide a recombinant in-vivo lysosomal protein for bacterial infection, in particular for multi-drug resistant acinetobacter baumannii infection.

Based on the unique advantages of phage antibiosis, the application takes a multi-drug-resistant Acinetobacter baumannii obtained by clinical separation as a host bacterium, separates a strain of Acinetobacter baumannii phage TC3 from sewage, discovers that lysozyme protein TCLys119 has the capability of cracking drug-resistant Acinetobacter baumannii and destroying the activity of a biological membrane through researching the biological characteristics of TC3, and hopes that lysozyme protein TCLys119 in TC3 has the opportunity to become a new drug-resistant bacteria therapeutic drug.

In a first aspect of the present application, there is provided a TCLys119 lysosomal protein having an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No. 2.

In other embodiments of the application, the protein is derived from the protein of SEQ ID No.1 or SEQ ID No.2 by substitution, deletion or addition of one or several amino acid residues and has the function of the protein of SEQ ID No.1 or SEQ ID No.2, the above-mentioned modifications do not disrupt the function of the protein and thus belong to the equivalent substitution forms of lysosomal proteins of the application.

In another preferred embodiment, the recombinant protein is a derivative protein formed by adding one or more amino acid residues to the protein of SEQ ID No.1 or SEQ ID No.2 (e.g., including a suitable expression tag, etc.), and having the function of the protein of SEQ ID No.1 or SEQ ID No. 2.

In another preferred embodiment, the derivative protein is a derivative protein which is formed by substitution, deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, most preferably 1 to 3 amino acid residues of the amino acid sequence of SEQ ID No.1 or SEQ ID No.2 and has the function of the oligopeptide of SEQ ID No. 2.

In another preferred embodiment, the protein is a structural protein derived from TC3 phage.

In a second aspect of the application there is provided a polynucleotide encoding a protein according to the first aspect of the application.

In a third aspect of the application there is provided an expression vector comprising a polynucleic acid as described in the second aspect of the application.

In a fourth aspect of the application there is provided a host cell comprising an expression vector according to the third aspect of the application, or having integrated on its genome a polynucleotide according to the second aspect of the application.

In another preferred embodiment, the host cell is E.coli.

In a sixth aspect of the present application, there is provided a method for preparing a protein according to the first aspect of the present application, comprising the steps of:

(1) Culturing a host cell according to the fourth aspect of the application under conditions suitable for production of said protein, thereby producing a protein according to the first aspect of the application; and

(2) Isolating the protein produced from the culture of step (1).

In a seventh aspect, the application provides the use of a protein according to the first aspect of the application, a polynucleotide according to the second aspect of the application, an expression vector according to the third aspect of the application or a host cell according to the fourth aspect of the application in the preparation of a pharmaceutical composition for the treatment of a disease or condition caused by acinetobacter baumannii infection.

In another preferred embodiment, the acinetobacter baumanii is multi-drug resistant acinetobacter baumanii.

In another preferred example, the acinetobacter baumanii is acinetobacter baumanii resistant to one or more antibiotics selected from the group consisting of: gentamicin, amoxicillin/clavulanic acid, aztreonam, ciprofloxacin, ceftriaxone, cefazolin, nitrofurantoin, imipenem, ampicillin, cefoxitin, levofloxacin, compound neotame, tobramycin, piperacillin/tazobactam, tigecycline, cefpiramide.

In an eighth aspect of the application, there is provided a pharmaceutical composition comprising a protein according to the first aspect of the application, a polynucleotide according to the second aspect of the application, an expression vector according to the third aspect of the application or a host cell according to the fourth aspect of the application, and a pharmaceutically acceptable carrier and/or adjuvant.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a plaque assay result;

FIG. 2 shows a phage genomic DNA restriction enzyme assay. Lane 1:1kb DNA ladder; lane 2, TC3 phage whole genome DNA without restriction enzyme cleavage; lanes 3-6, products of the cleavage of the whole genome DNA of the TC3 phage by the restriction enzymes DraI, ecoRI, hindIII and NdeI in sequence; lane 7 HindIII digested lambda phage whole genome DNA;

FIG. 3 shows SDS-PAGE gel electrophoresis of phage structural proteins. M is a known molecular weight protein standard; solid arrows point to three major protein bands; open arrows point to minor protein bands;

FIG. 4 is a SDS-PAGE map of TCLys 119;

FIG. 5 is a graph showing the antibacterial effect of TCLys119 recombinant protein measured by a plate method;

FIG. 6 shows the bacteriostasis of TCLys119 recombinant protein measured by broth dilution method.

Detailed Description

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

EXAMPLE 1 analysis of drug resistance of host bacteria

The host bacterium Ab3 is separated from sputum specimens of ICU patients transplanted by kidney of three hospitals of Hunan elegance, university of south China and is identified as Acinetobacter baumannii by a VITEK-2Compact full-automatic microbial analysis and identification system of French biology Mei Liai company.

1.1 drug sensitivity results of Acinetobacter baumannii Ab3

TABLE 1 drug sensitivity results for Ab3

The drug sensitivity results (Table 1) show that Ab3 is resistant to aminoglycosides, carbapenems, quinolones, cephalosporins, and mediates tigecycline in tetracyclines. Ab3 is a strain of multidrug-resistant Acinetobacter baumannii.

Example 2 isolation of Acinetobacter baumannii phages

2.1. Reagent(s)

(1) 3-fold LB liquid medium: 10g of tryptone, 10g of sodium chloride, 5.0g of yeast powder, 330ml of distilled water and sterilizing by high-pressure steam at 121 ℃ for 15min.

(2) LB agar: 10g of peptone, 5g of yeast powder, 5g of sodium chloride, 1g of glucose, 13g of agar and 1000ml of distilled water are added, and the mixture is sterilized by high-pressure steam for 15min at the temperature of 121 ℃.

(3) LB semisolid culture medium: mixing the LB liquid culture medium with LB agar in equal volume

2.2 phage isolation

(1) Preparation of host bacteria: acinetobacter baumannii preserved with glycerol is recovered by a three-zone streak method, single bacterial colony is picked and placed in 20ml of LB liquid culture medium for 5 hours, and bacterial liquid in logarithmic growth phase is obtained for later use.

(2) Collecting and treating sewage samples: taking 200ml of water in a western lake park, centrifuging at 6000rpm for 10min, centrifuging to remove most of bacteria and impurities, and taking the supernatant for later use.

Four Acinetobacter baumannii phages are separated from sewage by a double-layer agar plaque test, wherein a third strain is named as TC3 and has the ability of cracking Ab3, and the biological characteristics of TC3 and the ability of cracking Ab3 are verified and evaluated later.

2.3 plaque assay

Mixing 15ml of centrifuged sewage supernatant with bacterial liquid of Acinetobacter baumannii in equal volume, shaking and culturing for about 20 hours at 37 ℃, adding 1ml of chloroform for sterilization, shaking for ten minutes, centrifuging at 6000rpm for 10 minutes, mixing the supernatant with the prepared bacterial liquid of the corresponding Acinetobacter baumannii again, shaking and culturing for overnight at 37 ℃, repeating the steps for three times, and adding chloroform for sterilization for the third time to obtain the supernatant for standby. The LB semisolid culture medium is placed in a microwave oven and heated to boiling, poured into a 5ml sterile test tube, and the test tube is placed in a 42 ℃ water bath box for cooling, wherein the phage activity is affected by too high temperature, and the LB semisolid culture medium is coagulated by too low temperature. After the test tube is cooled to 42 ℃, 100ul of Acinetobacter baumannii bacterial liquid is added into the test tube, and after the mixture is uniformly mixed, the mixture is uniformly plated on an LB agar plate, a double-layer agar plate is prepared, and supernatant is dripped on the plate. The plate was incubated in a 37℃incubator, and the presence or absence of circular plaques at the site of the supernatant of the drop was observed the next day, and the results are shown in FIG. 1.

In the plaque assay, clear and transparent plaques were formed on the double-layered agar plates, indicating that the isolated phage had a lytic effect on the indicator bacteria. The single plaque is diluted by a multiple ratio and is plated with host bacteria, and circular, transparent and clear plaque with the diameter of about 1-2mm is formed on a double-layer agar plate (figure 1).

EXAMPLE 3 phage genomic DNA extraction and restriction enzyme assay

Referring to the molecular cloning Experimental guidelines, phage DNA extraction method to extract TC3 phage DNA, purified phage cultures were treated with DNaseI (100U/ml) and RNaseA (10. Mu.g/ml) at 37℃for 30 minutes to remove host bacterial nucleic acids; ethylenediamine tetraacetic acid (EDTA) (20 mM), sodium Dodecyl Sulfate (SDS) (0.5%), proteinase K (50. Mu.g/ml) and incubation at 56℃for 1 hour were then added to the mixture; an equal volume of phenol/chloroform (1:1) was then added to the mixture to extract the DNA. Phage DNA was precipitated with isopropanol (AR grade), the precipitate was washed with 70% ethanol, air dried and the genomic DNA was dissolved with deionized water.

The resulting phage genomic DNA was subjected to restriction digestion with several restriction enzymes: bamHI, draI, ecoRI, ecoRV, hindIII, ndeI, pstI, xbaI (NewEnglandBiolabs, USA). The digestion conditions were carried out according to the digestion instructions, and the digestion pattern was observed by a gel scanner after electrophoresis of the digested product in TAE running buffer. Lambda phage DNA digested with 1 kbDNAlader (FroggaBio, canada) and HindIII was used as molecular markers, and the results are shown in FIG. 2.

The restriction map was analyzed by a gel scanner after digesting the TC3 genomic DNA with 8 restriction endonucleases. The results showed that TC3 genomic DNA was digested with DraI, ecoRI, hindIII and NdeI, but not BamHI, ecoRV, pstI and XbalI. Agarose gel electrophoresis showed that the DraI, ecoRI, hindIII and NdeI enzymes cut the DNA into multiple DNA fragments (FIG. 2). Phage TC3 can be cleaved by the HindIII enzyme, which is a double-stranded DNase, thus indicating that the phage nucleic acid is double-stranded DNA, and when the restriction fragment sizes are summarized, the addition of the restriction fragments roughly estimates that the TC3 genome is about 48kb in length.

EXAMPLE 4 SDS-PAGE electrophoresis of phage structural proteins

Phage proteins include two broad classes of structural proteins and non-structural proteins. The nonstructural proteins are proteins encoded by phage genome and have a certain function in phage replication or gene expression regulation process, but are not bound in phage particles, including some regulatory proteins, auxiliary proteins and the like; structural proteins are proteins necessary for constructing a morphologically mature infectious phage particle, and include capsid proteins, tail proteins, and the like. The main functions of the structural protein are: (1) determining the appearance of phage and protecting the nucleic acid inside; (2) Participating in adsorption and invasion of phage, determining host specificity of phage; (3) surface antigens constituting phage. Separating the structural protein of phage TC3 by SDS-PAGE electrophoresis, and laying a foundation for subsequent bioinformatics research of the phage.

The purified TC3 phage proteins were subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant blue R-250, and the number and size of structural proteins were analyzed, and the results are shown in FIG. 3. As can be seen from FIG. 3, at least 10 protein bands appear on the gel within the standard range of medium molecular weight proteins, indicating that phage TC3 contains at least 10 structural proteins. Three major protein bands, seven minor protein bands, have molecular weights ranging from about 18 kDa to about 220kDa. Wherein the content of the structural protein corresponding to 45kD is maximum, which indicates that the protein occupies the largest copy number in the composition of TC3 particles and is the main structural protein. In general, however, the copy number of the phage capsid protein is highest, and thus the gene encoding the 45kD structural protein is presumed to be the major capsid protein of phage TC 3.

EXAMPLE 5 preparation and purification of recombinant protein of phage endolysin (designated TCLys 119)

Protein compositions include, but are not limited to, phage TC3 culture broth, phage TC3 extract, recombinant protein purified broth, and the like. The recombinant protein can also be prepared by a conventional technical means through a mode of introducing a gene sequence into engineering bacteria to ferment, separate and purify. The specific method for preparing the recombinant protein comprises the following steps:

the gene sequence of the recombinant protein is amplified by PCR, the sequence is cloned to a pET-28a vector by an enzyme digestion enzyme linkage method, an expression plasmid of the recombinant protein with an N-terminal fused 6 XHis tag is constructed, the escherichia coli engineering bacterium BL21 (DE 3) strain is utilized for carrying out heterologous expression, the strain is cultured on an LB+KanR resistant plate overnight, monoclonal antibodies are selected, transferred into a 5ml LB culture solution containing 100 mug/ml KanR, cultured for 4 hours at 37 ℃ and 180rpm, IPTG is added to a final concentration of 0.5-2 mM at 1:100 in an exponential metaphase (OD 600 = 0.6), and induction is carried out at 18 ℃ and 180rpm overnight. The bacterial liquid was centrifuged at 8,000rpm for 5 minutes to pellet cells, and the cells were stored at-20 ℃.

The thalli are evenly mixed in 10 times volume of ultrasonic lysate, 4ml of PMSF containing lysozyme, DNase/Rnase and final concentration of 1mM is added, evenly mixed, placed on ice for ultrasonic 90min for cracking and breaking until the solution is thoroughly clear, the cell lysate is centrifuged at 12,000rpm, 30min and 4 ℃, the supernatant is collected and placed on ice, the recombinant protein is obtained through Ni-16NTA His-Bind resin affinity chromatography purification, 209 amino acids are obtained, about 23KD is split-packed and stored at-80 ℃ for standby to prepare the proper concentration. SDS-PAGE patterns are shown in FIG. 4.

TCLys119 sequence (SEQ ID No. 1): MILLFKGVNAPKEVKQLQTLLKTLGYYSGRIDGVFGTGTEKAVIQYQKDNGLTPDGKVGKATAGRIGLHTGLEVSDEFVLNDASKKRLKGIHPDLVKVVERAIRISPIQFQVGEGLRTAERQKELMASGATQTLNSRHLTGHAVDLFAYPDGKLSWDWKYYYQIEEAVKQAAKELKVSIEWGGDWKSFKDGPHWQLPWNKYPK x

TCLys119 recombinant protein sequence (SEQ ID No. 2):

LVPRGSMILLFKGVNAPKEVKQLQTLLKTLGYYSGRIDGVFGTGTEKAVIQYQKDNGLTPDGKVGKATAGRIGLHTGLEVSDEFVLNDASKKRLKGIHPDLVKVVERAIRISPIQFQVGEGLRTAERQKELMASGATQTLNSRHLTGHAVDLFAYPDGKLSWDWKYYYQIEEAVKQAAKELKVSIEWGGDWKSFKDGPHWQLPWNKYPK*

EXAMPLE 6 antibacterial experiment of TCLys119 recombinant protein

Preparation of the culture medium: MH plates or MH broth manufactured by key trade company, jiang, are used.

Preparation of bacteria: the multi-drug resistant Acinetobacter baumannii Ab3 was subjected to bacteriostasis adjustment with physiological saline to obtain 0.5 Mitsubishi.

TCLys119 recombinant protein concentration gradient preparation: after purification, the original concentration of TCLys119 recombinant protein shown in SEQ ID No.2 was determined to be 48mg/ml, and TCLys119 recombinant protein was diluted with MH broth for double dilution according to the following table.

TABLE 2 double dilution of TCLys119 recombinant protein

Plate method: the bacterial solution of Ab3 with the turbidimetric end of 0.5 McAb is uniformly coated on a MH flat plate by a cotton swab, 10ul of TCLys119 recombinant proteins with different concentrations are added dropwise to the position marked by the corresponding concentration, and the antibacterial effect is observed after the culture in a 37-DEG incubator overnight, and the result is shown in FIG. 5.

Broth dilution method: the TCLys119 recombinant protein was diluted with MH broth to four concentrations of 12ug/ml,6ug/ml,3ug/ml, and 1.5ug/ml, respectively, and the recombinant protein without TCLys119 was used as a control, and then prepared bacterial solutions were added to each dilution well to give a final bacterial solution concentration of about 5X 109CFU/ml, and incubated in a 37℃incubator, and OD values at 595nm were measured per hour, and the results are shown in FIG. 6.

From the results shown in FIGS. 5 and 6, the TCLys119 recombinant protein has a good antibacterial effect on the multi-drug resistant Acinetobacter baumannii Ab3 at a protein concentration of 3ug/ml or above, which suggests that the protein can be used for inhibiting the multi-drug resistant Acinetobacter baumannii and treating diseases caused by infection of Acinetobacter baumannii.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. A TCLys119 lysosomal protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID No.1 or SEQ ID No. 2.

2. A polynucleotide encoding the protein of claim 1.

3. An expression vector comprising the polynucleic acid of claim 2.

4. A host cell comprising the expression vector of claim 3, or having integrated on its genome the polynucleotide of claim 2.

5. The host cell of claim 4, wherein the host cell is E.coli.

6. The method for producing a protein according to claim 1, comprising the steps of:

(1) Culturing the host cell of claim 4 under conditions suitable for production of the protein, thereby producing the protein of claim 1; and

(2) Isolating the protein produced from the culture of step (1).

7. Use of a protein according to claim 1, a polynucleotide according to claim 2, an expression vector according to claim 3 or a host cell according to claim 4 for the preparation of a pharmaceutical composition for the treatment of a disease or disorder caused by acinetobacter baumannii infection.

8. The use according to claim 7, wherein the acinetobacter baumannii is multi-drug resistant acinetobacter baumannii.

9. The use according to claim 7, wherein the acinetobacter baumanii is acinetobacter baumanii resistant to one or more antibiotics selected from the group consisting of: gentamicin, amoxicillin/clavulanic acid, aztreonam, ciprofloxacin, ceftriaxone, cefazolin, nitrofurantoin, imipenem, ampicillin, cefoxitin, levofloxacin, compound neotame, tobramycin, piperacillin/tazobactam, tigecycline, cefpiramide.

10. A pharmaceutical composition comprising the protein of claim 1, the polynucleotide of claim 2, the expression vector of claim 3 or the host cell of claim 4, and a pharmaceutically acceptable carrier and/or adjuvant.