CN108410840A - A kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application - Google Patents
A kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application Download PDFInfo
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Abstract
This hair discloses a kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application.The present invention provides it is following a) or b) shown in protein：A) protein that amino acid sequence forms shown in sequence in sequence table 1；B) amino acid sequence of sequence in sequence table 1 is passed through into the substitution of one or several amino acid residues and/or lacks and ors add and have the protein derived from sequence in sequence table 1 of cracking Gram-negative bacteria function.Protein provided by the invention can crack Escherichia coli, a variety of Gram-negative bacterias such as Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and salmonella typhimurium, the albumen acts synergistically with surfactant EDTA, and stronger inhibiting effect is generated to Gram-negative bacteria.Antibiotic preparation to research and develop new is laid a good foundation.
Description
Technical field
The invention belongs to biotechnology, it is related to a kind of Pseudomonas aeruginosa phage endolysin and its encoding gene
With application.
Background technology
In recent years, there is drug resistance in many bacteriums, or even multi-drug resistant bacteria occur.Multi-drug resistant bacteria is to people's
Health brings serious threat.Therefore, the task of top priority is a kind of novel antibiotic preparation of searching to cope with the prestige of drug-fast bacteria
The side of body.Bacteriophage has many advantages, such as specific high, without side-effects to human body paid attention to by scientist as a kind of bacterial virus.It bites
Thalline be it is a kind of can bacterial infection, fungi, actinomyces and conveyor screw virus, content is about 10 in nature31, or
It may be said that there are about 10 in environment7Bacteriophage/cm3.Bacteriophage can be divided into lysogenic phage and lytic phage.Lysogeny
Bacteriophage combines the genome of oneself with the genome of host strain and coexists；And lytic phage is that oneself genome is noted
It is replicated after entering host strain, be assembled into progeny phage and then crack host strain, discharge progeny phage.According to cracking performance phagocytosis
This characteristic of body, can be using bacteriophage as the treatment-related bacterium infection of antibiotic preparation.Some countries of Eastern Europe are sharp already
The bacterium infection of the mankind is treated with lytic phage.With the appearance of bacterial drug resistance in global range, people more pay attention to
The antibacterial characteristics of bacteriophage, correlative study are related to people doctor and veterinary clinic and the fields such as food security and the depollution of environment.
Bacteriophage will produce phage resistant strains, and biology as an independent life entity when as antibiotic preparation
Safety is constantly subjected to query.Bacteriophage endolysin (bacteriophage endolysins) is that dsDNA bacteriophages are infecting
A kind of hydrolase of bacterium later stage experssion.Researcher thinks that bacteriophage endolysin has the following advantages：1 high specificity is being killed
Normal flora will not be interfered while pathogen.2 are not easy to make bacterium to generate drug resistance, due to endolysin be bacteriophage with it is thin
It is generated during bacterium common evolutionary, highly conserved peptide glycan on directed toward bacteria cell wall, so generating the several of drug resistance
Rate very little.3 can kill in mucomembranous surface field planting pathogen.Endolysin may produce in vivo as a kind of protein macromolecule
Raw corresponding antibody neutralizes its sterilizing ability, but some experiments find that its antibody does not have neutralization to it.It is molten in bacteriophage
Element is not likely to produce drug resistance compared with bacteriophage, and Idelevich etc. has found chimaeric enzyme PRF-119 to the golden yellow of phage resistance
Color staphylococcus mutant strain, there is presently no the reports of relevant endolysin resistance.Endolysin clinical application safety and
The experiment of pharmacokinetic properties is prepared for, and if endolysin P128 comes into clinical II phase experiment, tests it to mankind's nose
The fungicidal effectiveness for the staphylococcus aureus that chamber carries.And endolysin as a kind of protein physicochemical property research very
Maturation can carry out modification transformation to it, so bacteriophage endolysin has the potentiality of novel antibacterial preparation.Recent study
Some animal models that person establishes also demonstrate the antibacterial activity of endolysin well.
Invention content
The object of the present invention is to provide a kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and applications.It should
Endolysin can degrade the protoplast of a variety of Gram-negatives (G-) bacterium, have synergistic effect with EDTA, enhance to G- bacterium
Inhibit.
Pseudomonas aeruginosa phage endolysin provided by the invention, entitled Lysin-G78 are derived from P. aeruginosa
Bacterium bacteriophage (Pseudomonas aeruginosa) vB_PaeM_G1 is following (a) or protein (b):
(a) protein that amino acid sequence forms shown in sequence in sequence table 1；
(b) amino acid sequence of sequence in sequence table 1 by the substitution of one or several amino acid residues and/or is lacked
It loses and/or adds and with the protein derived from (a) of cracking G- bacterium functions.
Wherein, sequence 1 is made of 187 amino acid in sequence table.
201710089442.1 application for a patent for invention《A kind of Pseudomonas aeruginosa phage and its application》Describe one
Kind bacteriophage vB_PaeM_QKL1, bacteriophage of the present invention can be selected the invention and be related to thalline.
For the ease of the purifying of Lysin-G78 albumen, can in sequence table the amino acid residue sequence composition of sequence 1
The upper label as shown in the table of amino terminal or carboxyl terminal connection of protein.
Table：The sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (b) can be artificial synthesized, also can first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of protein in above-mentioned (b) can will lack one in DNA sequence dna shown in sequence in sequence table 2
Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs.
The nucleic acid molecules for encoding the Lysin-G78 albumen also belong to protection scope of the present invention.
The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
It is RNA, such as mRNA, hnRNA or tRNA.
In one embodiment of the present of invention, the nucleic acid molecules are specially the gene for encoding the Lysin-G78 albumen
(being named as Lysin-G78), the Lysin-G78 genes be it is following it is any shown in DNA molecular：
1) DNA molecular shown in sequence 2 in sequence table；
2) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with the sequence 1) limited
The DNA molecular of homology and code for said proteins Lysin-G78.
Wherein, sequence 2 is made of 564 nucleotide, the as ORF of entire sequence 2, egg shown in polynucleotide sequence 1
White matter.
Recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecules also belong to protection scope of the present invention.
The recombinant vector can be recombinant expression carrier or recombinant cloning vector.
In one embodiment of the invention, it is inserted into institute between multiple cloning sites of the recombinant vector for pET-28a (+)
State the recombinant plasmid that gene obtains.
The expression cassette is by that can start the promoter of the gene expression, the gene and transcription terminator group
At.
In one embodiment of the invention, the recombinant bacterium is the Escherichia coli containing the recombinant vector；It is described big
Enterobacteria is specifically such as BL21 (DE3).
Application of the protein in inhibiting G- bacterium also belongs to protection scope of the present invention.
Application of the nucleic acid in inhibiting G- bacterium also belongs to protection scope of the present invention.
Application of recombinant vector, expression cassette or the recombinant bacterium of the nucleic acid molecules in inhibiting G- bacterium also belongs to the present invention
Protection domain.
Application of the protein in following (c1) or (c2) or (c1), (c2) combination also belongs to the protection of the present invention
Range：
(c1) cracking Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse are prepared
The product of salmonella typhi；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, onion Burkholder
The product of disease caused by bacterium and Salmonella Typhimurium Infection.
Application of the nucleic acid molecules in following (c1) or (c2) or (c1), (c2) combination also belongs to the guarantor of the present invention
Protect range：
(c1) cracking Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse are prepared
The product of salmonella typhi；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, onion Burkholder
The product of disease caused by bacterium and Salmonella Typhimurium Infection.
Recombinant vector, expression cassette or the recombinant bacterium of the nucleic acid molecules are in following (c1) or (c2) or (c1), (c2) combination
In application also belong to protection scope of the present invention：
(c1) cracking Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse are prepared
The product of salmonella typhi；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, onion Burkholder
The product of disease caused by bacterium and Salmonella Typhimurium Infection.
The method provided by the present invention for preparing Pseudomonas aeruginosa phage endolysin Lysin-G78, specific steps are such as
Under：
S1 designs specific primer according to sequence in sequence table 2, expands from Pseudomonas aeruginosa phage genomic DNA
Increase artificial Pseudomonas aeruginosa phage endolysin gene；
S2 builds the recombinant expression carrier of artificial Pseudomonas aeruginosa phage endolysin；
S3 converts recombinant expression carrier to competent escherichia coli cell, and screening obtains expressing artificial P. aeruginosa
The engineering bacteria of bacterium bacteriophage endolysin；
S4 induces Pseudomonas aeruginosa phage endolysin engineering bacteria using isopropyl-beta D-thio galactopyranoside
Expression obtains expression product；
S5 is by recombinant gene expression product, through affinity chromatography purifies and separates, the artificial P. aeruginosa that is recombinated
Bacterium bacteriophage endolysin.
Specifically, primer described in step S1 is：
Sense primer:5’-GCGGATCCATGATCACCGACAGAGAGTATCAG-3 ', wherein dashed part BamH
The restriction enzyme site of I
Downstream primer:5’-GCCTCGAGTCAGCCACTAGCTTCAGCATA-3 ', wherein dashed part is Xho I's
Restriction enzyme site.
The advantages and positive effects of the present invention are：
1 present invention isolates a Pseudomonas aeruginosa strain bacteriophage vB_PaeM_G1, and therefrom clone obtains endolysin
The encoding gene of Lysin-G78.Endolysin Lysin-G78 is produced using engineered strain, which passes through hydrolytic bacteria cell wall
The glycosidic bond between N-Acetyl-D-glucosamine and -acetylmuramic acid on peptide glycan finally makes host cell lysis, the albumen
With the advantages that good water solubility, activity is high, and antimicrobial spectrum is wide.
The 2 artificial endolysins of Pseudomonas aeruginosa phage provided by the invention can inhibit Escherichia coli, kerekou pneumonia
The growth of primary bacterium, pseudomonas aeruginosa, Burkholderia cepacia and salmonella typhimurium, this artificial pseudomonas aeruginosa
Bacteriophage endolysin has synergistic effect with surfactant EDTA, can improve the bactericidal activity to G- bacterium, prepare novel kill
Microbial inoculum.
Description of the drawings：
Fig. 1 is the plaque morphology of Pseudomonas aeruginosa phage vB_PaeM_G1；
Fig. 2 is the transmission electron microscope picture of Pseudomonas aeruginosa phage vB_PaeM_G1；
Fig. 3 is the PCR identifications for expressing bacterium BL21-G78 monoclonal colonies；
Fig. 4 is the SDS-PAGE qualification results of Lysin-G78 endolysins；
Fig. 5 is the lytic activity and fragmentation pattern of endolysin Lysin-G78；
Fig. 6 is the synergistic effect of endolysin Lysin-G78 and EDTA.
Specific implementation mode
The present invention is described in further detail below by specific implementation mode, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method used in the following example is conventional method unless otherwise specified.
Material used in the following example, reagent etc., are commercially available unless otherwise specified.
Embodiment 1：The separation and identification of bacteriophage
One, the separation of bacteriophage
CaCl is added in sewage 1L before taking hospital to handle2To 1mmol/L, 4 DEG C, 5000rpm centrifuges 10min, goes to decontaminate
Precipitation particle in water takes supernatant with 0.22 μm of filter membrane filtration sterilization；It takes filtrate 20mL to be mixed with 20mL 2 × LB culture mediums, presses
1% inoculum concentration is inoculated with the exponential phase pseudomonas aeruginosa of 400 μ L, 37 DEG C of enrichment culture 12h.Take the above-mentioned bacterium of 5ml
4 DEG C of liquid, 5000rpm centrifuge 10min, take 0.22 μm of filter membrane filtration sterilization of supernatant, obtain bacteriophage stoste.It is bitten what is obtained
10 times of gradient dilutions (10 of thalline stoste-1-10-8), the pseudomonas aeruginosa of the dilution and exponential phase of 300 μ L is taken respectively
1:1 mixing, 37 DEG C of culture 15min, mixes with 55 DEG C of LB liquid mediums of 4mL, is uniformly poured on solid LB agar tablet, cold
But 15min is inverted 37 DEG C and is incubated overnight.Next day obtains single plaque.Pipette tips picking is used on the tablet of Plaques assay
Larger, independent plaque is put into 1mL SM liquid, and (50mL 1M are added in gelatin 0.1g, MgSO47H2O 2g, NaCl 5.8g
Tris-HCl (pH 7.5), adds water to 1000mL) EP pipes in, be placed at room temperature for after 1h 4 DEG C overnight, next day takes 0.1mL by suitable
(10 after dilution-1-10-8, totally 8 gradients), the pseudomonas aeruginosa 1 with culture to exponential phase:1 mixing is done double-deck flat
Plate is purified, and next day obtains plaque of uniform size.After obtaining evenly sized plaque, this bacteriophage is cooked again
Double-layer plate is tested, and cultivates the form for 24 hours with observation plaque after 48h, plaque morphology such as Fig. 1 under the conditions of 37 DEG C respectively.
It was found that there is the translucent halo of a circle outside the transparent region of plaque, illustrate there is extracellular polymeric in the bacteriophage anal spine
Degrading enzyme, the enzyme energy degradation biological envelope extracellular polymeric.Bacteriophage diffuses out the clear area of plaque increase with time
And the bacterium around cracking, so halo can increase as time increases.It purified is bitten with sterilizing pipette tips picking is above-mentioned
Bacterial plaque is added in culture to the pseudomonas aeruginosa of exponential phase, 37 DEG C, and 140rpm cultivates 6h or so, the phase that culture solution becomes
To clarifications, 4 DEG C of culture solution, 8000rpm are centrifuged into 5min, take 0.22 μm of membrane filtration of supernatant, remove a small amount of bacterium and
Bacterial debris to get high-titer bacteriophage stoste.
Two, the identification of bacteriophage
Specific method is：The copper mesh of 350 mesh is immersed in bacteriophage refined solution, copper mesh is taken out after 10min, is inhaled with filter paper
Take extra liquid；Copper mesh is placed on the uranyl acetate drop of 5% (w/v), after dyeing take out be placed on it is to be measured on filter paper；It waits for
After whole samples are handled well, the tem observation morphology of phages that accelerating potential is 80kV is used.Electron microscope, which is shown, belongs to flesh tail disease
Malicious section (Myoviridae), head diameter are about 50nm, and tail length is about 65nm.The morphology of phages such as Fig. 2.
Embodiment 2：The extraction of phage genome
One, the concentration of phage particle
Pseudomonas aeruginosa overnight culture is transferred in 100mL LB liquid mediums, inoculum concentration 1%, amplification training
It supports to logarithmic phase (OD600 about 0.4), 5mL Pseudomonas aeruginosa phage culture solutions is added, after 37 DEG C of shaken cultivation 4-6h
To phage splitting liquid.DNase I and RNase A to final concentration of 5 μ g/mL are added into lysate, is stood for 37 DEG C after mixing
1h.NaCl to final concentration of 0.1mol/L is then added, ice bath 1h, 12000rpm centrifuge 20min after mixing dissolving.By supernatant
After liquid is gone in another centrifuge tube, PEG8000 to final concentration of 10% (w/v) is added, fully in 4 DEG C of standings after oscillation dissolving
Overnight, 12000rpm centrifuges 20min, abandons supernatant.With 500 μ L TM (0.05mol/L Tris-HCl pH7.5,0.2%
MgSO47H2O) precipitation is resuspended solution, is used in combination isometric chloroform primary, and 12000rpm centrifuges 10min, to remove
PEG8000 in re-suspension liquid finally obtains the crude extract of phage particle.
Two, the preparation of phage genome DNA
DNase I and RNase A, final concentration of 1 μ g/mL, 37 DEG C of standings are added in S1 in phage particle crude extract
1h, with the remaining host strain DNA or RNA that degrades.
Then 1mol/L EDTA (pH8.0) to final concentration 50mmol/L are added in S2, terminate DNase I and RNase A and live
Property；
S3 adds Proteinase K to final concentration of 50 μ g/mL, and SDS to final concentration of 0.5%, mixing, 56 DEG C of lh is added to digest egg
White matter；
Isometric phenol is added in S4：Chloroform：Isoamyl alcohol (25: 24: 1) mixing, 12000rpm centrifuges 10min, in collection
Clearly；
S5 steps 4 are repeated 3 times；
Isometric chloroform, mixing is added in S6, and 12000rpm, 10min collect supernatant；
95% ethyl alcohol that 1/10 volume 3mol/L NaAc are added in S7 and 2 times of volumes are pre-chilled, mixing, 12000rpm,
10min precipitates DNA；Add 70% ethyl alcohol (500 μ L) in precipitation, and the centrifuge tube covered tightly is reverse for several times, 12000rpm from
Heart 5min recycles DNA.
S8 removes supernatant, removes the alcohol drop on tube wall, and the centrifuge tube drying at room temperature 10min of opening is then used double
It steams water and DNA is resuspended.
Embodiment 3：The clone of endolysin gene Lysin-G78, the structure of expression vector
One, the acquisition of target fragment
For S1 according to sequence design a pair of specific primer of Lysin-G78 (sequence 2) gene code, primer sequence is as follows：
Sense primer：5’-GCGGATCCATGATCACCGACAGAGAGTATCAG-3 ', wherein dashed part BamH
The restriction enzyme site of I
Downstream primer：5’-GCCTCGAGTCAGCCACTAGCTTCAGCATA-3 ', wherein dashed part is Xho I's
Restriction enzyme site
Reflection system is：
PCR reaction conditions：95 DEG C of pre-degeneration 5min, (94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1.5min)
30 cycles of amplification；72 DEG C of extension 10min, 4 DEG C of preservations.Pcr amplification product has been seen whether through 1% agarose gel electrophoresis
Specific band.
Using phage genome DNA as the above-mentioned primer amplification Lysin-G78 genes of template, 1% agarose electrophoresis, mirror
Determine the size of amplified fragments.
PCR product is directly carried out double enzymes by S2 with QuickCut restriction enzymes (Takara) BamH I and Xho I
Cut, 37 DEG C of water-bath 5min, 1% agarose electrophoresis, plastic recovery kit recycles fragment purification, and by segment with use before
Connection, next day convert to large intestine bar overnight in 16 DEG C of T4 ligases for BamHI and XhoI double digestions and the pET-28a carriers of purifying
In bacterium DH5 ɑ competent cells.LB culture mediums, 37 DEG C of incubation 45min are added in the mixture of conversion.Mixture is coated with later
In the LB tablets containing kanamycins (50 μ g/ml), 37 DEG C of overnight incubations；
The identification of S3 positive colony bacteriums：Picking positive colony bacterium is inoculated in the training of the LB liquid containing kanamycins (50 μ g/mL)
It supports in base, after 37 DEG C of overnight incubations, kit extracts plasmid, serves the raw work sequencing in sea.Correct plasmid will be sequenced to be named as
pET-28a-G78。
S4 will be sequenced correct plasmid pET-28a-G78 and be transformed into expression strain Escherichia coli BL21 (DE3) competence.
Mixture is coated on the LB tablets containing kanamycins (50 μ g/ml), 37 DEG C of overnight incubations later；Bacterium is done with above-mentioned primer
Fall PCR identifications.Qualification result such as Fig. 3, the segment amplified is between 750bp and 500bp, size and target fragment size one
It causes.Expression bacterium containing positive recombinant plasmid is named as BL21-G78.
Embodiment 4：The induced expression of endolysin gene Lysin-G78 and purifying
Expression bacterium BL21-G78 single bacterium colonies containing recombinant plasmid are inoculated into the LB containing kanamycins (50 μ g/mL)
In culture solution, 37 DEG C of shaken overnight cultures；Next day is forwarded in 1: 100 ratio in 100mL LB culture mediums, 37 DEG C of oscillation trainings
It supports when being about 0.6 to OD600 values, IPTG (isopropylthiogalactoside) is added to final concentration 0.5mmol/L, 16 DEG C lure
16h.Thalline is collected, ultrasonic disruption cell (130W, super 3s stop 10s, ultrasonic 5min), 4 DEG C, 10000rpm centrifuges 10min,
Supernatant is collected, and by supernatant through 0.22 μm of membrane filtration, SDS-PAGE analyzes the protein expression situation in supernatant.By filtering
Supernatant is purified with His affinity chromatography nickel column (GE Healthcare, Sweden), is specifically illustrated that step carries out by kit, is obtained
It obtains albumen and is named as lyases Lysin-G78.
SDS-PAGE analysis results as shown in figure 4, the engineering bacteria BL21-G78 containing recombinant plasmid after IPTG is induced,
There are induced protein bands in the position of about 21kD in its supernatant, is consistent with expected 20.8KD, to show recombinant bacterium BL21-
G78 structures are correct, and the endolysin Lysin-G78 expressed is soluble protein
Embodiment 5：Endolysin Lysin-G78 cracks outer membrane permeability bacterial cell
S1 permeability bacterial cells
Permeabilized cells refer to changing in the case where not causing cell cracking and not destroying cell interior organic structure
Become cell wall and membrane passage so that the cell that small molecule and some bigger molecule substances can free in and out.
It is prepared as follows Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia, mouse
The permeability bacterial cell of salmonella typhi, staphylococcus aureus and staphylococcus epidermis.
The bacterial strain being incubated overnight is transferred in 250mL LB culture mediums, culture to OD600=0.6,4 DEG C of culture, and 4000
× g centrifuges 15min, collects thalline.Thalline is resuspended using the 0.05mol/L Tris buffer solutions that chloroform is saturated, at room temperature softly
Vibrate 45min.4000 × g, centrifuges 15min by 4 DEG C, abandons supernatant and collects sediment.Then use the phosphate of 10mmol/L slow
Fliud flushing is washed and is resuspended, and finally permeability bacterial cell is resuspended to OD600=0.6-1.0.
The cracking of S2 permeability bacterial cells
The S1 7 kinds of permeability bacterial cells prepared are cracked as follows.
The 30 μ l endolysins (final concentration of 2 μ g/mL) of final concentration of 2 μ g/mL and 170 μ l permeability bacterial cells (OD=
0.8) it is mixed to join in 96 orifice plates, at ambient temperature, the absorbance in each sample is measured using all-wave length microplate reader
OD600, and compared using 10mmol/L phosphate buffers.Absorbance OD600 values are significantly reduced than negative control value, are said
Detailed bacterium is cleaved.
As a result：Endolysin Lysin-G78 can under the concentration conditions of 2 μ g/ml efficient degradation Escherichia coli, pneumonia gram
The permeability bacterial cell of the primary bacterium of thunder, pseudomonas aeruginosa, Burkholderia cepacia and salmonella typhimurium.Such as Fig. 5 institutes
Show, OD600 values when acting on 2h from endolysin Lysin-G78 and protoplast judge, the G- of endolysin Lysin-G78 effects
Bacterium OD600 is significantly reduced, and endolysin Lysin-G78 is to G+ bacterium, such as staphylococcus aureus permeability bacterial cell and epidermis
Staphylococcus permeability bacterial cell is without splitting action.
The synergistic effect of embodiment 6 endolysin Lysin-G78 and surfactant EDTA
The bacterium solution being activated overnight is transferred to according to 1% inoculum concentration in 100ml LB culture mediums, culture to OD600=
0.6；100ul bacterium solutions are added in the semisolid LB (8% agar) of 50 DEG C of 10ml, sterile petri dish is then added together
In, 15 minutes are stood in super workbench, (diameter about 7mm) is punched with the sterile pipette tips of 200ul, it is spare.
Make negative control group with 0.9% physiological saline, EDTA, endolysin Lysin-G78, endolysin Lysin-G78 and
EDTA use in conjunction is as experimental group.It is added in 10ul samples to be tested to the hole of culture dish, 37 DEG C are incubated overnight, and observation is antibacterial
Loop diameter size.
The results are shown in Figure 6, in experiment, the final concentration of 2 μ g/ml of EDTA final concentration of 2.5uM, endolysin Lysin-G78
When have inhibition zone, and bacteriostasis is stronger (antibacterial circle diameter becomes larger) when the two use in conjunction.Illustrate surfactant EDTA with
Endolysin Lysin-G78 has synergistic effect, can further improve the bacteriostasis of Lysin-G78, is endolysin Lysin-G78
Application effective reference data is provided.
SEQUENCE LISTING
<110>Dalian University of Technology
<120>A kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application
<130> ZR181046LQ
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 187
<212> PRT
<213>Pseudomonas aeruginosa phage (Pseudomonas aeruginosa)
<400> 1
Met Ile Thr Asp Arg Glu Tyr Gln Gln Ala Ala Glu Met Leu Gly Val
1 5 10 15
Asp Val Pro Ala Ile Lys Ala Val Thr Lys Val Glu Ala Pro Val Gly
20 25 30
Gly Phe Gln Pro Thr Gly Glu Pro Thr Ile Leu Tyr Glu Arg His Gln
35 40 45
Met Tyr Arg Gln Leu Gln Ala Lys Gly Leu Pro Thr Glu Gly His Pro
50 55 60
Pro Asp Leu Val Asn Lys Val Ala Gly Gly Tyr Gly Lys Tyr Ser Glu
65 70 75 80
Gln His Ala Lys Leu Ala Arg Ala Val Lys Ile Asp Arg Asp Ser Ala
85 90 95
Leu Glu Ser Cys Ser Trp Gly Met Phe Gln Ile Met Gly Tyr His Trp
100 105 110
Lys Leu Met Gly Tyr Pro Thr Leu Gln Ala Phe Val Asn Ala Met Tyr
115 120 125
Ala Ser Glu Gly Ala Gln Met Asp Ala Phe Cys Arg Phe Ile Lys Ala
130 135 140
Gln Pro Thr Thr His Ala Ala Leu Lys Ala His Asp Trp Ala Lys Phe
145 150 155 160
Ala Arg Leu Tyr Asn Gly Pro Gly Tyr Ala Lys Asn Lys Tyr Asp Val
165 170 175
Lys Leu Glu Lys Ala Tyr Ala Glu Ala Ser Gly
180 185
<210> 2
<211> 564
<212> DNA
<213>Pseudomonas aeruginosa phage (Pseudomonas aeruginosa)
<400> 2
atgatcaccg acagagagta tcagcaagct gctgagatgt tgggggtaga tgtcccagcg 60
atcaaggcag tgaccaaggt ggaggccccg gtagggggct tccagcctac aggagagcca 120
acgatcctct acgagcgtca ccagatgtac cgacagctcc aggccaaagg gctcccaacg 180
gaaggtcatc ccccagacct ggtaaataag gtagctggtg ggtatggaaa atacagcgag 240
caacacgcta aactggcccg cgccgtaaag atcgacaggg acagcgccct ggagtcctgc 300
tcctggggga tgttccagat catgggctac cactggaagc tgatggggta ccctaccctt 360
caagctttcg taaacgccat gtacgccagc gaaggagccc agatggacgc cttctgccgg 420
ttcatcaagg cacaacccac cacgcatgct gccttgaaag cccatgattg ggccaagttt 480
gccagactgt acaacggtcc aggctacgcc aagaacaagt atgacgtgaa attggagaaa 540
gcatatgctg aagctagtgg ctga 564
Claims (14)
1. a kind of Pseudomonas aeruginosa phage endolysin, it is characterised in that：It is following (a) or protein (b):
(a) protein that amino acid sequence forms shown in sequence in sequence table 1；
(b) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or add and have the protein derived from (a) of cracking Gram-negative bacteria function.
2. encoding the nucleic acid molecules of protein described in claim 1.
3. nucleic acid molecules according to claim 2, it is characterised in that：The nucleic acid molecules are described in coding claim 1
The gene of protein, the gene be it is following it is any shown in DNA molecular：
1) DNA molecular shown in sequence 2 in sequence table；
2) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the sequence 1) limited
And encode the DNA molecular of protein described in claim 1.
4. according to claim 2, which is characterized in that the nucleic acid molecules are DNA or RNA.
5. recombinant vector, expression cassette or recombinant bacterium containing Claims 2 or 3 or 4 nucleic acid molecules.
6. according to claim 5, which is characterized in that the recombinant vector can be that recombinant expression carrier or recombinant clone carry
Body.
7. the preparation method containing Pseudomonas aeruginosa phage endolysin described in claim 1, which is characterized in that including as follows
Step：
S1 designs specific primer according to sequence in sequence table 2, is expanded from Pseudomonas aeruginosa phage genomic DNA artificial
Pseudomonas aeruginosa phage endolysin gene；
S2 builds the recombinant expression carrier of artificial Pseudomonas aeruginosa phage endolysin；
S3 converts recombinant expression carrier to competent escherichia coli cell, and screening obtains expressing artificial pseudomonas aeruginosa phagocytosis
The engineering bacteria of internal lysin；
S4 induces Pseudomonas aeruginosa phage endolysin engineering bacterium expression using isopropyl-beta D-thio galactopyranoside,
Obtain expression product；
S5 is by recombinant gene expression product, and through affinity chromatography purifies and separates, the artificial pseudomonas aeruginosa recombinated bites
Thalline endolysin.
8. the preparation method of Pseudomonas aeruginosa phage endolysin according to claim 7, which is characterized in that step S1 institutes
Stating primer is：
Sense primer:5’-GCGGATCCATGATCACCGACAGAGAGTATCAG-3’
Downstream primer:5’-GCCTCGAGTCAGCCACTAGCTTCAGCATA-3’.
9. application of the protein described in claim 1 in inhibiting Gram-negative bacteria.
10. application of the nucleic acid molecules according to claim 2 or 3 in inhibiting Gram-negative bacteria.
11. the recombinant vector of nucleic acid molecules described in claim 5, expression cassette or recombinant bacterium answering in inhibiting Gram-negative bacteria
With.
12. application of the protein described in claim 1 in following (c1) or (c2) or (c1), (c2) combination：
(c1) cracking Escherichia coli are prepared, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse typhus are husky
The product of door Salmonella；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse
The product of disease caused by salmonella typhi infection.
13. application of the nucleic acid molecules in following (c1) or (c2) or (c1), (c2) combination described in claim 3 or 4：
(c1) cracking Escherichia coli are prepared, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse typhus are husky
The product of door Salmonella；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse
The product of disease caused by salmonella typhi infection.
14. the recombinant vector of nucleic acid molecules described in claim 5, expression cassette or recombinant bacterium following (c1) or (c2) or (c1),
(c2) application in combining：
(c1) cracking Escherichia coli are prepared, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse typhus are husky
The product of door Salmonella；
(c2) it prepares for treating by Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa, Burkholderia cepacia and mouse
The product of disease caused by salmonella typhi infection.
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| CN114807104A (en) * | 2022-04-11 | 2022-07-29 | 西南大学 | Klebsiella pneumoniae phage lyase and preparation method and application thereof |
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