CN109666667A - The chimaeric enzyme antibiotic of killing staphylococcus aureus and its preparation and application - Google Patents
The chimaeric enzyme antibiotic of killing staphylococcus aureus and its preparation and application Download PDFInfo
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- CN109666667A CN109666667A CN201910060337.4A CN201910060337A CN109666667A CN 109666667 A CN109666667 A CN 109666667A CN 201910060337 A CN201910060337 A CN 201910060337A CN 109666667 A CN109666667 A CN 109666667A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Abstract
The present invention provides a kind of chimaeric enzyme antibiotic for killing staphylococcus aureus and its preparations and application.The chimaeric enzyme antibiotic includes cytoplasm transduction peptide CTP and the concatenated recombinant protein c TP-JDlys of staphylococcus aureus bacteriophage lyases JDlys.The preparation method of the chimaeric enzyme antibiotic includes: S1, design primer, respectively using lyases JDlys gene and cytoplasm transduction peptide CTP gene as template, expands JDlys genetic fragment and CTP genetic fragment；The CTP gene that amplification obtains, is inserted into the end 5` of JDlys gene in the recombinant plasmid by S2, the gene constructed recombinant plasmid of JDlys for obtaining amplification, constructs the recombinant plasmid of the CTP-JDlys of segment containing target gene；S3, the recombinant plasmid of the CTP-JDlys of segment containing target gene is expressed, after purification, obtains chimaeric enzyme avidin CTP-JDlys.CTP-JDlys albumen provided by the invention, which can enter, efficiently treats mouse skin abscess caused by resistant Staphylococcus aureus in skin keratinocytes.
Description
Technical field
The invention belongs to field of biotechnology, are the preparation and its treatment of a kind of novel enzyme antibiotic of field of biotechnology
The application of bacterium infection in epidermal keratinocyte, and in particular to it is golden yellow that one kind can efficiently enter killing drug resistance inside eukaryocyte
The biosynthesis of the staphylococcic Novel chimeric enzyme antibiotic of color and intracellular and dermapostasis bactericidal effect assessment；More specifically
Ground is related to a kind of chimaeric enzyme antibiotic for killing staphylococcus aureus and its preparation and application.
Background technique
Skin and skin structure infections (skin and skin structure infections, SSSIs) are by all kinds of
Diseases associated with inflammation caused by suppurative pathogenic bacteria infringement epidermis, corium and subcutaneous tissue.The pathogen of SSSIs is caused to have very much,
It mainly include staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa etc..Wherein, staphylococcus aureus is to draw
Play the most common pathogen of SSSIs.Skin keratinocytes are most important cells in skin epidermis, play an active part in and coordinate skin
Innate immune response.It has recently been demonstrated that horn cell can play similar early stage infection of staphylococcus aureus
The function of immunocyte identifies and transmits cause of disease signal, and can generate antibacterial peptide and pro-inflammatory cytokine resist it is golden yellow
The staphylococcic infection of color.Research shows that staphylococcus aureus can invade horn cell and continued survival in it, thus
Above-mentioned a series of immune response is escaped, the persistent infection of skin is caused.However, most of antibacterials are to Staphylococcus aureus
Microbial skin infection is helpless.For example, antibiotic is difficult freely to enter eukaryocyte, cell can not be effectively killed
Interior bacterium；And small molecule antibacterial peptide antimicrobial spectrum is wider, so as to cause the destruction of microecological balance in skin.In addition, in recent years,
Due to the excessive use of antibiotic, there is serious drug resistance situation (such as resistance to methoxy in the staphylococcus aureus being clinically separated
The separation of XiLin staphylococcus (MRSA)), this proposes the treatment of skin infection caused by staphylococcus aureus severeer
Challenge, therefore seek a kind of efficiently not drug resistant novel antibacterial preparation for resisting intracellular infection and be particularly important.
Bacteriophage is a kind of virus that can infect and crack bacterium, powerful to split after bacterium effect mainly has benefited from infection
The cytohydrolist of phase synthesis, the enzyme can be reached and quickly be split by the glycosidic bond between specific for hydrolysis cell wall amino sugar
Solve the purpose of bacterium, therefore also referred to as enzyme antibiotic or lyases (Lysin).Compared with antibiotic, enzyme antibiotic activity is strong, makees
With rapid, and be not easy Induction of bacterial and generate drug resistance, adverse effect will not be generated to humans and animals, be solve it is now increasingly tight
A kind of process for selective of the bacterial drug resistance of weight.As a kind of high molecular weight protein enzyme, which can not be freely accessible to
Inside eukaryocyte, therefore good therapeutic effect is had no to intracellular infection caused by bacterium.
Summary of the invention
It is an object of the invention to overcome the shortcomings of that existing antibiotic preparation treats intracellular bacterial infections, providing one kind can
Into the technical method of the enzyme antibiotic inside eukaryocyte.
The Intracellular delivery that cytoplasm transduction peptide (Cytoplasmic transduction peptide, CTPs) mediates is that have
Effect mediation foreign protein enters the novel strategy of eukaryocyte.CTPs is a kind of small peptide rich in cation, can successfully be transported
Functional protein enters cell interior.CTPs is many kinds of, and there is no be elaborated the mechanism of action of major part CTPs.Together
When, CTPs also differs greatly to the degree of injury and toxicity of cell because of cell category difference.In addition, CTPs and destination protein
There is also many technological difficulties, such as CTPs and destination protein series position and series system for expressing in series, it is possible to influence string
Join the space structure and biological activity of albumen.Therefore, selecting one from a variety of CTPs is suitble to cytoplasm of the invention to transduce
Peptide, and the optimal connection type for screening CTPs and series protein is technological difficulties of the invention.The present invention is by three kinds of CTP eggs
White (Tat type CTP: from 11 amino acid residues of inhibition of HIV；Ant type CTP: the feeler foot peptide fragment from drosophila is residual
Base；TP10 type CTP: the relevant neuropeptide galanin residue of wasp venom is derived from.) it is connected serially to the N of enzyme avidin respectively
End and C-terminal pass through enzymatic activity experiment, sterilization experiment intracellular and cytotoxicity experiment comprehensive assessment three kinds of CTP series proteins
Outer bactericidal effect intracellular finally found that wherein Tat type CTP (CTPTat) there is low cytotoxicity, after connecting with enzyme antibiotic not
It will affect its bactericidal activity, while there is the bactericidal effect intracellular better than conventional antibiotic, obtain a kind of efficiently into eukaryon
The Novel chimeric enzyme antibiotic of cell interior killing resistant Staphylococcus aureus.
The gene and CTP Tandem gene expression that the present invention encodes enzyme antibiotic.With the help of CTP, enzyme antibiotic into
Enter and efficiently kills resistant Staphylococcus aureus intracellular in epidermal keratinocyte.Furthermore by establishing mouse skin abscess sense
Dye-treatment model, the bactericidal effect of assessment CTP mediate cytolysis enzyme in animal body.
The purpose of the present invention is achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of chimaeric enzyme antibiotic for killing staphylococcus aureus, the chimaeric enzyme is anti-
Raw element includes the cytoplasm transduction peptide CTP and concatenated recombinant protein c TP- of staphylococcus aureus bacteriophage lyases JDlys
JDlys。
Preferably, the cytoplasm transduction peptide CTP is Tat type CTP, Ant type CTP or TP10 type CTP；The recombinant protein
CTP-JDlys is recombinant protein CTPTat-JDlys、CTPAnt- JDlys or CTPTP10-JDlys。
Preferably, the cytoplasm transduction peptide CTP is Tat type CTP；The recombinant protein c TP-JDlys is recombinant protein
CTPTat-JDlys。
Second aspect, the present invention provide a kind of preparation side of chimaeric enzyme antibiotic for killing staphylococcus aureus
Method includes the following steps:
S1, design primer, respectively with staphylococcus aureus bacteriophage lyases JDlys gene and cytoplasm transduction peptide CTP
Gene is template, expands JDlys genetic fragment and CTP genetic fragment；
The CTP gene that amplification obtains is inserted into described heavy by S2, the gene constructed recombinant plasmid of JDlys for obtaining amplification
The end 5` of JDlys gene, constructs the recombinant plasmid of the CTP-JDlys of segment containing target gene in group plasmid；
S3, the recombinant plasmid of the resulting CTP-JDlys of segment containing target gene of step S2 is expressed, after purification, is obtained
Cytoplasm transduction peptide CTP and staphylococcus aureus bacteriophage lyases JDlys concatenated chimaeric enzyme avidin CTP-
JDlys。
Preferably, in step S1, the staphylococcus aureus bacteriophage lyases JDLys gene and cytoplasm transduction peptide
CTPs gene refers to the gene sequence of the genome sequence of staphylococcus aureus bacteriophage and cytoplasm transduction peptide in GenBank respectively
Column synthesis.
Preferably, in step S1, the sequence such as SEQ of the upstream primer JDlys-F of the lyases JDlys gene
Shown in IDNO.1, the sequence of the downstream primer JDlys-R of the lyases JDlys gene is as shown in SEQ ID NO.2.
Preferably, in step S1, the transferring film Peptide C TP gene is transferring film Peptide C TPTP10Gene, transferring film Peptide C TPAntGene or
Transferring film Peptide C TPTP10Gene.
It is highly preferred that the transferring film Peptide C TPTatThe upstream primer CTP of geneTatThe sequence of-F as shown in SEQ ID NO.3,
The transferring film Peptide C TPTatThe downstream primer CTP of geneTatThe sequence of-R is as shown in SEQ ID NO.4；
The transferring film Peptide C TPAntThe upstream primer CTP of geneAntThe sequence of-F is as shown in SEQ ID NO.5, the transferring film
Peptide C TPAntThe downstream primer CTP of geneAntThe sequence of-R is as shown in SEQ ID NO.6；
The transferring film Peptide C TPTP10The upstream primer CTP of geneTP10The sequence of-F is as shown in SEQ ID NO.7, the transferring film
Peptide C TPTP10The downstream primer CTP of geneTP10The sequence of-R is as shown in SEQ ID NO.8.
Preferably, in step S2, the specific steps of the recombinant plasmid of the CTP-JDlys of segment containing target gene are constructed are as follows: with
Staphylococcus aureus bacteriophage lyases JDLys gene is template, constructs JDLys-pET-28a recombinant expression carrier；It will expand
Increase obtained CTP gene (CTPTat, CTPANT, CTPTP10) it is inserted into JDLys gene in JDLys-pET-28a recombinant expression carrier
The end 5`.
Preferably, in step S3, the expression, purifying the step of include: by the weight of the CTP-JDlys of segment containing target gene
Group plasmid is converted into e. coli bl21, and screening positive clone carries out the inducing expression and identification of albumen, is weighed after purification
Histone CTP-JDlys.
The third aspect, the present invention provide a kind of chimaeric enzyme antibiotic in infection of staphylococcus aureus horn cell mould
Application in type and infection of staphylococcus aureus mouse skin abscess model.
Preferably, the staphylococcus aureus is MRSA bacterial strain USA300.
Fourth aspect, the present invention provide a kind of chimaeric enzyme antibiotic and split bacterium system preparing resistant Staphylococcus aureus
Purposes in agent.
The gene and CTP that the present invention encodes enzyme antibioticTatTandem gene expression.In CTPTatWith the help of, enzyme antibiosis
Element, which enters in epidermal keratinocyte, efficiently kills resistant Staphylococcus aureus intracellular.Furthermore by establishing mouse skin purulence
Swollen infection-treatment model, assesses CTPTatThe bactericidal effect of mediate cytolysis enzyme in animal body.
The content of present invention includes: step 1: through prokaryotic expression, purifying, obtaining three kinds of cytoplasm transduction peptides and golden yellow respectively
The concatenated PROTEIN C TP of staphylophage lyasesTat-JDlys、CTPAnt- JDlys and CTPTP10-JDlys；
Step 2: it determines the lytic activity of three kinds of chimeric proteins in step 1 and splits bacterium spectrum；
Step 3: it establishes MRSA bacterial strain USA300 infection the sticking of horn cell, invade and Survival model；
Step 4: recombinant protein c TP is usedTat- JDLys carries out MRSA infection sterilization experiment intracellular and assesses its bactericidal effect；
Step 5: the abscess model of MRSA bacterial strain USA300 infecting mouse epidermis is established；
Step 6: recombinant protein c TP is usedTat- JDLys carries out the treatment of abscess locally injecting and assesses therapeutic effect.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the present invention will efficiently crack the enzyme avidin and eukaryon cytoplasm transduction peptide egg of resistant Staphylococcus aureus
White expressing in series obtains to have and splits the active recombinant expression protein of bacterium, the present invention provides one kind can by optimization series connection strategy
Mouse skin purulence caused by killing specific pathogen bacterium and efficiently treatment resistant Staphylococcus aureus is efficiently entering in eukaryocyte
It is swollen novel to split bacteria preparation.
2, it is respectively compared CTPTat- JDlys and JDLys albumen and the staphylococcus aureus lysozyme of commercialization, through the ages
Mycin and chloramphenicol find the bactericidal effect of bacterium in horn cell: CTPTat- JDlys can enter under transferring film Peptide C TP help
Intracellular bacteria is killed in horn cell, and the lyases JDlys for lacking transferring film peptide can hardly kill bacterium intracellular.CTPTat-
The JDlys horn cell state that treated infects is restored rapidly, under the pro-inflammatory cytokine and cytotoxicity of cell are significant
Drop.Further, since epidermis is mainly made of the horn cell of different differential periods, persistent infection of the bacterium in horn cell
It induces dermapostasis and ulcer is formed.CTP provided by the inventionTat- JDlys albumen can enter efficiently treated in horn cell it is resistance to
Mouse skin abscess caused by medicine staphylococcus aureus.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 be embodiment 2 in using 10%SDS-PAGE analyze and identify purifying recombinant expression protein CTP-JDlys and
The schematic diagram of JDlys；
Fig. 2 is the horn cell of staphylococcus aureus USA300 infection in embodiment 5 through CTPTatTreated by-JDlys
Co-focusing imaging；Wherein, Fig. 2A is staphylococcus aureus fluorescence imaging figure；Fig. 2 B is CTPTat- JDlys fluorescence imaging figure；Figure
2C is horn cell image under visible light；Fig. 2 D is Fig. 2A, 2B and 2C stacking image figure；
After Fig. 3 is three classes transferring film peptide recombinant protein in embodiment 5 and JDlys processing, MRSA bacterial strain is deposited in horn cell
Viability；
Fig. 4 is to compare CTP in embodiment 5TatThe survival ability intracellular of MRSA bacterial strain after-JDlys and chloramphenicol are handled；
Fig. 5 is recombinant protein c TP in embodiment 6TatAfter-JDlys and JDlys processing, horn cell pro-inflammatory cytokine
And cytotoxicity variation；In figure, Fig. 5 A is the transcriptional level of 3h cell pro-inflammatory cytokine IL-6 after handling, TNF-α；Fig. 5 B
For the emission levels of 3h cytotoxicity after processing；
Fig. 6 is recombinant protein c TP in embodiment 7TatMouse skin abscess area after-JDlys and JDlys treatment；
Fig. 7 is recombinant protein c TP in embodiment 7TatBacterial population variation in mouse skin after-JDlys and JDlys treatment.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, several changes and improvements can also be made.These belong to the present invention
Protection scope.
Whole staphylococcus aureus according to the present invention is that the separation of this laboratory saves (Wang Z, Zheng P, Ji
W,Fu Q,Wang H,Yan Y,Sun J.2016.SLPW:AVirulent Bacteriophage Targeting
Methicillin-Resistant Staphylococcus aureus In vitro and In vivo.Front
Microbiol 7:934.), possess independent intellectual property right.Such as pET-28a-CTP-JDlys's according to the present invention containing plasmid
E. coli bl21 is established by this laboratory.
In following example, test method without specific conditions, usually according to normal condition, such as Sambrook etc.
Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
Condition, or according to the normal condition proposed by manufacturer.
1 design primer of embodiment, PCR amplification target gene fragment
With reference to the genome of staphylococcus aureus bacteriophage, cytoplasm transduction peptide and green fluorescent protein GFP in GenBank
Sequence is respectively synthesized lyases JDlys gene, three kinds of transferring film peptide (CTPTat、CTPANTAnd CTPTP10) gene and green fluorescence egg
White GFP gene；Design primer, respectively using the gene of synthesis as template, using round pcr amplifying target genes, wherein JDlys base
Because size is 1488bp, CTPTatGene size is 33bp, CTPANTGene size is 48bp, CTPTP10Gene size be 63bp,
GFP gene size is 720bp.Concrete operations are as follows:
Design primer, primer sequence are shown in Table 1.It is glimmering with the lyases JDlys gene, transferring film Peptide C TP gene and green of synthesis
The DNA of photoprotein GFP is template, expands JDlys genetic fragment with primer JDlys-F1/JDlys-R；Primer CTP is used respectivelyTat-
F/CTPTat-R、CTPAnt-F/CTPAnt- R and primer CTPTP10-F/CTPTP10- R expands three transferring film peptide gene segments；Use primer
GFP-F/GFP-R expands GFP genetic fragment.PCR after reaction, takes 10 μ L products to carry out 1% agarose gel electrophoresis, examines
Purpose band size.PCR product is recycled using plastic recovery kit.
Table 1PCR primer sequence
The building of embodiment 2 recombinant plasmid pET-28a (+)-CTP-JDlys, expression and purifying
1 gained JDlys gene product construction recombination plasmid of embodiment is obtained using prokaryotic expression system pET-28a (+)
To recombinant plasmid pET-28a-JDLys, the end 5` of JDlys gene carries out digestion in pET-28a-JDlys later, is inserted into respectively
CTPs or GFP gene.Sequence analysis and comparison are then carried out, determines the recombinant plasmid pET-28a-CTP for obtaining correct sequenceTat-
JDlys、pET-28a-CTPAnt-JDlys、pET-28a-CTPTP10- JDlys and pET-28a-GFP-CTPTat-JDlys.It will be upper
Recombinant plasmid transformed is stated into expressed receptor bacterium-e. coli bl21 (DE3), screening positive clone carries out the inducing expression of albumen
And identification, obtain recombination JDlys albumen and chimeric lytic enzyme recombinant protein c TPTat-JDlys、CTPAnt-JDlys、CTPTP10-
JDlys and GFP-CTPTat- JDlys, molecular weight are respectively 54.7kDa, 56.1kDa, 56.6kDa, 57.1kDa and 82.1kDa；
By the recombinant protein of inducing expression through ni-sepharose purification and PBS dialysis treatment, the protein formulation that is purified；Concrete operations are as follows:
The glue recycling PCR product of JDlys gene in embodiment 1 is digested with BamHI/XhoI and is cloned into pET-
In 28a (+) carrier.Three CTP sequences and GFP sequence are then inserted into the BamHI of pET-28a (+) carrier respectively
(amino terminal of JDlys sequence) site forms CTPs-JDlys and GFP-CTPTatThe recombinant vector of-JDlys.By recombinant products
It is transferred in cloning vector e. coli bl21 (DE3), 37 DEG C are incubated overnight, and picking positive colony is identified through PCR, the positive of identification
Bacterial strain send to Sheng Gong bioengineering limited liability company and is sequenced, and the correct positive recombinant plasmid of sequencing is inoculated into 5ml respectively
It is incubated overnight in the LB liquid medium of the kanamycins positive (1:1000).Be incubated overnight bacterium solution by 1:100 be forwarded to 1L card that
Mycin the positive (1:1000) LB liquid medium in, 37 DEG C 200rpm shaken cultivation 3 hours, until OD600About 0.6.It is added eventually
Concentration is 1mmol/L isopropyl-β-D-thiogalactoside (IPTG), and 25 DEG C of 120rpm induce 12h.Later 4 DEG C of 5000rpm from
It washes three times, is finally resuspended with 25mL PBS, the bacterial suspension after resuspension uses ultrasonic wave repeatedly after heart 10min, 20mLPBS resuspension
It is broken.Broken bacterium solution 8000rpm under the conditions of 4 DEG C be centrifuged 20min take supernatant to cross 0.45 μm of filter membrane to obtain albumen respectively thick
Extract.With the Bindingbuffer rinse Ni column of 10 times of column volumes, sample is added in column, coutroi velocity < 5 drops/min.With
200mM imidazoles elutes destination protein, collects eluent, the protein formulation as purified.1L bacterium solution is able to obtain 20mg's
CTPTat- JDlys albumen, the CTP of 16mgAnt- JDlys albumen, the CTP of 6mgTP10- JDlys albumen, 25mg JDlys albumen and
The GFP-CTP of 10mgTat- JDlys albumen.
Fig. 1 is the recombinant expression protein schematic diagram that purifying is analyzed and identified using 10%SDS-PAGE；In figure, 1 is standard egg
White Marker；2 be the JDlys (54.7kDa) of purifying；3 be the CTP of purifyingTat- JDlys albumen (56.1kDa)；4 be purifying
CTPAnt- JDlys albumen (56.6kDa)；5 be the CTP of purifyingTP10- JDlys albumen (57.1kDa)；6 be the GFP- of purifying
CTPTat- JDlys albumen (82.1kDa).
Embodiment 3 recombinates the external minimal inhibitory concentration of lyases (MIC) measurement
The recombinant expression protein that embodiment 2 obtains is carried out to split bacterium experiment in vitro, inspection splits bacterium activity and splits bacterium spectrum.Specifically
It operates as follows:
By the aureus strains culture of separate sources to logarithm early stage (OD600 is 0.2 to 0.4).After PBS washing three times
100 μ l bacterial suspensions are taken (to be diluted to 5 × 106Cfu/mL) with 100 μ l difference final concentration (10,20,40,80,160,320 Hes
640 μ g/mL) recombination lyases mixing, in 96 hole microtiter plates measure lyases MIC.Plate is incubated at 37 DEG C
20 hours, and (OD is read with 96 plate readers600)。
Experimental result is as shown in table 2, the chimeric recombination lyases CTP of Tat typeTatThe cracking of-JDlys and not chimeric transferring film peptide
Enzyme JDlys is compared, and the bactericidal effect (CTP of slight decrease is presented to fraction bacterial strainTatFraction bacterial strain after-JDlys processing
MIC is higher than JDlys), but CTP as the result is shownTat- JDlys has antimicrobial spectrum identical with JDlys.However, other two kinds chimeric
Lyases CTPAnt- JDlys and CTPTP10- JDly shows that significantly reduced bactericidal effect is (above two embedding to most strains
The MIC for closing most strains after cracking enzymatic treatment is higher than JDlys).
The lytic activity of the bacterial strain of the present invention of table 2 and lyases to each bacterial strain
aMLST parting: Multilocus sequence typing；bBacterium source: I, No.1 People's Hospital Shanghai City's clinical separation strain；II,
Shanghai City Kingsoft cattle farm mazoitis milk cow clinical separation strain；III is bought from U.S.'s ATCC reference strain；IV, laboratory save
Bacterial strain.
Embodiment 4 recombinates the burnt positioning of lyases copolymerization intracellular
Horn cell (RPMI 1640 and 5%FBS) is passaged in 50 millimeters of culture vessel with glass bottom, in 37 DEG C of 5%CO2Item
After cultivating 12 hours under part, when horn cell reaches 90% coverage, the staphylococcus aureus of logarithmic growth phase is added
USA300(OD600=0.4~0.6) it, is incubated for 1 hour for 37 DEG C.Later, sample is washed 3 times with PBS, and is celebrated with containing 50 μ g/ml
1640 culture medium of RPMI of big mycin acts on 1 hour.By sample and staphylococcus aureus SPA anti-mouse monoclonal antibody (1:
200；Ab37644, Abcam) it is incubated for 30 minutes at 37 DEG C.The PBS washing goat anti-mouse IgG that rear and TRITC is coupled three times
(1:600；Ab6786, Abcam) it incubates 30 minutes at 37 DEG C.After washing, 80 μ g/ml are added and melt with green fluorescent protein (GFP)
Recombinant C TP-JDlys (the GFP-CTP of conjunctionTat- JDlys albumen) effect 30 minutes.PBS is washed 3 times, and the copolymerization then used is burnt
Fluorescence microscope (60X amplification factor) observation.
Bacterial strain USA300 (red in Fig. 2A) and CTP as the result is shownTat- JDlys (Fig. 2 B Green) is in horn cell
Common location state (yellow in Fig. 2 D) (Fig. 2) is in single z- plane.
Embodiment 5CTPTat- JDlys sterilization experiment intracellular
MRSA bacterial strain USA300 is grown 3 hours in BHI culture medium 37 DEG C, and 5,000 × g is centrifuged 5 minutes and collects carefully
Bacterium cell, PBS are washed 3 times, are resuspended in spare in not antibiotic fresh serum free RPMI1640 culture medium.Horn cell is equal
It is even to be laid in 12 hole tissue culturing plates, staphylococcus aureus (10 is added6The hole CFU/), bacterium is with horn cell infection multiplicity
10, it is cultivated 1 hour at 37 DEG C.Keratinocyte removal extracellular bacteria is washed with PBS later, is added big containing 50 μ g/ml celebrating
The RPMI1640 culture medium of mycin washs cell three times to remove with PBS after 1 hour to kill extracellular staphylococcus aureus
Gentamicin maintains cell to grow 3 hours in the presence of 50 μ g/ml gentamicins of control group.The CTP of purifyingTat-JDlys(40、
80,160 and 320 μ g/ml；Respectively 1,2,4 and 8 × MIC), JDlys (40 μ g/ml；2 × MIC), commercialization molten grape ball
Rhzomorph (40 μ g/ml；2 × MIC), vancomycin (5 μ g/ml；2 × MIC) and chloramphenicol (12.5,25,50 and 100 μ g/ml；Point
Wei 1,2,4 and 8 × MIC) it is added separately to maintain 3 hours in treated cell.Take cell sample respectively later.With
PBS is washed three times, is then incubated for 15 minutes with 0.02%Triton X-100 at 37 DEG C to dissolve keratinocyte and discharge thin
Intracellular bacteria.Each diluted concentration of lysate is spread evenly across on BHI agar plate, and is incubated overnight at 37 DEG C.System
Bacterium colony number is counted to determine the quantity of Intracellular bacterial.Statistical method is as follows: opposite intracellular bacteria number (rCFU)=each time
Point intracellular bacteria number/0h intracellular bacteria number.
As the result is shown: CTPTatMRSA Strain survival rate intracellular is remarkably decreased after-JDlys processing.And CTPAnt- JDlys and
CTPTp10- JDlys can not kill intracellular MRSA bacterial strain (Fig. 3).At the lysostaphin and vancomycin of commercialization
The decline of intracellular bacterial population is not obvious (Fig. 3) after reason.In addition, CTPTatIt is dense with chloramphenicol when-JDlys concentration is 2 times of MIC
When degree is 8 times of MIC quite (Fig. 4) to the Scavenging activity of intracellular MRSA bacterial strain.It should be the result shows that CTPTat- JDlys is intracellular to be killed
Bacterium ability is better than bactericide-chloramphenicol intracellular.
Embodiment 6CTPTat- Lys bactericidal effect assessment intracellular
Horn cell (RPMI 1640 and 5%FBS) is passaged in 50 millimeters of culture vessel with glass bottom, in 37 DEG C of 5%CO2Item
After cultivating 12 hours under part, when horn cell reaches 90% coverage, the staphylococcus aureus of logarithmic growth phase is added
USA300(OD600=0.4~0.6) it, is incubated for 1 hour for 37 DEG C.Later, sample is washed 3 times with PBS, and is celebrated with containing 50 μ g/ml
1640 culture medium of the RPMI effect of big mycin, collects different time points cell, detects cell pro-inflammatory cytokine and thin respectively
The emission levels of cellular toxicity assess CTPTatThe variation of cell state after the processing of-JDlys albumen.Concrete operations are as follows:
Step 1: cytokines measurement
CTP is collected respectivelyTatHorn cell supernatant after the processing of-JDlys, JDlys albumen with untreated (control), uses
RNA extracts kit (Qiagen) extraction purification RNA from supernatant.According to specification, RNA reverse transcription reagent box is used
(TaKaRa) cDNA synthesis is carried out.The mRNA level in-site of the cDNA of synthesis two-step method qRT-PCR test sample.By beta-actin
As reference gene.The primer sequence of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and beta-actin in
In table 3.Use compares cycle threshold value (2-ΔΔCT) method analysis mRNA level in-site.
Step 2: cytotoxicity detection.
CTP is collected respectivelyTatHorn cell supernatant after the processing of-JDlys, JDlys albumen with untreated (control), such as says
Shown in bright book, cell lactic dehydrogenase is detected using 96 non-radioactive cell toxicity test kit (Promega) of CytoTox
Release.Final (the OD under microplate reader of sample492) measurement absorbance.The LDH of the cell release of space management is decided to be spontaneous release
It puts；Maximum release is defined as with the LDH that final concentration of 1% Triton X-100 lytic cell discharges.Cytotoxicity calculates such as
Under: % cytotoxicity=(LDH release-spontaneous release to be measured)/(maximum release-spontaneous release).
The results show that being used compared with JDlys processing and control treatment (cell of untreated after MRSA strain infection)
CTPTatThe pro-inflammatory cytokine IL-6 of 12h cell after-JDlys processing, the transcriptional level of TNF-α decline obvious (Fig. 5 A)；
CTPTatThe release of cell various time points LDH is remarkably decreased (Fig. 5 B) after-JDlys processing.This shows to use CTPTatThe place-JDlys
Reason can mitigate inflammatory reaction caused by intracellular MRSA bacterial strain and reduce the toxicity of cell.
3 fluorescence quantification PCR primer sequence of table
Embodiment 7CTP-JDlys treats mouse skin abscess caused by MRSA
The Balb/C female mice of 6 week old is selected to be used for the building of subcutaneous abscess model, later respectively with such as CTPTat-
The continuous three days locally injectings treatment of JDlys, JDlys albumen, concrete operations are as follows:
Mouse anesthesia by its abdomen two sides shaving and sterilizes, random to be grouped (every group of 20 mouse).Then, by 50 μ
LMRSA bacterial strain USA300 (5 × 107The side CFU/) and isometric (131~220 μm of autoclaved Cytodex-1 bead；
Sigma-Aldrich it) mixes, subcutaneous injection.Start within 24 hours after infection, for three days on end respectively by the CTP of purifyingTat- JDlys and
JDlys albumen is subcutaneously injected near the skin of infection.Subcutaneous individually injection CTP-JDlys (not attacking poison) and individually injection PBS
Buffer (attacking poison not treat) is as a control group.
It is observed continuously later 13 days, and therapeutic effect is assessed, the specific method is as follows:
Mouse skin damage location area is monitored, and is recorded.The results show that the CTP compared with non-treatment groupTat- JDlys egg
Mouse skin abscess area (Fig. 6) is substantially reduced after white treatment.Wherein CTPTatMaximum abscess area after-JDlys treatment is about
For 0.72cm2(the 5th day), hence it is evident that be less than control group (the 11st day about 2.68cm2)；CTPTatThe the 3rd, 5 and 7 day after-JDlys treatment
Abscess area be significantly less than JDlys treatment group.
In addition, put to death after respectively being anaesthetized mouse at the 3rd, 6,9 and 12 day, by abdomen two sides skin injury region without
Bacterium, which cuts off and sterile PBS is added, carries out tissue homogenate.By suspensions of tissues serial dilution in sterile PBS, it is coated on BHI agar
On plate, 37 DEG C of incubations.Bacterial population is recorded after 24 hours.Experimental result is as shown in fig. 7, CTPTatMouse after-JDlys treatment
Abscess local bacterial number be below control group and JDlys treatment group in various time points；The result shows that CTPTat- JDlys albumen
Mouse skin abscess caused by MRSA bacterial strain can effectively be treated.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
SEQUENCE LISTING
<110>Shanghai Communications University
<120>the chimaeric enzyme antibiotic of killing staphylococcus aureus and its preparation and application
<130> DAG34348
<160> 16
<170> PatentIn version 3.5
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Claims (10)
1. a kind of chimaeric enzyme antibiotic for killing staphylococcus aureus, which is characterized in that the chimaeric enzyme antibiotic includes born of the same parents
The matter transduction peptide CTP and concatenated recombinant protein c TP-JDlys of staphylococcus aureus bacteriophage lyases JDlys.
2. the chimaeric enzyme antibiotic according to claim 1 for killing staphylococcus aureus, which is characterized in that the cytoplasm
Transduction peptide CTP is Tat type CTP, Ant type CTP or TP10 type CTP；The recombinant protein c TP-JDlys is recombinant protein CTPTat-
JDlys、CTPAnt- JDlys or CTPTP10-JDlys。
3. the chimaeric enzyme antibiotic according to claim 1 or 2 for killing staphylococcus aureus, which is characterized in that described
Cytoplasm transduction peptide CTP is Tat type CTP；The recombinant protein c TP-JDlys is recombinant protein CTPTat-JDlys。
4. a kind of preparation method of the chimaeric enzyme antibiotic according to claim 1 for killing staphylococcus aureus, special
Sign is, includes the following steps:
S1, design primer, respectively with staphylococcus aureus bacteriophage lyases JDlys gene and cytoplasm transduction peptide CTP gene
For template, JDlys genetic fragment and CTP genetic fragment are expanded；
The CTP gene that amplification obtains is inserted into the recombination matter by S2, the gene constructed recombinant plasmid of JDlys for obtaining amplification
The end 5` of JDlys gene in grain, constructs the recombinant plasmid of the CTP-JDlys of segment containing target gene；
S3, the recombinant plasmid of the resulting CTP-JDlys of segment containing target gene of step S2 is expressed, after purification, obtains cytoplasm
Transduction peptide CTP and staphylococcus aureus bacteriophage lyases JDlys concatenated chimaeric enzyme avidin CTP-JDlys.
5. the preparation method of the chimaeric enzyme antibiotic according to claim 4 for killing staphylococcus aureus, feature exist
In in step S1, the sequence of the upstream primer JDlys-F of the lyases JDlys gene is described as shown in SEQ ID NO.1
The sequence of the downstream primer JDlys-R of lyases JDlys gene is as shown in SEQ ID NO.2.
6. the preparation method of the chimaeric enzyme antibiotic according to claim 4 for killing staphylococcus aureus, feature exist
In in step S1, the transferring film Peptide C TP gene is transferring film Peptide C TPTP10Gene, transferring film Peptide C TPAntGene or transferring film Peptide C TPTP10
Gene.
7. the preparation method of the chimaeric enzyme antibiotic according to claim 6 for killing staphylococcus aureus, feature exist
In the transferring film Peptide C TPTatThe upstream primer CTP of geneTatThe sequence of-F is as shown in SEQ ID NO.3, the transferring film peptide
CTPTatThe downstream primer CTP of geneTatThe sequence of-R is as shown in SEQ ID NO.4；
The transferring film Peptide C TPAntThe upstream primer CTP of geneAntThe sequence of-F is as shown in SEQ ID NO.5, the transferring film peptide
CTPAntThe downstream primer CTP of geneAntThe sequence of-R is as shown in SEQ ID NO.6；
The transferring film Peptide C TPTP10The upstream primer CTP of geneTP10The sequence of-F is as shown in SEQ ID NO.7, the transferring film peptide
CTPTP10The downstream primer CTP of geneTP10The sequence of-R is as shown in SEQ ID NO.8.
8. the preparation method of the chimaeric enzyme antibiotic according to claim 4 for killing staphylococcus aureus, feature exist
In, in step S3, the expression, purifying the step of include: using prokaryotic expression system pET-28a (+), by fusion segment
CTP-JDlys construction recombination plasmid, by recombinant plasmid transformed into e. coli bl21, screening positive clone carries out luring for albumen
Expression and identification are led, obtains recombinant protein c TP-JDlys after purification.
9. one kind is based on chimaeric enzyme antibiotic described in claim 1 in infection of staphylococcus aureus keratinocyte model and gold
Application in staphylococcus aureus infecting mouse epidermis abscess model；The staphylococcus aureus is MRSA bacterial strain USA300.
10. a kind of chimaeric enzyme antibiotic according to claim 1 is split in bacteria preparation preparing resistant Staphylococcus aureus
Purposes.
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| NL2034252B1 (en) * | 2022-07-22 | 2024-01-29 | Univ Kunming Science & Technology | Phage lytic enzyme with fluorescent marker and application thereof |
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| NL2034252B1 (en) * | 2022-07-22 | 2024-01-29 | Univ Kunming Science & Technology | Phage lytic enzyme with fluorescent marker and application thereof |
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