CN104726429A

CN104726429A - Bacteriophage lysin with improved antibacterial effect

Info

Publication number: CN104726429A
Application number: CN201510002678.8A
Authority: CN
Inventors: 任慧英; 张灿; 王元超; 卢国民; 孙虎芝; 王婷; 邹玲; 刘文华
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2015-01-05
Filing date: 2015-01-05
Publication date: 2015-06-24
Anticipated expiration: 2035-01-05
Also published as: CN104726429B

Abstract

The invention discloses a bacteriophage lysin with animproved antibacterial effect. The 99th leucine and the 102nd methionine of lyase Bp7e amino acid are respectively mutated to obtain alanine and glutamic acid, by using a prokaryotic expression technique, mutant protein can be obtained, and the mutant protein is identified by using Western-blot and is named Bp7c mutant protein. Bp7e and Bp7e mutant protein are purified, the concentration of Bp7e and Bp7e mutant protein is detected, and the in-vitro pyrolysis experiment and the pyrolysis spectrum detection show that purified Bp7e and Bp7e mutant protein have a broad-spectrum antibacterial function and have pyrolysis effects on micrococcus lysodeikticus, staphylococcus aureus, salmonellae and multiple serotype escherichia colis, and the pyrolysis effect of the Bp7e mutant protein is wholly superior to that of natural lyase Bp7e.

Description

A kind of bacterial virus catenase with the fungistatic effect of enhancing

Technical field

The present invention relates to biomedicine technical field, particularly relate to a kind of bacterial virus catenase being prepared the method with the bacterial virus catenase of the fungistatic effect of enhancing and the sudden change obtained by rite-directed mutagenesis.

Background technology

Along with the continuous appearance of resistance, phage has become one of focus of software engineering researchers invent novel biological agent.But phage itself can not leave Host Strains existence, resistant gene, the virulence that may cause may be brought to propagate, people are made to carry out this problem of prevention and therapy bacteriological infection still there is dispute to phage preparation whether should be developed.Therefore, people are in the urgent need to finding a kind of method of safer and more effective prophylactic treatment bacteriological infection.Bacterial virus catenase also receives the favor of investigator as the plurality of advantages that a kind of new bio antibiotic preparation shows.Bacterial virus catenase (bacteriophage lysins) is the enzyme produced in host bacterial latter stage at lytic cycle by double-stranded DNA phage, except the filamentous phages of single stranded DNA is external, most of phage relies on the peptidoglycan in lyase hydrolysis Host Strains cell walls thus discharges progeny phage.

The bacterial virus catenase of purifying can act on the distinctive peptidoglycan of bacteria cell wall, causes bacteria cell cracking simultaneously.But due to the textural difference of bacteria cell wall, lyase is to G ⁺and G ^-the outer fragmentation effect of bacterium of bacterium is different, to G ⁺the effect of bacterium is apparently higher than to G ^-the effect of bacterium.However, the sterilization feature of bacterial virus catenase uniqueness and Action advantage still receive the favor of investigator, and to be undergone technological transformation to bacterial virus catenase by genetic engineering means and obtain the novel lyase albumen of broad host range and also become possibility.

High efficiency also plays effect at short notice to kill bacterium is the significant sterilization feature of bacterial virus catenase.The mixing of several lyase or lyase also can produce synergetic antibacterial effect when using with Antibiotic combination, raising sterilization effect.Meanwhile, relatively high sterilization specificity and affect the another large advantage that normal microflora is bacterial virus catenase hardly.In general, staphylococcus phagocytosis lyase only kills specific staphylococcus, and bacteriophage lyase of streptococcus pneumonia is only responsive to specific suis, and they affect hardly on body normal microflora.Certainly, part bacterial virus catenase is also had also to show certain broad spectrum.

Current lot of domestic and international investigator has done research in various degree to multiple bacterial virus catenase.The application of the reported first such as Schuch bacterial virus catenase in treatment bacteriological infection.One section of lyase gene in lactobacillus phage LL-H is cloned in intestinal bacteria by Vasala etc., has given expression to the enzyme with the effect of dissolved cell wall.Kikkawa etc. study anthrax bacillus lyase PlyG after expression and purification, and utilize ELISA quantitative and qualitative analysis to detect and find, lyase PlyG has the effect of cracking category-B anthrax bacillus, but fragmentation pattern is narrow.Djurkovic etc. utilize suis lyase Cpl-1 and microbiotic acting in conjunction in penicillin-fast suis, found that synergy given play to by lyase and penicillin.Also someone started to utilize the genes such as bacterial virus catenase to carry out cloning and expressing in recent years in China.Bacteriophage lyase of streptococcus pneumonia CPL21 gene is inserted pET28a carrier by Lu Hairong etc., solution expression with high efficiency is obtained in e. coli bl21, through DEAE affinity chromatography, obtain the albumen of high purity 97%, bacteriostatic test shows, the CPL21 albumen of purifying has obvious germicidal action to clinical streptococcus pneumoniae, and inhibition zone size is proportional with protein concentration.After Phage PhiX174 lyase E is cloned into pBV220 carrier by Liu Shuan etc., then recombinant vectors is transferred to intestinal bacteria O ₁₅₇: H ₇in, after abduction delivering, find intestinal bacteria O ₁₅₇: H ₇bacterium surface defines many apertures, and thalline content is flowed out by duct, forms bacterium shell, reaches 98.4% to colibacillary lysis efficiency, and this test is also for the preparation of bacterium shadow vaccine provides thinking.Mycobacteriophage D29 lyase gene gene10 after large intestine-mycobacterial shuttle vector clone, through mycobacterium abduction delivering, and is confirmed that gene10 albumen has lytic activity by Yang Wenhui etc.

Although bacterial virus catenase is compared with microbiotic and Phage therapy there is many advantages, but still there are some problems, wherein the security of antibacterials is problems of people's special concern.Due to the efficient feature rapidly of phage splitting enzyme and sterilizing, can toxic substance, Inflammatory substances etc. that when people also worry that lyase destroys bacteria cell wall and cytolemma again, bacteria lysis produces produce harm to body, but about lyase, body are not produced to the report of harm at present.Clearance rate is in vivo the another aspect affecting lyase germicidal action, because bacterial virus catenase is protein, when being applied to mucous membrane or whole body, body can be stimulated to produce immunne response, thus reduce its activity, and accelerate clearance rate in its body.In addition, lyase also may be hydrolyzed enzyme liberating and inactivation in vivo, and this also accelerates its speed removed in vivo, become lyase be widely used in clinical before need the problem that solves.

Along with the mankind enter the genome times afterwards comprehensively, the structure design of protein and the progressive development simultaneously also having promoted lyase transformation field of proteomic techniques.Some investigators change specificity and the catalytic activity of natural cleavage enzyme by the method for the structural domain replacing lyase.By to the useful modification of lyase and transformation, not only can increase the lytic activity of lyase, also may expand its fragmentation pattern and then reach the object optimizing lyase.

In recent years along with the attention that investigator studies phage and lyase thereof, increasing bacterial virus catenase prepares purifying by methods such as prokaryotic expressions, and is applied to the treatment of the aspects such as bacteriological infection.Have lysis efficiency based on bacterial virus catenase high, not easily produce the advantages such as Resistant strain, the biotechnological formulation particularly killing antibiotic resistant bacteria for software engineering researchers invent newtype drug provides vast potential for future development.Theory and practical value is had more with its high performance lyase.At present, bacterial virus catenase is studied just towards following future development: gene order and the organic evolution relation of probing into important pathogenic bacteria phage and lyase thereof; Obtain the bacterial virus catenase of large-scale purification with molecular biology and culture of microorganism and detect its bacteriostatic activity; By methods such as genetically engineereds, directed beneficial mutation is carried out to natural cleavage enzyme, develop the sterilization potential of lyase to greatest extent.

Summary of the invention

Due to the appearance of current increasing resistant organism, phage and lyase therapy is made to enter the visual field of investigator once again.Along with the theory and technology trend of artificial reconstructed phage is ripe, the artificial reconstructed phage of using gene engineering technique and lyase thereof also receive the favor of investigator, and phage and lyase treatment also achieve breakthrough progress.

Amino acid whose for lyase Bp7e (SEQ ID No:1) the 99th leucine and 102 methionine(Met) carry out sporting L-Ala and L-glutamic acid by the present invention respectively, obtain mutant protein through prokaryotic expression technology and through Western-blot, it identified, called after Bp7e mutant protein (SEQ ID No:2).Purifying and concentration determination have been carried out to Bp7e and Bp7e mutant protein, tested by external cracking and fragmentation pattern detect learn, Bp7e and Bp7e mutant two kinds of albumen of purifying have the bacteriostatic action of wide spectrum, all have lytic effect to micrococcus lysodeikticus, streptococcus aureus, Salmonellas and various serotype intestinal bacteria, and Bp7e mutant lytic effect entirety is better than natural cleavage enzyme Bp7e.

Accompanying drawing explanation

The SDS-PAGE electrophoretic analysis of Fig. 1 recombinant phage lyase Bp7e mutant: 1, the abduction delivering of empty plasmid; 2, containing the abduction delivering of the recombinant plasmid of lyase Bp7e mutant; M. standard protein molecular mass.

The Western-blot qualification of Fig. 2 abduction delivering product (lyase Bp7e mutant): 1, abduction delivering product; M, standard protein molecular mass.

Fig. 3 lyase Bp7e and mutant thereof are to the fungistatic effect of micrococcus lysodeikticus: left figure is lyase Bp7e; Right figure is lyase Bp7e mutant.

Fig. 4 lyase Bp7e and mutant thereof are to colibacillary fungistatic effect: left figure is lyase Bp7c; Right figure is lyase Bp7e mutant.

Fig. 5 lyase Bp7e and mutant thereof are to the fungistatic effect of streptococcus aureus: left figure is lyase Bp7e; Right figure is lyase Bp7e mutant.

Fig. 6 lyase Bp7e and mutant thereof are to the fungistatic effect of Salmonellas: left figure is lyase Bp7e; Right figure is lyase Bp7e mutant.

Fig. 7 egg white lysozyme is to the fungistatic effect of micrococcus lysodeikticus.

Fig. 8 egg white lysozyme is to the fungistatic effect of intestinal bacteria, Salmonellas and streptococcus aureus: be followed successively by intestinal bacteria, Salmonellas and streptococcus aureus from left to right.

Fig. 9 lyase Bp7e and mutant thereof are to the lysis efficiency of different strains: the lysis efficiency of (A) lyase Bp7e: the lysis efficiency of (B) lyase Bp7e mutant.

Embodiment

Embodiment 1: the prokaryotic expression of bacterial virus catenase Bp7e mutant

1.1 material

Bacterial virus catenase Bp7e protein expressing plasmid pET28a-Bp7e, e. coli strain bl21 are preserved by Animal Science And Technology Microbiological Lab of Qingdao Agricultural University;

1.2 method

1.2.1 the amino acid whose first run jump reaction of bacterial virus catenase Bp7e

1.2.1.1 for the mutational site design primer of goal gene

Carry out rite-directed mutagenesis to Bp7e (SEQ ID No:1) amino acid, make its 99th leucine sport L-Ala, the 102nd methionine(Met) sports L-glutamic acid.According to the design of primers principle of site-directed mutagenesis kit, design two pairs of primers, primer is synthesized by Shanghai biotechnology Services Co., Ltd.

Primer one upstream: 5 ' AAGTTCGTCGTTGTGCTGCAATTAACATGGTCTTC3 '

Primer one downstream: 5 ' GAAGACCATGTTAATTGCAGCACAACGACGAACTT3 '

Primer two upstream 5 ' TGTGCTGCAATTAACGAGGTCTTCCAAATGGG3 '

Primer two downstream 5 ' CCCATTTGGAAGACCTCGTTAATTGCAGCACA3 '

With recombinant expression plasmid pET28a-Bp7e for template, with primer one and test kit set up the reaction system of 50 μ L with reagent:

1.2.1.2 the selection of mutant plasmid

PCR reaction terminates rear use Mutazyme ^tMenzymic digestion methylates plasmid thus select mutant plasmid DNA.Concrete steps are, first prepare above-mentioned PCR reaction product, add 1 μ L (10U/ μ L) MutazymeTM enzyme, 37 DEG C of incubations 1 hour.

1.2.1.3 mutant plasmid is transformed in E. coli expression strains BL21

(1) 10 μ L mutant plasmids are added in 50 μ L bacillus coli DH 5 alpha competent cells, gently after mixing, ice bath 30min;

Heat shock 90s in (2) 42 DEG C of water-baths, is placed in rapidly 2 ~ 3min on ice, adds 1000 μ LB liquid nutrient mediums, 37 DEG C of shaking culture 90min;

(3) the centrifugal 5min of 4000r/min discards suspension bacteria liquid after 600 μ L supernatant liquors;

(4) get 100 μ L bacterium liquid even spread containing on the LB flat board of Kana, absorb rearmounted 37 DEG C completely and be inverted overnight incubation.

1.2.1.4 sequential analysis

Then prove to there occurs orthomutation when LB flat board growing white colony, object band is checked through agarose gel electrophoresis after extracting plasmid, and by again recovering containing the white colony of mutant plasmid of detecting, 37 DEG C spend the night shake bacterium after deliver to Shanghai Sheng Gong biotechnology company limited and check order; Sequencing result shows successfully the 99th leucine to be sported L-Ala

1.2.2 the amino acid whose next round jump reaction of bacterial virus catenase Bp7e

The lyase Bp7e mutant plasmid suddenling change correct with the first run is masterplate, sets up next round PCR amplification system with primer two.Method is the same, confirms, obtain the lyase Bp7e mutant as shown in SEQ ID No:2 through order-checking.Namely through two-wheeled mutation process, lyase Bp7e is made finally to sport object lyase Bp7e mutant.

1.2.3 the abduction delivering of recombinant expression plasmid bacterium pET28a-Bp7e mutant protein

The correct white colony of picking order-checking sudden change, inoculation is containing the LB liquid nutrient medium of Kana, and 37 DEG C of shaking culture are spent the night, next day by 1: 100 volume ratio namely get 70 μ L and inoculate 7mL and contain in the LB liquid nutrient medium of Kana, 37 DEG C of shaking culture are about the OD that 2h makes nutrient solution ₆₀₀be about 0.6 ~ 0.8, add the IPTG that final concentration is 1.0mmol/L, 37 DEG C of centrifugal 10min of shaking culture 3h, 4000r/min collect bacterial sediment, by bacterial sediment with being transferred in 1.5mL centrifuge tube after 700 μ L PBS Eddy diffusions, add 1 × SDS sample-loading buffer of 70 μ L, boil 15min, the centrifugal 3min of 4000r/min, get supernatant and carry out SDS-PAGE electrophoretic analysis, by recombinant protein called after lyase Bp7e mutant, and under establishing same condition, the empty plasmid pET28a of abduction delivering compares.Recombinant expressed lyase Bp7e mutant protein is carried out Western-blot qualification.As shown in Figure 1, Western-blot result as shown in Figure 2 for electrophoresis result.From Fig. 1 and Fig. 2, the abduction delivering of recombinant plasmid pET28a-Bp7 mutant is in 22KD place appearance one band; Not there is band at object band place in empty vector control group, conforms to expected results.

Embodiment 2: the bacteriostatic activity of bacterial virus catenase Bp7e and mutant protein thereof detects

1 materials and methods

1.1 material

Micrococcus lysodeikticus, intestinal bacteria, Salmonellas, streptococcus aureus, 01,015,024,078,088 serotype intestinal bacteria are preserved by this laboratory.

1.2 method

1.2.1 the purifying of bacterial virus catenase Bp7e and mutant protein thereof

(1) chromatography column is assembled: add chromatography column after being mixed by 1mL filler, room temperature leaves standstill 10min, after layering, the liquid outlet of bottom is opened, allows ethanol slowly be flowed out by action of gravity.

(2) respectively the bacterial strain picking list colony inoculation 37 DEG C of shaking culture in 5mL is containing the LB liquid culture of Kana expressing lyase Bp7e and mutant thereof are spent the night.

(3) take out 2.5mL next day respectively and add the LB liquid culture (each recombinant bacterium cultivate 2 bottle) of 250mL containing Kana, it is about 0.6 ~ 0.8 that 37 DEG C of joltings are cultured to OD600, then adding IPTG makes IPTG concentration in the protein induced system of recombinant bacterium be 1.0mmol/L, 37 DEG C of centrifugal 10min of shaking culture 3h, 4000r/min collect thalline.

(4), after collecting thalline, every 100mg thalline (weight in wet base) adds 1-5mL bacterial lysate (having added 10 μ LPMSF in every 1mL bacterium extraction agent), ultrasonic degradation thalline.

(5) keep bacterium liquid to be in ice bath in ultrasonic procedure, the ultrasonic apparatus power 220Hz that ultrasound condition is, period avoids solution overheated, ultrasonic 3s, interval 1s, and when bacterium liquid is more limpid, about 30min stops ultrasonic.

(6) 10000rpm, 4 DEG C of centrifugal 3min, collect the soluble proteins in supernatant.

(7) with Binding Buffer by cellular lysate liquid equimultiple dilution back loading upper prop, flow velocity be 10 times of column volumes/hour, collect stream and wear liquid, carry out coutroi velocity by the speed controlling the cellular lysate liquid added.

(8) use the Binding Buffer of 15 times of column volumes to rinse pillar, wash away foreign protein.

(9) use Elution Buffer wash-out, collect elution peak.Elution peak can be in charge of collection, and every 1mL collects 1 pipe.

(10) after wash-out, use the deionized water wash pillar of 10 times of column volumes successively, then balance (ethanol will by filler submergence), 4 DEG C of preservations with 20% ethanol of 3 times of column volumes.

(11) SDS-PAGE electrophoresis detection is passed through to the elution peak collected.

Obtain bacterial virus catenase Bp7e purified accordingly and mutant protein thereof by finally merging to retain afterwards to the electrophoresis detection of solution in collection tube and protein concentration analysis, purifying protein is for subsequent use through dialysed overnight.

1.2.2 the dull and stereotyped bacteriostatic test of lyase Bp7e albumen and mutant thereof

Respectively by intestinal bacteria, Salmonellas, Staphylococcus aureus and micrococcus lysodeikticus recovery, picking list bacterium colony 37 DEG C of 180rpm in LB meat soup cultivate about 10h, getting appropriate bacterium liquid sterile saline doubling dilution is respectively about 0.13 to OD600, makes its bacterial concentration reach 10 ⁸cfu/mL.Get cotton swab even spread in ordinary flat of 200uL bacterium liquid sterilizing, put sterilizing Oxford cup, purifying lyase Bp7e albumen and mutant protein albumen thereof that 200 μ L concentration are 1mg/mL is added respectively in the cup of Oxford, cultivate about 12h in 37 DEG C of incubators observe and measure inhibition zone size, simultaneously detectable level be the fungistatic effect of the standard egg white lysozyme of 1mg/mL as controlled trial, result is as shown in table 1 and Fig. 3-8.

Table 1 lyase Bp7e albumen and mutant thereof are to the inhibition zone size detecting bacterial classification

From table 1 and Fig. 3-8 result, lyase Bp7e albumen and mutant thereof all have obvious fungistatic effect to micrococcus lysodeikticus, intestinal bacteria, Salmonellas, streptococcus aureus, maximum to colibacillary inhibition zone, and lyase Bp7c mutant protein is all large than Bp7e to the inhibition zone of these 4 kinds of bacterium, standard egg white lysozyme is obvious to micrococcus lysodeikticus fungistatic effect, and does not all have obvious fungistatic effect to intestinal bacteria, Salmonellas and streptococcus aureus.

1.2.3 the detection of Bp7 lyase and mutant lysis efficiency and fragmentation pattern

By resuspended for the centrifugal rear PBS damping fluid of using respectively of 01,015,024,078,088 serotype intestinal bacteria and micrococcus lysodeikticus growing to logarithmic phase, being adjusted to absorbance OD600 is about 0.5.The lyase Bp7c albumen of purifying identical for concentration and mutant protein thereof are respectively got 100 μ L add in equivalent bacterial suspension, record the OD600 value of initial albumen and bacterial suspension, every strain bacterium is cooked 3 repetitions.37 DEG C of incubators are cultivated, and mix the OD600 of suspension when measuring 2h and 6h respectively.Decline 50% for cracking positive criteria with bacterial suspension turbidity, detect the splitting action of phage Bp7e and mutant above-mentioned bacterial strains, the results are shown in Table 2 and Fig. 9.

The bacterium spectrum of splitting of table 2 lyase Bp7e albumen and mutant thereof measures (+for positive: ± be the weak positive)

Result shows, survey bacterial strain suspension OD600 value decline all to some extent, the fragmentation pattern of lyase Bp7e and mutant thereof is consistent, the lysis efficiency of mutant all higher than natural cleavage enzyme, and detects the OD600 value decline more than 50% of micrococcus lysodeikticus, intestinal bacteria 015 and 088 after 2h, and to intestinal bacteria 01,024, detect OD600 value after 078,2h and be declined by less than 50%, and 6h detects its 0D600 value all declines about 50%.Illustrate that the fragmentation pattern of lyase Bp7e and mutant is comparatively wide and fragmentation pattern is identical, and the lytic effect of lyase Bp7e mutant is better, in table 2, but two kinds of albumen are different to the lysis efficiency of different strains, the highest to the lysis efficiency of intestinal bacteria 015 and 088, see Fig. 9.

Claims

1. a N,O-Diacetylmuramidase Bp7e, is characterized in that: it has the aminoacid sequence shown in SEQ ID No:1.

2. a N,O-Diacetylmuramidase Bp7e mutant, is characterized in that: its mutant obtained for carrying out amino acid whose for the N,O-Diacetylmuramidase Bp7e shown in claim 1 the 99th leucine and 102 methionine(Met) respectively sporting L-Ala and L-glutamic acid.

3. N,O-Diacetylmuramidase Bp7e mutant according to claim 2, is characterized in that: it has the aminoacid sequence shown in SEQ ID No:2.

4. the nucleic acid of coding N,O-Diacetylmuramidase Bp7e according to claim 1 or the N,O-Diacetylmuramidase Bp7e mutant described in Claims 2 or 3.

5. comprise the expression plasmid of nucleic acid described in claim 4.

6. comprise the host cell of nucleic acid according to claim 4 or expression plasmid according to claim 5.

7. host cell according to claim 6, is characterized in that: described cell is intestinal bacteria.

8. N,O-Diacetylmuramidase Bp7e according to claim 1, N,O-Diacetylmuramidase Bp7e mutant described in the Claims 2 or 3 purposes in the medicine of preparation treatment or prevention bacteriological infection.

9. purposes according to claim 8, is characterized in that: described bacterium is micrococcus lysodeikticus, streptococcus aureus, Salmonellas and intestinal bacteria.