CN106854247A - A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella - Google Patents
A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella Download PDFInfo
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- CN106854247A CN106854247A CN201611139628.5A CN201611139628A CN106854247A CN 106854247 A CN106854247 A CN 106854247A CN 201611139628 A CN201611139628 A CN 201611139628A CN 106854247 A CN106854247 A CN 106854247A
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Abstract
The invention belongs to animal organism pharmaceutical engineering field, specifically disclose a kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella, by designing primer, PCR Successful amplifications go out the lyase gene of phage E CGD1, and are transferred to expression system acquisition recombinant bacterium.Recombinant bacterium is expressed with IPTG inductions, and cellular lysate is obtained the restructuring lyases of soluble phage E CGD1 after induction, and the restructuring lyases can crack many plants of Escherichia coli and salmonella, i.e., with fragmentation pattern wider.Additionally, restructuring lyases shows with PB synergistic result, the presence for recombinating lyases reduces the minimum inhibitory concentration of PB, i.e., both has collaboration fungistatic effect.Recombinate the identification of lyases lytic activity, and its fragmentation pattern wide identification, disclose the restructuring lyases of phage E CGD1 in the prevention and control of Escherichia coli and salmonella infection with potential application value.
Description
Technical field
The present invention relates to animal organism pharmaceutical engineering technical field, Escherichia coli can be cracked more particularly, to one kind
And the preparation method of the bacterial virus catenase of salmonella.
Background technology
Escherichia coli（Escherichia coli）It is both that one kind in humans and animals enteron aisle normally perches bacterium, while being also
A kind of important pathogenic bacteria.Escherichia coli are broadly divided into enteron aisle non-pathogenic symbiotic bacterial strain, enteropathic bacterial strain and enteron aisle
Outer pathogenic bacterial strains three major types.Used as the pathogenic bacteria that a kind of humans and animals are common, it can cause enteric infection, urinary tract infection, complete
The sexy dye of body and other clinical infections etc..Wherein parenterally pathogenic bacterial strains can cause serious extra intestinal infection, especially urinate
Road infects, and is played an important role in antibiotics resistance spreads through sex intercourse.In bacillus coli multiple endurance strain, parenterally
Pathogenic bacterial strains ratio is in the majority.The appearance of multidrug resistant sex chromosome mosaicism, many problems are added to the prevention and control of coli-infection.
Salmonella（Salmonella）It is Gram-negative enteric bacterium, is a kind of cause of disease of important food origin disease,
Worldwide cause great economic loss and seriously threaten public health security.Caused by salmonella infection
Salmonellosis be a kind of food-borne zoonosis, clinical symptoms include enterogastritis, bacteremia, typhoid fever and focal infection
Deng；Enterogastritis symptom can be dehydrated from mild abdominal discomfort to vomiting, or even occur dead.People is often because intake is contaminated
Animal-derived food and infect salmonella, such as intake by salmonella-polluted egg.In the world, every year by Salmonella
Enterogastritis case is estimated up to 93,800,000 caused by bacterium infection, death toll up to 155, as many as 000 people, wherein by food
The case that propagation causes accounts for 85% or so.
In Escherichia coli resistance present situation, beta-lactam antibiotic resistance problems are particularly acute.The resistance of this kind of bacterial strain
Mechanism is primarily due to the presence of beta-lactamase, and the enzyme can degrade beta-lactam antibiotic.In recent years, caused in parenterally
New beta-lactamase is occurred in that in characteristic of disease bacterial strain, such as plasmid-mediated AmpC beta-lactamases, wide spectrum beta-lactamase and carbon
Penem enzyme etc..Most famous novel ss-lactam enzyme is reported first in nineteen eighty-three, and is named as wide spectrum beta-lactamase.
This fermentoid can degrade PCs, cephalosporins and monocycle beta-lactam antibiotics, but can not degrade cephamycin and carbon
Penems antibiotics, its activity can be suppressed by typical beta-lactamase inhibitor.Carbapenem resistance is that occur recently
New problem, mainly caused by plasmid-encoded carbapenem enzyme, and at present carbapenem resistance mainly appear on doctor
The coli strain of institute, no small problem is caused to the preventing and treating of coli-infection.
Salmonella drug resistance problems are also increasingly serious, and multiple antibiotic resistant strain emerges in an endless stream, to erythromycin, ammonia benzyl mould
The drug resistance of element, SMZco, Ciprofloxacin etc. is appeared in the newspapers.The salmonella drug resistance problems of large-scale pig farm also compare
Sternness, certain researcher carries out drug resistance analysis to the salmonella for being isolated from pig farm excrement sample, finds isolated strains to tetracycline, ring
Third husky star, Enrofloxacin, Amoxicillin, kanamycins, Florfenicol and ampicillin etc. show height resistance.At present, it is many
Weight antibody-resistant bacterium is widely current, and epidemic link constantly produce to clinics such as quinolones, Third generation Cephalosporins
The drug resistance of important antibiotic, as thorny problem emerging in world wide.Therefore, the research and development of novel antibacterial preparation seem pole
For urgent.
Bacteriophage is widely present in environment, is the virus with microorganisms such as fungi, bacteriums as host, virulent phage tool
There is efficient sterilizing ability.Bacteriophage synthesizes a kind of bacterial virus catenase in the latter stage in the cycle of infecting, lyases destruction host's
Cell membrane so that host cell osmotic unbalances simultaneously finally rupture and reach the purpose for discharging progeny phage.Bacteriophage and its
Lyases equally has killing action to antibiotic resistant bacteria, by technique for gene engineering, can artificial synthesized phagocytosis in large quantities
Body lyases.Therefore, bacteriophage and its lyases have huge application prospect.
The content of the invention
The technical problems to be solved by the invention be seek it is new can be while suppressing the antibacterial of Escherichia coli and salmonella
Preparation, to reduce the use with substitute antibiotics, there is provided expression can crack the bacteriophage restructuring of Escherichia coli and salmonella
The preparation method of the recombination bacillus coli BL21/pET-32a-lysin of lyases TrxA-lysin.
Second object of the present invention is to provide the restructuring lyases TrxA-lysin that the above method is prepared.
Third object of the present invention is to provide the restructuring lyases TrxA-lysin to be used to crack Escherichia coli and sand
Door Salmonella.
The purpose of the present invention is achieved by the following technical programs：
A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella, comprises the following steps：
S1. the lyases lysin genes of phage E CGD1 are expanded；It is connected with plasmid, and transgene engineering bacteria is contained
The genetic engineering bacterium of lysin recombinant plasmids；
S2. the OD values for cultivating the genetic engineering bacterium containing lysin recombinant plasmids to bacterium solution are 0.6~0.8, are induced with IPTG
Genetic engineering bacterium containing lysin recombinant plasmids, obtains soluble recombinant protein TrxA-lysin；The lyases lysin genes
Sequence such as SEQ ID NO：Shown in 1.
Preferably, lyases lysin genes described in S1 are expanded by primer P1 and P2 and obtained, the sequence of the primer P1 and P2
Such as SEQ ID NO：2 and SEQ ID NO：Shown in 3.
PET-32a expression systems have advantages below：Belong to prokaryotic expression system, it is adaptable to phage splitting enzyme gene
Expression, carries sulphur hydrogen reduction albumen TrxA genes, can increase the disulfide formation of foreign protein, increases the solubility of destination protein
Expression.
Used as a kind of specific embodiment, present invention design primer obtains cleavable Escherichia coli using the method for PCR
And the bacterial virus catenase lysin genes of salmonella.The gene is connected in pET-32a expression plasmids and builds recombination expression
Plasmid pET-32a-lysin, with ITPG induced expression recombinant proteins TrxA-lysin.
Preferably, the condition of IPTG inductions is described in S2：The final concentration of 1mM of IPTG, induction time is 2h, inducing temperature
It is 37 DEG C.
As a kind of more preferably implementation method, the S1 of the preparation method of bacterial virus catenase of the present invention, including with
Lower step：
（1）Design primer, using round pcr to the lyases of the phage E CGD1 of Escherichia coli and salmonella can be cracked
Lysin genes are expanded.
（2）The lysin genes of acquisition are connected to pET-32a and screening obtains recombinant plasmid pET-32a-lysin；Using
Heat-shock transformed method imports in e. coli bl21 pET-32a-lysin recombinant plasmids, and acquisition recombination bacillus coli BL21/
pET-32a-lysin。
Preferably,（1）The response procedures of the PCR are：98 DEG C of min of predegeneration 3；98 DEG C of 10 s of denaturation, 55 DEG C of annealing 10
S, 72 DEG C of 10 s of extension, completes 30 and circulates；Extend 5 min after 72 DEG C.
Preferably,（2）The heat-shock transformed method is：E. coli bl21 competent cell is taken, pET-32a-lysin is added
Recombinant plasmid, mixes the min of ice bath 30, and in after 42 DEG C of heat-shock transformed 90 s, the min of ice bath 2 adds LB fluid nutrient mediums recovery 30
Min, plate screening positive transformant.
Preferably, the preparation method of bacterial virus catenase of the present invention also includes purification of soluble recombinant protein TrxA-
The step of lysin.
It is highly preferred that the step of purification of soluble recombinant protein TrxA-lysin is：After induced expression is collected by centrifugation
BL21/pET-32a-lysin somatic cells, washing, it is resuspended after filtering, with Ni NTA Beads load column chromatography recombinate
Albumen TrxA-lysin.
The present invention also provides the bacterial virus catenase recombinant protein TrxA-lysin that the above method is obtained.
The present invention also provides applications of the bacterial virus catenase recombinant protein TrxA-lysin in terms of bacterium is cracked.
The present invention also provides the bacterial virus catenase recombinant protein TrxA-lysin and is preparing prevention and/or treatment bacterium
Application in terms of the medicine of infection.
Preferably, in above-mentioned application, the bacterium is Escherichia coli and/or salmonella.
Compared with prior art, the invention has the advantages that：
The invention provides a kind of preparation method of the recombination bacillus coli for expressing phage E CGD1 lyases lysin genes.I.e.
UsingE.coliBL21/pET-32a escherichia expression systems, are weighed to phage E CGD1 lyases lysin genes
Group expression, reducing albumen TrxA by sulphur iron increases the formation of destination protein disulfide bond and the optimization of expression condition, successfully obtains
The soluble protein of high activity, protein purification needs not move through the denaturation renaturation process of complexity.Meanwhile, by Escherichia coli after induction
Between -80 DEG C and room temperature after freeze thawing 3 times, bacterium re-suspension liquid becomes clarification, sticky to BL21/pET-32a-lysin re-suspension liquids, i.e.,
By simple freeze thawing, cell rupture can be made and most of destination protein is discharged.Lyases is recombinated in the present invention slightly to carry
The preparation of thing can be obtained by simple freeze thawing, not only easy to operate, with low cost, and not be easily caused destination protein denaturation,
It is suitable for a large amount of preparations of lyases.
The recombinant protein TrxA-lysin that will be prepared is used to crack the pretreated Escherichia coli of chloroform and Salmonella
Bacteria strain, it is found that TrxA-lysin has splitting action to many plants of Escherichia coli and Salmonella strains, that is, confirm TrxA-
Lysin can simultaneously crack Escherichia coli and salmonella.Simultaneously the fragmentation pattern wide of TrxA-lysin disclose it in Escherichia coli and
There is potential application value in the prevention and control of salmonella infection.
Brief description of the drawings
Fig. 1 is phage E CGD1 electromicroscopic photographs（Amplify 49,000 times）.
Fig. 2 is the PCR amplifications of phage E CGD1 lyases lysin genes：M is the DNA Marker of DL 2 000；1
It is negative control；2 and 3 is the pcr amplification product of lyases lysin genes.
Fig. 3 is the double digestion qualification result of recombinant plasmid pET-32a-lysin：M1 is DL10000 DNA Marker；M2
It is the DNA Marker of DL 5 000；1 is recombinant plasmid pET-32a-lysin；2 pass through for recombinant plasmid pET-32a-lysinNco I、HindIII carries out double digestion qualification result.
Fig. 4 is the purification result of lyases recombinant protein TrxA-lysin：M is protein standard molecular weight；1 is BL21/
PET-32a induces the total bacterial protein of 4 h；2 and 3 be respectively BL21/pET-32a-lysin induce 2 h total bacterial proteins and
Its thalline freeze thawing supernatant is through the sample after 0.22 μm of filtering；4 is the lyases recombinant protein after ni-sepharose purification.
Fig. 5 is the lytic effect of the lyases recombinant protein TrxA-lysin DH5 α cells pretreated to chloroform.
Specific embodiment
With reference to Figure of description and specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate
The present invention rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in lower example embodiment, generally according to
This area normal condition or the condition advised according to manufacturer.Unless otherwise defined, all specialties used in text and section
Learn term identical with meaning familiar to the person skilled in the art.
E. coli bl21 of the embodiment 1 containing lysin genes expresses the structure of bacterial strain
Comprise the following steps：
S1. the structure of recombinant plasmid pET-32a-lysin
S11. the acquisition of lysin genes：Isolated one plant can simultaneously crack Escherichia coli and Salmonella from sanitary sewage
The bacteriophage of bacterium（ECGD1 is named as, its electromicroscopic photograph is shown in Fig. 1）, the genome of the bacteriophage is extracted after mass propgation bacteriophage,
And carry out the splicing and analysis of high-flux sequence and sequencing result.
The extraction of phage E CGD1 genomes：The bacteriophage concentrate after 600 μ l filtration sterilizations is taken, DNase I are added
1 μ g/ml are with RNase A to final concentration, 37 DEG C of digestion are overnight（Thoroughly remove Host Strains nucleic acid）, 80 DEG C of 15 min of inactivation
（Note：The temperature can not inactivate RNase A）；To the 0.5 mol/L EDTA that 24 μ l are separately added into concentrate（Final concentration 20
mmol/L）, the 20 mg/mL Proteinase Ks of 1.5 μ l（The μ g/mL of final concentration 50）, the 10%SDS of 30 μ l（Final concentration 0.5%）, 56
DEG C h of water-bath 1；Plus isometric balance phenol, gentle concussion 1 min, 12 000 rpm are centrifuged 5 min, shift upper strata aqueous phase in new
In centrifuge tube；Plus isometric phenol-chloroform-isoamyl alcohol（25：24：1）, gentle concussion 1 min, 12 000 rpm 5 min of centrifugation,
Transfer upper strata aqueous phase is in new centrifuge tube；Plus isometric isopropanol, -70 DEG C of 3 h of placement；4 DEG C, 13 000 rpm centrifugations 20
Min, slowly outwells supernatant；Isometric 75% ice ethanol is added, 1 min is stood, 7 000 g is centrifuged 1 min, slowly outwells ethanol,
Room temperature is uncapped and places 10 min, ethanol is volatilized completely, with appropriate deionized water dissolving DNA, -20 DEG C of preservations.
Phage E CGD1 complete genome sequences are uploaded to GenBank（Serial No.：KU522583.1）, noted by sequence
Release and comparison obtains lysin genes, size is 501 bp.Primer P1, P2 are designed according to the gene order, in sense primer P1
Middle insertion restriction enzymeNcoI restriction enzyme sites,NcoI is the amalgamation and expression restriction enzyme site containing initiation codon ATG,
Its 5 ' end adds 4 protection base CATG.Restriction enzyme is inserted in anti-sense primer P2HindThe restriction enzyme site of III,
The end of anti-sense primer 5 ' adds 3 protection base CCC.Lysin gene orders such as SEQ ID NO：Shown in 1；P1, P2 primer of synthesis
Sequence such as SEQ ID NO：Shown in 2~3；Genome with 1 μ L phage Es CGD1 is template, adds high-fidelity DNA polymerase
Prime STAR Max（2×）25 μ L, 10 μM of each 1 μ L of primer P1, P2, use ddH2O is mended to 50 μ L, and response procedures are
98 DEG C of min of predegeneration 3；98 DEG C of denaturation 10 s, 55 DEG C of annealing 10 s, 72 DEG C of 10 s of extension, complete 30 and circulate；Extend 5 after 72 DEG C
min.PCR reactions are completed, and product are carried out the observation of 1% Ago-Gel and are reclaimed, it is seen that the amplified band of the bp of size about 500,
It is consistent with expected results（Such as Fig. 2）.
S12. the structure of recombinant plasmid pET-32a-lysin：The PCR primer that will be reclaimed in S11 is usedNco I、Hind III
Carry out double digestion treatment, the band of the bp of glue reclaim size about 500；Double enzymes are carried out to pET-32a empty plasmids in the same way
Cut, the band of the bp of glue reclaim size about 5900.The lysin genes for taking glue reclaim after 4 μ L double digestions respectively completely modify fragment
With the pET-32a empty plasmids of glue reclaim after 1 μ L double digestions, add 1 μ L T4 DNA ligases and 1 μ L 10 ×
Buffer, uses ddH2O is mended to 10 μ L, is connected under the conditions of 16 DEG C are placed in after mixing overnight, and connection product is convertedE.coli DH5
α competent cells, are containing 100 μ g/mL ampicillins（Ampicillin）LB agar plates in, 37 DEG C were cultivated
Night, then picking single bacterium colony.By colony inoculation in containing 100 μ g/mL ampicillins（Ampicillin）LB liquid training
Support incubated overnight in base.PCR identified with bacterium solution to be checked as template, adds the μ of archaeal dna polymerase 2 × EsTaq Master Mix 10
Each 0.5 μ L of L, primer P1, P2, the μ L of template 0.5, use ddH2It is 95 DEG C of min of predegeneration 5 that O is mended to 20 μ L, PCR response procedures,
95 DEG C of denaturation 30 s, 55 DEG C of annealing 30 s, 72 DEG C of 1 min of extension, 30 circulations；Extend 10 min after 72 DEG C.PCR primer uses 1%
Agarose gel electrophoresis is detected, it is seen that the amplified band of the bp of size about 500, consistent with expected results（Such as Fig. 1）.To inspection
Survey positive bacterium solution carries out plasmid extraction with DNA extraction agent box, and carries out double digestion identification and sequencing, double digestion
There are two purpose bands of about 500 bp and 5900 bp in rear electrophoresis（Such as Fig. 3）, and sequencing identification result is consistent with expection, i.e.,
Obtain recombinant plasmid pET-32a-lysin.
S2. the e. coli bl21 containing lysin genes expresses the structure of bacterial strain
S21. the preparation of e. coli bl21 competent cell：By what is frozenE.coliBL21 is in line on LB plating mediums
Recovery, picking single bacterium falls within LB fluid nutrient mediums overnight Amplification Culture.According to 1：100 ratio meets the BL21 of incubated overnight
Plant in the fresh LB fluid nutrient mediums of 25 ml, culture to logarithmic phase, OD600nmValue about 0.6.Nutrient solution is transferred in centrifuge tube,
10 min are placed on ice, and 10 min are centrifuged in 3000 g at 4 DEG C.Supernatant discarded, with the CaCl of 0.05 mol/L of precooling2It is molten
The mL of liquid 10 gently suspension cells, after placing 15-30 min on ice, 3000 g are centrifuged 10 min at 4 DEG C.Supernatant discarded, adds 4
The CaCl of 0.05 mol/L of the mL precoolings containing 15% glycerine2Solution, gently suspension cell, places a few minutes on ice, impression
State cell suspension.
S22. the PCR identifications of the conversion of e. coli bl21 and transformant：Take 50 μ L e. coli bl21 competence thin
Born of the same parents, melt on ice bath, add 1 μ L pET-32a-lysin recombinant plasmids, gently mix；By the min of thing ice bath 30 mixed above
After be put into 42 DEG C of s of heat shock 90, rapidly by the min of mixture ice bath 3, add 100 μ L LB fluid nutrient mediums, in 37 DEG C, 140
The min of condition of culture culture 45 of rpm.Bacterium solution after culture is spread evenly across containing 100 μ g/mL ampicillins
（Ampicillin）LB solid mediums in, in 37 DEG C of overnight incubations, picking single bacterium colony.By colony inoculation in containing 100 μ g/
ML ampicillins（Ampicillin）LB fluid nutrient mediums in incubated overnight.PCR identified with bacterium solution to be checked as template, plus
Enter archaeal dna polymerase 2 × EsTaq Master Mix 10 μ L, primer P1, P2 each 0.5 μ L, the μ L of template 0.5, use ddH2O mend to
20 μ L, PCR response procedures are 95 DEG C of predegenerations 5 min, 95 DEG C of denaturation 30 s, 55 DEG C of 30 s of annealing, and 72 DEG C extend 1 min, 30
Individual circulation；Extend 10 min after 72 DEG C..PCR primer is detected with 1% agarose gel electrophoresis, it is seen that the bp's of size about 500
Amplified band.
S3. lysin genes are containedE.coliBL21/pET-32a-lysin recombinantly expresses bacterium induced expression process：
First it is the determination of optimal induction time, PCR is accredited as the BL21/pET-32a-lysin line overnight incubations of the positive；
Picking single bacterium colony is inoculated in 3 mL LA fluid nutrient mediums, 37 DEG C, and 220 rpm cultivate 10 h, takes out bacterium solution, puts 4 DEG C of refrigerator mistakes
Night；Above-mentioned bacterium solution is inoculated in 3 mL LA fluid nutrient mediums, 37 DEG C, 220 rpm shaking cultures by 1% volume ratio；Work as OD600
For 0.6~0.8 when, add final concentration of 1 mmol/L IPTG to carry out induced expression.Take induction different time bacterium solution, 12,
000 rpm is centrifuged 2 min, removes supernatant, and 100 μ L 1 × SDS sample-loading buffers are added in bacterial precipitation, 5 min is boiled in boiling water left
The right side, 12,000 rpm are centrifuged 2 min, and taking supernatant carries out the optimal induction time of SDS-PAGE electrophoresis detections.Meanwhile, set up pET-
Induced expression bacterium solution of the 32a empty plasmids in BL21 is used as control.Before loading, gel is put into Vertial electrophorestic tank, voltage is set
Being set to 80 V carries out the min of prerunning 30 to eliminate the influence of unpolymerized material in gel；Setting voltage is 80 V after loading, is treated
Bromophenol Blue dye is pressed into a straight line for level, is brought the voltage up to 160 V when will enter separation gel；Dyestuff is close to gel bottom
Stop electrophoresis during portion.Gel is removed, in dyeing 1 h or so with coomassie brilliant blue staining liquid on horizontal shaker, then is entered with destainer
Row decolourize, period change destainer, decolourize to zone of protein it is clear, gel is taken a picture with gel imaging system.
Result shows that optimal induction time is 2 h.Using the optimal induction time of above-mentioned determination, luring for destination protein is carried out
Lead expression.Be collected by centrifugation induction after thalline, with 1 mL PBS it is resuspended after, multigelation 3 times between -80 DEG C and room temperature, 4 DEG C
Under the conditions of 10,000 rpm 2 min are centrifuged, take supernatant, add isometric 2 × SDS sample-loading buffers, 5 min are boiled in boiling water
Left and right, 12,000 rpm are centrifuged 2 min, and taking supernatant carries out the expression-form of SDS-PAGE electrophoresis detection recombinant proteins, as a result shows
Show destination protein predominantly solubility expression.
S4. recombinant protein TrxA-lysin purge processes：The BL21/pET-32a-lysin after induced expression is collected by centrifugation
Somatic cells, are washed twice with PBS.According to thalline：Lysis buffer = 1：10（W/V）Thalline is suspended, in -80
DEG C multigelation 3 times and room temperature between, 10,000 rpm is centrifuged 20 min under the conditions of 4 DEG C, takes supernatant, and filtered with 0.22 μm
Membrane filtration, collects filtrate, for the purifying of destination protein.
Ni NTA Beads are loaded into suitable chromatographic column, pillar is balanced with 5 times of Lysis buffer of column volume, make to fill out
Material is under identical buffer system with destination protein, plays a part of protected protein.Sample is added to the Ni NTA for having balanced
In Beads（Ensure destination protein and Ni2+It is fully contacted, improves the rate of recovery of destination protein）, efflux is collected, for SDS-
The combination situation of PAGE analysing proteins.Cleaned with the Wash buffer of 10~15 times of column volumes, removal is non-specific to inhale
Miscellaneous liquid is washed in attached foreign protein, collection.Pillar is eluted using 5~10 times of Elution buffer of column volume, eluent is collected, i.e.,
Destination protein component.Follow-up desalination and concentration are carried out to the albumen for purifying using 10 kDa ultra-filtration centrifuge tubes.Purification result
As shown in Figure 4.
The Activity determination of the recombinant protein TrxA-lysin of embodiment 2
Comprise the following steps：
S1. the pretreated DH5 α of chloroform are cracked with recombinant protein TrxA-lysin：Test strains are inoculated in LB in 1% ratio
In culture medium, incubated overnight adds final concentration of 5% chloroform, the min of shaken cultivation 15 thalline to be collected by centrifugation, is used in combination in bacterium solution
Deionized water is cleaned twice, and -80 DEG C save backup.Before determining activity, the bacterial precipitation Tris-HCl of 50 mmol/L（PH
8.2, containing 0.1% Triton X-100）It is resuspended.By the cracking enzyme solutions of 10 μ L（Recombinant protein TrxA-lysin solution）Add
To in 90 μ L bacterium re-suspension liquids, it is stored at room temperature to lytic effect substantially, determines OD450.Experiment sets three repetitions, and will
The treatment of Tris-HCl buffer solutions is used as negative control.Because phage E CGD1 can crack Recombinant organism strain DH5 α,
Therefore the lytic activity of restructuring lyases TrxA-lysin is first detected as test strains with DH5 α.Using the turbid of chloroform pretreatment
The lytic activity of degree method detection restructuring lyases TrxA-lysin, it is found that bacterium solution becomes rapidly clear in the presence of TrxA-lysin
Clearly, OD4500.79 is reduced in 10 min（Fig. 5）, illustrating the TrxA-lysin of this experiment expression has lytic activity.
S2. PB cracks viable bacteria with recombinant protein TrxA-lysin combinations：The experiment is divided into two groups, including glues more
Rhzomorph B and recombinant protein synergy group and PB control group.Synergy group test process substantially, with 2 times of multiple proportions
Dilution method is prepared and contains PB final concentration of 210、29、28、27……2-4、2-5The LB culture mediums of μ g/mL（Each gradient 3
mL）, 100 μ L recombinant protein TrxA-lysin solution are added toward each dilution gradient, then bacillus coli DH 5 is inoculated with 1% ratio
α, 37 DEG C of shaken cultivations are overnight.PB control group is processed ibid, but without recombinant protein TrxA-lysin solution.
Result shows, in PB control group, PB is 2 μ g/mL to DH5 α minimum inhibitory concentrations；Lyases TrxA-
In lysin and PB synergy group, PB is 1 μ g/mL to DH5 α minimum inhibitory concentrations.That is lyases
The presence of TrxA-lysin reduces the minimum inhibitory concentration of PB, so as to prove that both has collaboration fungistatic effect.
S3. the measure of recombinant protein TrxA-lysin fragmentation patterns：The method detection pre-processed using the chloroform described in S1 is split
Solution enzyme recombinant protein observes bacteria lysis effect and its OD to the lytic activity of test strain450Value changes.Test strain includes
Escherichia coli and salmonella Clinical isolation, and Recombinant organism strain.Lyases splits to test strains
Solution value=Δ 450nm（Test group decreasing value）—Δ450nm（Tris-HCl control group decreasing value）.With respect to lytic activity=lyases
To the cracking value/lyases of test strains to the cracking value of DH5 α.It is cracking positive criteria with bacillus coli DH 5 alpha, using chloroform
The nephelometry of pretreatment detects that lyases to Escherichia coli and the lytic activity of salmonella, as a result shows that lyases is to difference
The Escherichia coli and salmonella in source have lytic activity, show to live the broad-spectrum sterilization of Escherichia coli and salmonella
Property.Learn that lyases is generally higher than salmonella to the lytic activity of Escherichia coli by relative lytic activity measurement result.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 501
<212> DNA
<213>Lyases lysin gene orders
<400> 1
atggctaaag tagttgatgt gtttgatatg ttacgttttg atgaaggact aaagctaact 60
gtatatactg ataccgaagg gtattggacg gttggtattg gacaccttct gacaaaactt 120
aaagataaat cagaagctat acgcattctt gataacttag taggtagaaa aactaatggg 180
gttattactg aagcggaagc tagaaaaatc tttgagggtg acgtaaagaa agcgatacaa 240
caaatccatt caagtaccat attatctcct atttacgata aagtaagtcc taatcgtaaa 300
atggctatta tcaatatggt atttcaaatg ggattgaaag gtgcagaatc tttcaaaaat 360
agcttgactt tagtgagtaa ttcatattat actcaagcct ctataaattt acgtaaaagt 420
aaatggtatc gccaaacgcc taatcgcgca gagcgtgtaa ttcaggtgct taaaactgga 480
acattagacg cttataacta a 501
<210> 2
<211> 26
<212> DNA
<213>Primer P1 sequences
<400> 2
catgccatgg ctaaagtagt tgatgt 26
<210> 3
<211> 29
<212> DNA
<213>Primer P2 sequences
<400> 3
cccaagcttt tagttataag cgtctaatg 29
Claims (8)
1. a kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella, it is characterised in that including
Following steps：
S1. the lyases lysin genes of phage E CGD1 are expanded；It is connected with plasmid, and transgene engineering bacteria is contained
The genetic engineering bacterium of lysin recombinant plasmids；
S2. the OD values for cultivating the genetic engineering bacterium containing lysin recombinant plasmids to bacterium solution are 0.6~0.8, are induced with IPTG
Genetic engineering bacterium containing lysin recombinant plasmids, obtains soluble recombinant protein TrxA-lysin；The lyases lysin genes
Sequence such as SEQ ID NO：Shown in 1.
2. the preparation method of bacterial virus catenase according to claim 1, it is characterised in that lyases lysin described in S1
Gene is expanded by primer P1 and P2 and obtained, the sequence such as SEQ ID NO of the primer P1 and P2：2 and SEQ ID NO：Shown in 3.
3. the preparation method of bacterial virus catenase according to claim 1, it is characterised in that the bar of IPTG inductions described in S2
Part is：The final concentration of 1mM of IPTG, induction time is 2h, and inducing temperature is 37 DEG C.
4. the preparation method of bacterial virus catenase according to claim 1, it is characterised in that also including purification of soluble weight
The step of histone TrxA-lysin.
5. the bacterial virus catenase recombinant protein TrxA-lysin that any one of Claims 1-4 method is obtained.
6. applications of the bacterial virus catenase recombinant protein TrxA-lysin described in claim 5 in terms of bacterium is cracked.
7. bacterial virus catenase recombinant protein TrxA-lysin described in claim 5 is preparing prevention and/or treatment bacterium infection
Medicine in terms of application.
8. the application according to claim 6 or 7, it is characterised in that the bacterium is Escherichia coli and/or salmonella.
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| CN109568494A (en) * | 2019-01-22 | 2019-04-05 | 浙江中医药大学 | A kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim |
| CN110527655A (en) * | 2019-07-15 | 2019-12-03 | 江苏省家禽科学研究所 | A kind of duck source Escherichia coli probiotics strain and its screening preparation method and purposes |
| CN110592056A (en) * | 2019-09-19 | 2019-12-20 | 昆明理工大学 | Phage lyase composite powder and preparation method and application thereof |
| CN112175928A (en) * | 2020-10-13 | 2021-01-05 | 华中农业大学 | Application of protein encoded by salmonella bacteriophage gene as gram-negative bacteria lyase |
| CN114292836A (en) * | 2021-11-05 | 2022-04-08 | 广东医科大学 | Lyase of endoproteolyticenza salmonella bacteriophage, encoding gene thereof, preparation method and application thereof |
| WO2023279983A1 (en) * | 2021-07-05 | 2023-01-12 | 中国科学院武汉病毒研究所 | Antibacterial peptide p104 and lyase lysp53 having broad-spectrum cracking activity, and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109568494A (en) * | 2019-01-22 | 2019-04-05 | 浙江中医药大学 | A kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim |
| CN110527655A (en) * | 2019-07-15 | 2019-12-03 | 江苏省家禽科学研究所 | A kind of duck source Escherichia coli probiotics strain and its screening preparation method and purposes |
| CN110592056A (en) * | 2019-09-19 | 2019-12-20 | 昆明理工大学 | Phage lyase composite powder and preparation method and application thereof |
| CN112175928A (en) * | 2020-10-13 | 2021-01-05 | 华中农业大学 | Application of protein encoded by salmonella bacteriophage gene as gram-negative bacteria lyase |
| WO2023279983A1 (en) * | 2021-07-05 | 2023-01-12 | 中国科学院武汉病毒研究所 | Antibacterial peptide p104 and lyase lysp53 having broad-spectrum cracking activity, and applications thereof |
| CN114292836A (en) * | 2021-11-05 | 2022-04-08 | 广东医科大学 | Lyase of endoproteolyticenza salmonella bacteriophage, encoding gene thereof, preparation method and application thereof |
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