CN102703491A - Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof - Google Patents
Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof Download PDFInfo
- Publication number
- CN102703491A CN102703491A CN2012101626403A CN201210162640A CN102703491A CN 102703491 A CN102703491 A CN 102703491A CN 2012101626403 A CN2012101626403 A CN 2012101626403A CN 201210162640 A CN201210162640 A CN 201210162640A CN 102703491 A CN102703491 A CN 102703491A
- Authority
- CN
- China
- Prior art keywords
- cracking
- bacteria
- preparation
- protein
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a bacteria-cracking preparation for effectively cracking escherichia coli as well as a cracking method and an application thereof. The bacteria-cracking preparation is prepared by the following steps of: designing a primer; using PCR (polymerase chain reaction) to amplify catenase gene in lysogenic escherichia coli for inducing lytic phage to obtain a target segment; connecting the target segment with pET-28a(+) carrier to obtain recombinant expression plasmid; introducing the recombinant expression plasmid to escherichia coli BL21(DE3) to express, thus obtaining positive transformant; expressing the recombinant fusion protein by induction; performing purification and induction to obtain purified recombinant protein, and screening the recombinant expression protein with cracking activity to obtain the bacteria-cracking preparation. The cracking method comprises the step of adopting 0.8% of beta-mercaptoethanol or 10mM of cysteine as reducing agent at pH of 8.0 and temperature of 37 DEG C, and 20mM of sodium acetate buffer liquid at pH of 5.2. The bacteria-cracking preparation obtained by the method provided by the invention has strong cracking specificity and high cracking efficiency for various serotype escherichia coli, and can effectively kill the escherichia coli.
Description
Technical field
The present invention relates to a kind of preparation method of splitting bacteria preparation of biological technical field, be specifically related to colibacillary bacteria preparation and cleavage method thereof, the purposes split of a kind of efficient cracking.
Background technology
The intestinal bacteria main parasitic is in the enteron aisle of humans and animals; Great majority are normal microfloras of enteron aisle; But can become pathogenic bacterium under given conditions; It is interior pathogenic pathogenic outward with enteron aisle to cause enteron aisle, shows as airsacculitis, arthrosynovitis and the granuloma etc. of diarrhoea, hemorrhagic enteritis, septicemia, bird.Wherein morbific typical serotype representative is O157 in the enteron aisle; It is EHEC (Enterohemorrhagic Escherichia coli; EHEC) a main serotype is the former bacterium of food source property Amphixenosis, and it can cause people's food poisoning through the food that pollutes; But therefore serious causing death develops the killing colon bacillus high-efficiency preparation and aspect public health, has great importance.
At present can kill bacteria mainly comprise multiple sterilizing agent, sanitas, microbiotic, metabolic antagonist etc.Yet, along with the widespread use of these medicines, not only having produced a large amount of drug residues, the multidrug resistant of bacterium, high dosage resistance etc. are also more and more serious, and the propagation of the Resistant strain of bacterium further aggravates, thereby have increased environment and human threat.Therefore scientists is being explored the preparation of new kill bacteria always.Phage is the virus of infringement bacterium, not only can mediate the change of host bacterium biological characteristics, can also influence the breeding cycle of host bacterium, the molten former and cracking state of decision host bacterium.Lyase has specificity hydrolysis host bacteria cell walls in the lytic phage, destroys the integrity of bacteria cell wall, thus kill bacteria.Phage-coded lyase can only be attacked specific bacterium as phage, has higher specificity.And the speed that lyase acts on bacterium is exceedingly fast; So that bacterium can not produce resistance to this enzyme; And the lyase of phage has than phage antimicrobial spectrum more widely, therefore screens the preparation of kill bacteria from the angle of bacterial virus catenase, is the new direction in the present antibiotic preparation.
The research of bacterial virus catenase so far mainly concentrates on the bacterioclasis to gram-positive microorganism; For example the research of the lyase of suis, anthrax bacillus, mycobacterium tuberculosis etc. is more relatively; And to Gram-negative bacteria, particularly the research of coliphage lyase does not have report as yet.Because different phages has different lyase sequences; Even identical gene order also can be brought into play different effects in different phages; And the gene that has also can receive the restriction of a lot of conditions at vivoexpression, screens the microbial inoculum that splits that really has bacterioclasis thereby hindered.The present invention is through comparing the genome series of Escherichia coli O 157 phage Min27; Screen a plurality of specific sequence fragments,, carry out prokaryotic expression, induce and purifying through modifying; Obtained the activated bacteria preparation that splits, can have splitting action the intestinal bacteria of multiple serotype.
Summary of the invention
The objective of the invention is to screen gene with specific bacterioclasis; Through making up prokaryotic expression system; Screen recombination expression product with lytic activity; Confirming influences the active optimum cracking condition of expression product, and a kind of can cleaving various serotype colibacillary bacteria preparation and cleavage method thereof, purposes split is provided.
The objective of the invention is to realize through following technical scheme:
The present invention relates to the colibacillary bacteria preparation that splits of a kind of efficient cracking, the said bacteria preparation that splits gets through following method preparation:
Step 1 through the method for phage induction, is induced the intestinal bacteria lysogenic strain, obtains the lytic phage particle, and purifying obtains phage, extracts the DNA of phage;
Step 2, the design primer is a template with step 1 gained DNA, the Using P CR corresponding gene fragment that increases;
Step 3 adopts prokaryotic expression system pET-28a (+), with the step 2 gained DNA construction recombination plasmid that increases;
Step 4, to expressing bacterium BL21 (DE3), the screening positive transformant carries out induction expression of protein and evaluation, obtains recombinant protein with the recombinant plasmid transformed of step 3;
Step 5, the recombinant protein of purifying abduction delivering, screening has the recombinant expression protein of lytic activity, obtains protein formulation, the promptly efficient colibacillary bacteria preparation that splits of cracking.
Preferably, the nucleotides sequence of primer described in the step 2 is classified as, the upper reaches: CGCGGATCCATGAGCAGGAAACTCCG, downstream: CCCAAGCTTTTATCTGTCGATTCCCC.
Preferably, the fragment of amplification gene described in the step 2 is 534bp.
Preferably, the recombinant protein that obtains in the step 4 is 23kDa.
Preferably; The recombinant expression protein that screening described in the step 5 has lytic activity is specially: the bacterial lysate of the purified recombinant albumen that obtains, positive transformant is carried out dull and stereotyped cracking experiment; Relatively split the bacterium effect, thereby screening has the recombinant expression protein of lytic activity.
The invention still further relates to the colibacillary cleavage method that splits bacteria preparation of a kind of aforesaid efficient cracking; The reductive agent that said cleavage method adopts is that mass percent concentration is 0.8% beta-mercaptoethanol or the halfcystine of 10mM; Damping fluid is a 20mM pH5.2 sodium-acetate buffer; The pH value of cracking bacterium liquid is 8.0, and temperature of reaction is 37 ℃.
The invention still further relates to the colibacillary purposes of splitting bacteria preparation of a kind of aforesaid efficient cracking, said cracking preparation is as the cracking preparation of cracking different serotypes coli strain.
Preferably, said serotype coli strain is intestinal bacteria O46, O118, O138, O157 and MC1061 bacterial strain.
Principle of the present invention is: through sequence alignment, design 2 pairs of possible primers, adopt the possible lyase gene of inductive lytic phage Min27 among the pcr amplification lysogeny intestinal bacteria Min27; Obtain the purpose fragment, again the purpose fragment is connected with pET-28a (+) carrier, obtain recombinant expression plasmid pET-28a (+); Import escherichia coli BL21(DE3) expression, obtain positive transformant BL21 (DE3)/pET-28a (+)-P1 and BL21 (DE3)/pET-28a (+)-P2, through inducing; The fusion rotein of express recombinant through Ni column purification and adding reductive agent renaturation, obtains purified recombinant albumen; Has the bacterium of splitting activity, the intestinal bacteria that called after LysEC, said preparation can the multiple different serotypes of cracking; And other entero-bacte of not cracking, and lytic activity is high.
Compared with prior art; The present invention has following beneficial effect: the present invention is split the bacterium experiment through prokaryotic expression system and flat board; Filter out and have the active recombinant expression protein LysEC of the bacterium of splitting, can be the intestinal bacteria of external efficient, specificity cleaving various serotype.
Description of drawings
Fig. 1 is the synoptic diagram of the recombinant expression protein LysEC of employing SDS-PAGE purification Identification;
Fig. 2 is the dull and stereotyped cracking experiment of a purified recombinant albumen LysEC synoptic diagram.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.Among the following embodiment; The experimental technique of unreceipted actual conditions; Usually according to normal condition; Sambrook equimolecular clone for example: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment 1, phage induce the preparation with phage DNA
Through the method for phage induction, induce the intestinal bacteria lysogenic strain, obtain the lytic phage particle, purifying obtains phage, extracts the DNA of phage; Concrete operations are following:
With Escherichia coli O 157 Min27 strain inoculation 5mL LB liquid nutrient medium, after 37 ℃ of shaken overnight, add in the fresh LB liquid nutrient medium with 1: 4 volume ratio; Add ametycin to final concentration 1.0 μ g/mL again; After continuing jolting 14h, add behind 0.1% the chloroform jolting 15min again, the centrifugal 10min of 4000g; The supernatant liquid filtering degerming promptly contains the Stx2 phagocytosis in the filtrating.MC1061 added carry out doubling dilution 10 in the LB substratum
-2, 10
-4, 10
-6, get 200 μ l phage diluents and 200 μ l host bacterium mixings respectively, and add 1mol/L CaCl
2To final concentration 0.1mol/L, 37 ℃ hatch 15min after, mixed solution is added mixing in the top-agar with oneself fusing, pour on the flat board that contains bottom-layer agar, treat that top-layer agar solidifies after, place 37 ℃ of incubators to cultivate 10-12h, observe the plaque form.After single plaque enlarged culturing, collect the phage suspension.
Go into to add Proteinase K to final concentration 50 μ g/ml in the phage suspension, 37 ℃ of incubations 30 minutes add SDS again to final concentration 0.5%, mixing, 56 ℃ of incubations 1 hour.Carry out extracting with equal-volume Tris balance phenol (pH8.0), centrifugal 10 minutes of 4000g collects the upper strata water.With the extracting once more of equal-volume chloroform, centrifugal 10 minutes of 5000g collects the upper strata water, and is transferred in another centrifuge tube.Add the 3mol/L sodium acetate to final concentration 0.3mol/L at the aqueous phase that reclaims, the absolute ethyl alcohol that adds two volumes again washs gently, and centrifugal 10 minutes of 12000g abandons supernatant, with an amount of TE dissolution precipitation, obtains phage DNA.
Embodiment 2, design 2 pairs of primer primers, the pcr amplification target gene fragment
Designing 2 couples of primer P1 and P2, is template with embodiment 1 gained DNA, the Using P CR corresponding gene fragment that increases, and the amplified fragments of confirming gene is 534bp and 501bp; Concrete operations are following:
Dna sequence dna (NC_010237) according to Escherichia coli O 157 Min27 phage among the GenBank; Design 2 pairs of primer primers; Primer sequence is seen table 1,2 constant gene segment Cs that increase respectively, and the position of primer 1 amplification is at 1-534; The 34-534 amino acids place, position of primer 2 amplification introduces BamH I and Hind III restriction enzyme site respectively at 5 ' end of upstream and downstream primer.With the phage DNA is template, carries out the amplification of target gene fragment according to ordinary method, and reaction is got 5 μ L products and carried out agarose gel electrophoresis after finishing.Utilize the PCR product to reclaim test kit and reclaim the PCR product.
Table 1PCR primer sequence
Embodiment 3, construction of recombinant plasmid
Adopt prokaryotic expression system pET-28a (+),, carry out sequential analysis and comparison, confirm to obtain recombinant plasmid pET-28a (+)-P1 and pET-28a (+) P2 of correct sequence embodiment 2 amplification gained DNA construction recombination plasmids; Concrete operations are following:
The pcr amplification product of embodiment 2 is connected to upward order-checking of pMD-18T carrier (Takara company).The institute designed primer upper reaches comprise BamH I restriction enzyme site, and downstream comprise Hind III restriction enzyme site.BamHI and Hind III enzyme are cut PCR product and pET-28a (+), 10～16 ℃ of connections of spending the night of T4DNA ligase enzyme (Takara company).Connect product and change in the bacillus coli DH 5 alpha, 37 ℃ of incubated overnight are extracted plasmid.Cut evaluation with BamH I and Hind III enzyme, the positive recombinant plasmid order-checking of evaluation obtains positive recombinant plasmid pET-28a (+)-P1 and pET-28a (+)-P2 respectively.
Embodiment 4, the prokaryotic expression of recombinant plasmid
Recombinant plasmid pET-28a (+)-P1 and pET-28a (+)-P2 of embodiment 3 are transformed into expression bacterium BL21 (DE3); Screening positive transformant BL21 (DE3)/pET-28a (+)-P1 and BL21 (DE3)/pET-28a (+)-P2 carry out induction expression of protein and evaluation; The recombinant protein that obtains confirms that its molecular weight of albumen is respectively 23kDa and 22kDa; Concrete operations are following:
Extract positive recombinant plasmid dna, change in the e. coli bl21 (DE3).Obtain positive transformant BL21 (DE3)/pET-28a (+)-P1 and BL21 (DE3)/pET-28a (+)-P2 respectively, respectively 1 colony inoculation 2ml of picking kantlex LB substratum incubated overnight.Get 5mL incubated overnight bacterium liquid inoculation 1L kantlex LB substratum, 37 ℃ of 140rpm shaking culture 3 to 4 hours are to OD
600Be about 1.0.Isopropyl-(IPTG) the 100 μ L that add 1000mmol/L are 1mmol/L to final concentration, 27 ℃ of 80rpm shaking culture 4h.Get the centrifugal 1min of 1ml bacterium liquid 12000rpm; Abandon supernatant, it is resuspended that deposition adds 20 μ L zero(ppm) water, adds 20 μ L, 2 * SDS-PAGE sample-loading buffer mixing again; Carry out the SDS-PAGE gel electrophoresis behind 100 ℃ of boiling water bath 10min, through SDS-PAGE electrophoresis detection expression of recombinant proteins situation.Inductive contains pET-28a empty carrier bacterium to be done as above and to handle the back and carry out the SDS-PAGE electrophoresis as contrast, and the fusion recombinant protein of acquisition is respectively 23kDa and 22kDa.
Embodiment 5, the purifying of fusion rotein
The recombinant protein of purifying abduction delivering obtains protein formulation; Concrete operations are following:
IPTG inductive bacterium 1L be dissolved in the 25mL precooling lysis buffer (contain the 50mM sodium phosphate buffer, pH8.0), ultrasonication.8000g centrifuging and taking supernatant.Wash the Ni post with the lysis buffer of 8 column volumes, with on the sample to post, wash post with the lysis buffer of precooling again, until OD
280＜0.1.Cleaning buffer solution A (lysis buffer adds the 5mM imidazoles) with precooling washes post, until OD
280＜0.1.Cleaning buffer solution B (lysis buffer adds the 20mM imidazoles) with precooling washes post, until OD
280＜0.1.Use dissolution fluid (lysis buffer adds the 250mM imidazoles) to wash post at last, collect elutriant, be purified recombinant albumen.
Fig. 1 is the synoptic diagram of the recombinant expression protein LysEC of employing SDS-PAGE purification Identification, can be known by Fig. 1: the 1st, and the expression product of inductive bacterium; The 2nd, standard Marker albumen; The 3rd, inductive bacterial product not; The 4th, the purifying of bacteria-induction product obtains recombinant protein, can obtain the very high purification of recombinant proteins of purity can be found out the induced product purifying of positive transformant BL21 (DE3)/pET-28a (+) P1 by figure after.
Embodiment 6, and flat board splits the bacterium experiment
The bacterial lysate of purified recombinant albumen, positive transformant BL21 (DE3)/pET-28a (+) P1 and BL21 (DE3)/pET-28a (+)-P2 that embodiment 5 is obtained carries out dull and stereotyped cracking and tests; Relatively split the bacterium effect; Screening has the recombinant expression protein of lytic activity; The expression product of confirming BL21 (DE3)/pET-28a (+)-P1 has the bacterium of splitting activity, with its called after LysEC; Concrete operations are following:
200ml express bacterium BL21/pET28a-P1, BL21/pET28aP2 and empty carrier bacterium BL21/pET28a 27 ℃ centrifugal after inducing 4 hours, with the resuspended ultrasonication afterwards of the aseptic PBS damping fluid (pH 7.2) of 5ml 10mM.Ultrasonic power is 300-400W, work 5s, interval 15s, 120 circulations.Obtain lysate and do dull and stereotyped breaking test.With mixing in the agarose of bacterium adding 0.65% thawing after the intestinal bacteria MC1061 bacterium cleaning of fresh culture, mixture is poured in the plate, and every bore dia 10mm is punched in the cooling back on plate.Difference Dropwise 50 μ l empty carrier lysate, the bacterial lysate of 50 μ l primers 1, the bacterial lysate of primer 2,37 ℃ of incubators are cultivated until observing the transparent inhibition zone that occurs on the substratum.The result shows that the prokaryotic expression product of BL21 (DE3)/pET-28a (+)-P1 shows and splits the bacterium effect; And the prokaryotic expression of BL21 (DE3)/pET-28a (+)-P2 does not split the bacterium effect; Recombination expression product called after LysEC with BL21 (DE3)/pET-28a (+)-P1 is and splits bacteria preparation.
Fig. 2 is the dull and stereotyped cracking experiment of a purified recombinant albumen LysEC synoptic diagram, and can be known by Fig. 2: the bacterial lysate of A:BL21 (DE3)/pET-28a (+)-P2 does not have splitting action to intestinal bacteria indicator bacterium; The bacterial lysate of B:BL21 (DE3)/pET-28a (+)-P1 has splitting action to intestinal bacteria indicator bacterium; The purifying thing of the abduction delivering product of B:BL21 (DE3)/pET-28a (+)-P1 is the strongest to the splitting action of intestinal bacteria indicator bacterium.
Embodiment 7, split confirming of the best cracking condition of bacteria preparation LysEC
The purification of recombinant proteins LysEC that explores embodiment 5 acquisitions brings into play active top condition; Concrete operations are following:
The mensuration of best cracking condition comprises: the test of successively decreasing of measuring methods such as reductant concentration, damping fluid composition, pH value, cracking temperature employing turbidity, confirm best cracking condition.1mg/ml LysEC purifying protein adds 0.5%, 0.8%, 1.0%, 3.0% and 5.0% (V/V) beta-mercaptoethanol respectively; 1mg/ml LysEC purifying protein adds 1mM, 2mM, 4mM, 5mM, 8mM, 10mM, 15mM halfcystine respectively; Or use 20mM/L to carry out resuspended bacterium liquid, and furnishing OD as pH4.0, pH6.0, pH8.0, pH10.0, pH12.0 and pH14.0 sodium phosphate buffer
600Be about 1.0; Or with 20mMpH5.2 sodium-acetate buffer, 10mM pH6.8 phosphate buffered saline buffer, 10mM pH7.2 phosphate buffered saline buffer, the resuspended bacterium liquid of 20mM pH8.5Tris-Cl damping fluid; Or the cracking agent behind bacterial suspension 100 μ l and the 100 μ l purifying mixed, be placed on 4 ℃, 18 ℃, 25 ℃, 37 ℃, 42 ℃ respectively.3 repetitions are respectively done in each experiment, and lysis buffer is as blank.Behind the reaction 30min, read absorbancy under the room temperature at the 600nm place.Get absorbancy and reduce the best cracking top condition of maximum conduct.Beta-mercaptoethanol concentration is 0.8%; Halfcystine: optimum concn is 10mM; The optimum response damping fluid is a 20mM pH5.2 sodium-acetate buffer; The pH value for cracking bacterium liquid of best lytic effect is 8.0; The lyase optimal reaction temperature is 37 ℃.
Embodiment 8, split bacteria preparation LysEC lytic activity
Measure the enzymic activity of the purification of recombinant proteins LysEC of embodiment 5 acquisitions; Concrete operations are following:
The centrifugal 2min of intestinal bacteria 12000rmp with fresh culture abandons supernatant, and it is resuspended that the 10mM PBS (pH7.2) of precooling washes twice back, transfers OD
600Be 1.0.The concentration of purification of recombinant proteins is the LysEC of 1mg/ml; In 96 orifice plates, carry out doubling dilution with 10mM PBS (pH7.2), final volume is 100 μ l, adds 100 μ l bacterial suspensions in each dilution holes; 3 repetitions of each extent of dilution; Bacterial suspension adds 10mM PBS (pH7.2) as blank, reads absorbancy at the 600nm place, the record initial value.96 orifice plates are put 37 ℃, place 50min, and 600nm reads absorbancy in place, writes down reacted light absorption value, calculate turbidity and descend, and can make the inverse of high dilution of the enzyme of bacterial turbidity decline 50% promptly be defined as this protease activities (unit/mg).Under 37 ℃ of conditions, when original content is the LysEC extension rate of 1mg/ml when being 1:3200, the OD600 value of Escherichia coli O 157 descends 50%, and therefore, the activity of LysEC is 3200u/mg, and the contained LysEC amount of per unit is 0.31 μ g.
Embodiment 9, split bacteria preparation LysEC and split the bacterium spectrum
Measure the scope of splitting the bacterium spectrum of the purification of recombinant proteins LysEC of embodiment 5 acquisitions; Concrete operations are following:
Use 20mM pH7.2PBS (sodium phosphate) damping fluid resuspended respectively after centrifugal bacterial strains such as the Salmonellas that grows to logarithmic phase, golden yellow grape ball, suis, intestinal bacteria O46, O118, O138, O157 and MC1061, be adjusted to OD
600Be 1.0.Bacterial suspension after the dilution adds respectively in 96 orifice plates, and every hole 100 μ l do four repetitions.With final concentration is that the LysEC of 1.25 μ g/ml splits bacteria preparation 100 μ l and adds in the bacterial suspension.Every strain bacterium is cooked 3 repetitions, and a remaining hole adds 20mM pH7.2 sodium phosphate buffer 100ul as blank.After 37 ℃ 50min is reacted in concussion down, read absorbance at extinction wavelength 600nm place.With bacterial turbidity decline 50% is the cracking positive criteria, detects the splitting action of LysEC to bacterial strains such as Salmonellas, golden yellow grape ball, suis, intestinal bacteria O46, O118, O138, O157 and MC1061.Lyase LysEC1 only has splitting action to the coli strain of different serotypes as a result, and Gram-negative bacteria such as Salmonellas, 3 kinds of gram-positive microorganisms are comprised that golden yellow grape ball and suis all do not have lytic activity (seeing table 2).LysEC can reach 51%～73% to the colibacillary lysis efficiency of 6 strains, and all is not higher than 10% for other bacterial strain lysis efficiency.
The bacterium spectrum of splitting of table 2LysEC is measured
| Bacteria name | Bacterial strain number | Initial OD 600 | Effect back OD 600 | Lytic activity |
| Subtilis | 0.335 | 0.323 | - | |
| Streptococcus aureus | ATCC | 0.298 | 0.297 | - |
| Swine streptococcus | SS2-H | 0.286 | 0.267 | - |
| The white dysentery salmonella | 7200407 | 0.256 | 0.233 | - |
| Salmonella typhimurtum | C79-32-015 | 0.255 | 0.261 | - |
| Intestinal bacteria O46 | E036 | 0.288 | 0.127 | ?+ |
| Intestinal bacteria O115 | 30801 | 0.276 | 0.134 | ?+ |
| Intestinal bacteria O138 | 30917 | 0.291 | 0.268 | ?- |
| Escherichia coli O 157 | ATCC43895 | 0.285 | 0.076 | ?+ |
| Escherichia coli O 157 | Min-27 | 0.279 | 0.113 | ?+ |
| Escherichia coli O 157 | DG03511 | 0.276 | 0.261 | ?+ |
| Intestinal bacteria MC1061 | MC1061 | 0.283 | 0.115 | ?+ |
Claims (8)
1. the colibacillary bacteria preparation that splits of efficient cracking is characterized in that, the said bacteria preparation that splits gets through following method preparation:
Step 1 through the method for phage induction, is induced the intestinal bacteria lysogenic strain, obtains the lytic phage particle, and purifying obtains phage, extracts the DNA of phage;
Step 2, the design primer is a template with step 1 gained DNA, the Using P CR corresponding gene fragment that increases;
Step 3 adopts prokaryotic expression system pET-28a (+), with the step 2 gained DNA construction recombination plasmid that increases;
Step 4, to expressing bacterium BL21 (DE3), the screening positive transformant carries out induction expression of protein and evaluation, obtains recombinant protein with the recombinant plasmid transformed of step 3;
Step 5, the recombinant protein of purifying abduction delivering, screening has the recombinant expression protein of lytic activity, obtains protein formulation, the promptly efficient colibacillary bacteria preparation that splits of cracking.
2. the colibacillary bacteria preparation that splits of efficient cracking according to claim 1 is characterized in that the nucleotides sequence of primer described in the step 2 is classified as, the upper reaches: CGCGGATCCATGAGCAGGAAACTCCG, downstream: CCCAAGCTTTTATCTGTCGATTCCCC.
3. the colibacillary bacteria preparation that splits of efficient cracking according to claim 2 is characterized in that the fragment of amplification gene described in the step 2 is 534bp.
4. according to claim 1 and the colibacillary bacteria preparation that splits of 2 described efficient cracking, it is characterized in that the recombinant protein that obtains in the step 4 is 23kDa.
5. the colibacillary bacteria preparation that splits of efficient cracking according to claim 1; It is characterized in that; The recombinant expression protein that screening described in the step 5 has lytic activity is specially: the bacterial lysate of the purified recombinant albumen that obtains, positive transformant is carried out dull and stereotyped cracking experiment; Relatively split the bacterium effect, thereby screening has the recombinant expression protein of lytic activity.
6. colibacillary cleavage method that splits bacteria preparation of efficient cracking according to claim 1; It is characterized in that; The reductive agent that said cleavage method adopts is that mass percent concentration is 0.8% beta-mercaptoethanol or the halfcystine of 10mM; Damping fluid is a 20mM pH5.2 sodium-acetate buffer, and the pH value of cracking bacterium liquid is 8.0, and temperature of reaction is 37 ℃.
7. the colibacillary purposes of splitting bacteria preparation of efficient cracking according to claim 1 is characterized in that, said cracking preparation is as the cracking preparation of cracking different serotypes coli strain.
8. purposes according to claim 7 is characterized in that, said serotype coli strain is intestinal bacteria O46, O118, O138, O157 and MC1061 bacterial strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101626403A CN102703491A (en) | 2012-05-23 | 2012-05-23 | Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101626403A CN102703491A (en) | 2012-05-23 | 2012-05-23 | Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102703491A true CN102703491A (en) | 2012-10-03 |
Family
ID=46896538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101626403A Pending CN102703491A (en) | 2012-05-23 | 2012-05-23 | Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102703491A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830825A (en) * | 2014-09-28 | 2015-08-12 | 中国海洋大学 | Endolysin sourced from salmonella bacteriophage and application thereof |
| CN105112393A (en) * | 2015-05-05 | 2015-12-02 | 吉林大学 | Bacteriophage fusion lyase capable of lysing Escherichia coli |
| CN107502603A (en) * | 2017-09-06 | 2017-12-22 | 江苏省农业科学院 | A kind of Escherichia coli lyases and preparation method and application |
-
2012
- 2012-05-23 CN CN2012101626403A patent/CN102703491A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 夏炉明等: "丝裂霉素c对溶源性大肠杆菌0157 stx毒素", 《中国农业科学》 * |
| 杨曦等: "大肠杆菌0157 STX噬菌体裂解酶的原核表达及活性研究", 《中国优秀硕士学位论文全文数据库》 * |
| 陈蔚青等: "链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测", 《生物工程学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830825A (en) * | 2014-09-28 | 2015-08-12 | 中国海洋大学 | Endolysin sourced from salmonella bacteriophage and application thereof |
| CN105112393A (en) * | 2015-05-05 | 2015-12-02 | 吉林大学 | Bacteriophage fusion lyase capable of lysing Escherichia coli |
| CN107502603A (en) * | 2017-09-06 | 2017-12-22 | 江苏省农业科学院 | A kind of Escherichia coli lyases and preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jabrane et al. | Characterization of serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen | |
| Schuch et al. | A bacteriolytic agent that detects and kills Bacillus anthracis | |
| Mayer et al. | Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin | |
| Haq et al. | Isolation and partial characterization of a virulent bacteriophage IHQ1 specific for Aeromonas punctata from stream water | |
| Frana et al. | Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype Typhimurium phage type DT104 | |
| Fernandes et al. | Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus | |
| Varani et al. | The role of prophage in plant-pathogenic bacteria | |
| Shanks et al. | Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii | |
| CN108486089A (en) | Derived from the wide range lyases and its antibacterial applications of salmonella bacteriophage | |
| Drebes Dörr et al. | Interbacterial competition and anti‐predatory behaviour of environmental Vibrio cholerae strains | |
| Fuente et al. | Growth inhibition of bacterial fish pathogens and quorum-sensing blocking by bacteria recovered from Chilean salmonid farms | |
| Foysal et al. | Molecular characterization and interactome analysis of aerolysin (aer) gene from fish pathogen Aeromonas veronii: The pathogenicity inferred from sequence divergence and linked to histidine kinase (cheA) | |
| CN107058265A (en) | Pseudomonas aeruginosa phage lyases and application | |
| CN106854247A (en) | A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella | |
| Valenzuela et al. | Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation | |
| CN104830825A (en) | Endolysin sourced from salmonella bacteriophage and application thereof | |
| Zampara et al. | Developing innolysins against Campylobacter jejuni using a novel prophage receptor-binding protein | |
| CN102181420A (en) | Expression method of lactococcus lactis of porcine streptococcus phage catenase | |
| CN102703491A (en) | Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof | |
| CN105112393A (en) | Bacteriophage fusion lyase capable of lysing Escherichia coli | |
| Jiang et al. | AcuC, a histone deacetylase, contributes to the pathogenicity of Aeromonas hydrophila | |
| Hasegawa et al. | TetR-type transcriptional regulator VtpR functions as a global regulator in Vibrio tubiashii | |
| Lim et al. | Bactericidal effect of Cecropin A fused endolysin on drug-resistant gram-negative pathogens | |
| CN104593346A (en) | Endolysin from vibrio parahaemolyticus phage and application of endolysin | |
| Boyer et al. | Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20121003 |