CN102181420A - Expression method of lactococcus lactis of porcine streptococcus phage catenase - Google Patents
Expression method of lactococcus lactis of porcine streptococcus phage catenase Download PDFInfo
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Abstract
The invention discloses an expression method of lactococcus lactis of porcine streptococcus phage catenase in the technical field of biology. A pyrolyzed gene of porcine streptococcus virulent phage SMP is augmented by utilizing polymerase chain reaction (PCR) to obtain a target fragment which is connected with a pNZ8148 vector so as to obtain a recombined expression plasmid; lactococcus lactis L9000 expression is adopted so s to obtain a fusion protein the molecular weight of which is 52kDa, so as to obtain activated catenase which has a pyrolyzed actioin on multiple clinically separated porcine streptococcus in vitro.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically is a kind of Lactococcus lactis expression method of porcine streptococcus phage lyase.
Background technology
Streptococcus suis is that a kind of people beast suffers from transmissible disease altogether, can cause piglet to suffer from meningitis, septicemia, sacroiliitis, endocarditis, pneumonia and people's meningitis.Streptococcus suis 2-type is popular the widest, and is also the strongest to the virulence of pig, causes enormous economic loss to pig industry, aspect public health, relevant practitioner's life security constituted a serious threat.Treatment for Streptococcus suis mainly is an antibiotic therapy, yet along with the widespread use of antibacterials, the propagation of bacterial resistance bacterial strain further aggravates.And lyase is by the phage gene group coding, enzyme that can the hydrolytic bacteria cell walls.Bacterial virus catenase is as phage attack bacteria only, and do not influence zooblast, and has higher specificity, so this enzyme does not influence harmless or useful zoobiotic.It is generally acknowledged that the speed that lyase acts on bacterium is exceedingly fast, to such an extent as to bacterium can not produce resistance to this enzyme.The lyase of phage has than phage antimicrobial spectrum more widely.So lyase has certain advantage as the novel antibacterial medicine.But though use escherichia expression system great expression lyase, expression product can be applied to animal after needing further to purify.The present invention uses the lysis genes of the Lactococcus lactis expression swine streptococcus virulent phage SMP of aliment security level, obtained activated lyase, can splitting action be arranged to the clinical isolating swine streptococcus of many strains external, provide possibility for directly carrying out clinical application.
Find through retrieval prior art, Liping Wangs etc. have been delivered " 32 kinds of antibacterials are to the streptococcic antibacterial activity in vitro in clinical separation pig source ", the document has been put down in writing the chemical sproof result of study demonstration of the isolating swine streptococcus in some pig farms to 32 kinds of antibacterials, the clinical isolates strain is based on resistant organism, and mostly be multidrug resistant, especially to sulfa drugs, but woods amine, tetracyclines, Macrolide, the resistance of aminoglycosides medicine is the most serious, resistance is not only general, and mostly be the height resistance, seek another kind of antibacterial mechanisms or antibacterials and seem particularly important.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of Lactococcus lactis expression method of porcine streptococcus phage lyase is provided, utilize the lyase gene of pcr amplification swine streptococcus virulent phage SMP, obtain the purpose segment, again the purpose segment is connected with the pNZ8148 carrier, obtain recombinant expression plasmid, importing Lactococcus lactis L9000 expresses, obtain the fusion rotein that molecular weight is 52kDa, obtain activated lyase, the lyase that obtains can have splitting action to the clinical isolating swine streptococcus of many strains external.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of porcine streptococcus phage SMP lyase, its dna sequence dna is shown in Seq ID No.3.
The present invention relates to the Lactococcus lactis expression method of above-mentioned porcine streptococcus phage SMP lyase, comprise the steps:
Described purifying is meant: with SS 2-H is indicator, adopts double-deck agar method to cultivate phage.
Described double-deck agar is meant: bottom platform is a 1.5wt% agar, and top layer is the 0.7wt% agarose.
Described double-deck agar method is meant: phage SMP is done 10 with suis THB substratum (Todd-Hewitt broth, THB substratum)
-2, 10
-4, 10
-6Above-mentioned phage diluent of 200 μ L and 200 μ L host bacterium SS2-H mixings are got in the constant gradient dilution, add 1mol/L CaCl again
2To final concentration 0.1mol/L, said mixture is positioned over 37 ℃ hatches behind the 15min and 50 ℃ of top-agar mixings that melted, be cast on the bottom platform, room temperature is placed to top-layer agar solidify after, be inverted in 37 ℃ of incubators and cultivate 10-12h, get the flat board that grows up to intensive continuous plaque, every plate adds 5mL SM liquid (prescription: NaCl 5.8g, MgSO47H2O 2g, 1M TrisCL (pH7.5) 50ml, 2% gelatin 5ml adds deionized water to 1000ml), rocked 3 hours at 4 ℃ of shaking tables, collection contains the SM liquid of phage, place aseptic centrifuge tube, 4000g removed cell debris in centrifugal 10 minutes, obtained the phage of purifying.
Described extraction is meant: add the 20mg/mL Proteinase K to final concentration 50 μ g/mL in the resuspended liquid of phage, 37 ℃ of incubations 30 minutes, add again 10%SDS (sodium laurylsulfonate) to final concentration be 0.5%, behind the mixing with digestion mixture in 56 ℃ of incubations 1 hour, be cooled to room temperature.With equal-volume phenol/chloroform extracting, 3000g separated two-phase in centrifugal 5 minutes, and aqueous favoring is collected in another centrifuge tube.Add the 3mol/L sodium acetate to final concentration 0.3mol/L in the aqueous favoring that reclaims, the dehydrated alcohol that adds two volumes behind the mixing washs gently, and centrifugal 2 minutes of 13000g abandons supernatant, reclaims the DNA precipitation.TE (nucleic acid dissolving damping fluid) dissolving phage DNA with proper volume.
Described pcr amplification specifically may further comprise the steps:
2.1) according to the SMP dna sequence dna (EF116926) of Genbank, design two primer P1 and P2, at the restriction enzyme site of 5 ' end and 3 ' end insertion restriction enzyme XbalI and HindIII, wherein: primer P1 is shown in Seq ID No.1, and P2 is shown in Seq ID No.2 respectively.
2.2) with primer P1, P2 by the following pcr amplification that carries out: the PCR system is the P21 μ L of P11 μ L, the 10 μ M/L of 2 * PCR mix, 25 μ L, 10 μ M/L, SM solution 5 μ L, the water 18 μ L of phage; The PCR parameter is that 35 circulations are (behind 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 58 ℃ of annealing 30s, 72 ℃ are extended 1min 45s; 72 ℃ are extended 10min).
2.3) after reaction finishes, get 5 μ L products and be used for 1% agarose gel electrophoresis, the PCR product reclaims test kit with the PCR product and reclaims.
Described expression specifically may further comprise the steps:
3.1) target gene fragment is connected with the pMD18-T carrier
Use the pMD18-T reaction kit of TAKARA company to connect target gene fragment and pMD18-T carrier (carrying carrier pMD18-T, Solution I), ligation system following (the μ L of unit):
16 ℃ of reaction 30min connect product and will be used to transform DH5 α competent cell behind the mixing.Get 2 μ L and connect in the product adding DH5 α competent cell, place 30min on ice.42 ℃ of circulator bath effect 90s, ice bath 2min.Add 37 ℃ of 120 rev/mins of shaking culture 45min of 800 μ L intestinal bacteria substratum (Luria-Bertani broth, LB substratum) again.Get the LB flat board that the coating of 100 μ L shake cultures contains 50 μ g/mL kantlex, be inverted dull and stereotyped 37 ℃ and cultivate 12-16h.
3.2) evaluation of pMD18-T gene recombination plasmid
Several are inoculated in the LB substratum (containing 50 μ g/mL kantlex) with single bacterium colony picking of growing on the flat board, cultivate 12h for 37 ℃.(day root biochemical technology company limited) extracts plasmid with plasmid extraction kit, and concrete steps are seen product description.The plasmid that extracts is used Xbal I, Hind III double digestion (Xbal I, Hind III enzyme, 10 * damping fluid are purchased the company in Fermentas), 1% agarose gel electrophoresis respectively.If the plasmid of two bands is the pMD18-T gene recombination plasmid.Cut the target gene fragment of glue recovery 1500bp.
Double digestion reaction system following (the μ L of unit):
3.3) target gene fragment is connected with the pNZ8148 carrier
Extract the pNZ8148 plasmid with plasmid extraction kit, adopt and use Xbal I, Hind III double digestion respectively.
Double digestion reaction system following (the μ L of unit):
Mixture places 37 ℃ of incubator effect 2h.
Enzyme is cut product and is dyeed with EB, carry out electrophoresis after, reclaim test kit with gel and reclaim, the operation steps part is seen product description.
The target gene fragment of connecting band toughness end and Lactococcus lactis expression vector, 16 ℃ are spent the night, ligation system following (the μ L of unit):
Connect product and be directly used in conversion.
3.4) preparation of Lactococcus lactis competent cell
Single bacterium colony of picking fresh culture is inoculated in 5mL GM17 liquid nutrient medium, adds in the 150mLGM17 substratum after 30 ℃ of incubated overnight, and 30 ℃ are cultured to OD
6000.5; Shift nutrient solution in the 500mL centrifuge tube of precooling, place 10min on ice; 4 ℃, the centrifugal 10min of 5000r/m, the electricity conversion damping fluid suspension with the 15mL precooling repeats 3 times; 4 ℃, the centrifugal 10min of 5000r/m transforms damping fluid with the ice-cold electricity of 1.5mL and suspends; The every pipe 40 μ L of packing ,-80 ℃ frozen.
3.5) electricity is transformed into Lactococcus lactis
Get the Lactococcus lactis electricity transformed competence colibacillus cell of preparation, place 5min on ice; Add in every pipe competent cell and connect product 20 μ L, the soft back ice bath 1min that mixes; With competent cell be connected precooling electricity that mixture of products changes 2mm over to and transform in the cup Gene Pulser with Bio-Rad company conversion instrument (the 25 μ F that shock by electricity that shock by electricity, 12.5KV/cm, 200 Ω), recovery media SGM17MC, containing on the GM17 solid medium flat board of paraxin 30 ℃ of insulation 24 72h.The picking transformant is inoculated in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of overnight incubation, PCR reaction, 1% agarose gel electrophoresis.Positive recombinant plasmid called after pNZ8148-lys.Simultaneously, electricity changes pNZ8148 and goes into Lactococcus lactis electricity transformed competence colibacillus cell, obtains
3.6) the LySMP induction expression of protein
Clone's positive bacteria 0.8mL of selection incubated overnight is inoculated in 10mL and contains in the M17 substratum of 5 μ g/mLCm, and 30 ℃ of 120 rev/mins of shaking culture are to OD
6000.4.Add nisin to final concentration be 1ng/mL.30 ℃ of shaking culture 3h.
Compared with prior art, the present invention has following beneficial effect: the present invention obtains activated lyase by the lysis genes of Lactococcus lactis expression swine streptococcus virulent phage SMP, and it can have splitting action to many strains swine streptococcus external.In addition, the used Lactococcus lactis Lactococcus of the present invention lactis 9000 is the Gram-positive probiotic bacterium, and is significant for the external secretion expression and the clinical application that realize lyase LySMP.
Relatively the fragmentation pattern of phage and lyase is found, the SMP phage only can cracking 2 strain streptococcus suis 2-types, but the lyase cleavable 7 strain streptococcus suis 2-types that Lactococcus lactis is expressed, in addition, can also cracking swine streptococcus 7 type SH040805,9 type SH040817, intestinal bacteria E.coli MC1061 and Salmonellas NATSEL0114.
Porcine streptococcus phage SMP involved in the present invention separates, identifies (Ma for this laboratory, Y.L., Lu, C.P., Isolationand identification of a bacteriophage capable of infecting Streptococcus suis type 2 strains.Veterinary Microbiology, 2008,132:340-3471.) (whole genome sequence has been submitted GenBank, accession number EF116926. to).
Engineering strain involved in the present invention: the e. coli bl21 (Wang.Y that contains plasmid pET-28a-lys, Sun.J.H., Lu.C.P., Purified Recombinant Phage Lysin LySMP:An Extensive Spectrum of Lytic Activityfor Swine Streptococci.Current of Microbiology.2009 58:609-615.) is preserved by this laboratory with the Lactococcus lactis L9000 that contains recombinant plasmid pNZ8148-lys.
Swine streptococcus S.suis 2-3 involved in the present invention, S.suis 2-4, S.suis 2-6, S.suis 2-h, S.suis 2-9, S.suis 2-N, S.suis HA9801, S.suis HA9802, S.suis HA9803, S.suis zy05719, S.suis 006731, S.suis 13, S.suis2-1, S.suis zy05721, S.suis zy05722, S.suis SH040805, S.suis SH040817 (Liping Wang, Lu Chengping, Tang Jiaqi, 32 kinds of antibacterials are to the streptococcic antibacterial activity in vitro in clinical separation pig source, the microorganism journal, 2004,44 (6): 794～799; Zhu Haodan, Gu Hongwei, Lu Chengping. the new gene trag of expression in vivo is in distribution and the immunoreactivity analysis thereof of swine streptococcus, microorganism journal, 2008,48 (12): 1642-1648; Wang.Y, Sun.J.H., Lu.C.P., PurifiedRecombinant Phage Lysin LySMP:An Extensive Spectrum of Lytic Activity for SwineStreptococci.Current of Microbiology.2009,58:609-615) and horse animal doctor streptococcus spp ATCC35246 (Fan Hongjie, Lu Chengping, Tang Jiaqi. the amalgamation and expression of streptococcus equi epizootic disease subspecies class M gene and streptococcus suis 2-type mrp gene fragment and piglet immunological test, Agricultural University Of Nanjing's journal, 2003,26 (4): 78-81) preserve for this laboratory.
Streptococcus aureus (Song Shiping, Xie Xiaozhen, Xi Daoyou, Deng the resistance monitoring and analysis of .158 strain streptococcus aureus, Chinese hospital infection magazine, 2003,13 (4): 384-385), Salmonellas NATSEL0114 and intestinal bacteria E.coli MC1061 (Zhang Hezhan. the classification of Salmonellas, name and Chinese Salmonellas bacterial type distribute, microbiology immunology progress, 2002,30 (2): 74-76) preserve for this laboratory.
Lactococcus lactis 9000 involved in the present invention (Igor Mierau.Michiel Kleerebezem, 10 years of thenisin-controlled gene expression system (NICE) in Lactococcus lactis.Appl MicrobiolBiotechnol, 2005,68:705-717) available from Dutch Nizo Food Research Inst. and Belgian bacterial classification center.
Description of drawings
Fig. 1 is an embodiment effect synoptic diagram;
Among the figure: A is that the proteic Western-blotting of recombinant expressed lySMP analyzes, wherein: 1, protein molecular marker; 2, nisin induces the Lactococcus lactis L9000 that contains recombinant plasmid pNZ8148-lys; 3, IPTG induces the BL21 that contains recombinant plasmid pET-28a-lys; 4, nisin induces the Lactococcus lactis L9000 that contains plasmid pNZ8148-;
B is that the proteic SDS-PAGE of recombinant expressed lySMP analyzes, and wherein: 1, nisin induces the Lactococcus lactis L9000 that contains recombinant plasmid pNZ8148-lys; 2, the Lactococcus lactis L9000 that contains recombinant plasmid pNZ8148-lys without inductive; 3, nisin induces the Lactococcus lactis L9000 that contains plasmid pNZ8148; 4, the Lactococcus lactis L9000 that contains plasmid pNZ8148 without inductive; 5, protein molecular Marker; 6, IPTG induces the BL21 that contains recombinant plasmid pET-28a-lys.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, detailed embodiment and concrete operating process have been provided, the experimental technique of unreceipted actual conditions, principle of work, usually carry out according to normal condition, Sambrook equimolecular clone for example: the condition described in the laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989), or the condition of advising according to manufacturer.Protection scope of the present invention is not limited to following embodiment.
One, the expression of the preparation of the purification of phage, DNA and lyase:
(1) clone of swine streptococcus virulent phage SMP lysis genes: the gene order design primer according to swine streptococcus virulent phage SMP carries out pcr amplification.Be connected to pMD-18T carrier (Takara company) and go up order-checking.Designed primer upstream comprises Xbal I restriction enzyme site, and the downstream comprises Hind III restriction enzyme site.
(2) structure of expression vector: Xbal I and Hind III enzyme respectively cut pMD-18T carrier and pNZ8148 carrier, after cutting glue and reclaiming, and 10～16 ℃ of connections of spending the night of T4DNA ligase enzyme (Takara company).Connect product and change among the Lactococcus lactis L9000,30 ℃ of incubated overnight, screening positive clone extracts plasmid.Cut evaluation with Xbal I and Hind III enzyme.The positive recombinant plasmid order-checking of identifying.
(3) Expression of Fusion Protein: clone's positive bacteria 0.8mL of selection incubated overnight is inoculated in 10mL and contains in the M17 substratum of 5 μ g/mLCm, and 30 ℃ of 120 rev/mins of shaking culture are to OD
6000.4.Add nisin to final concentration be 1ng/mL.30 ℃ of shaking culture 3h.Collect bacterium liquid, ultrasonication can get the lys crude extract.
(4) SDS-PAGE electrophoresis: the separation gel of preparation 12% and 5% concentrated glue, sample boils 4min with sample-loading buffer boiling water, concentrates glue voltage 80V, separation gel voltage 136V, electrophoresis 4～5h, Coomassie brilliant blue dyeing 2h.Decolouring is observed, and lighter band (Fig. 1) occurs at the 52kDa place.
WESTERN-BLOT immunoblotting: carry out SDS electrophoresis gained SDS-PAGE gel, NC film (nitrocellulose filter) and filter paper balance 30min in changeing the film damping fluid with embodiment 1 step.Stack according to the order of sequence from anode to negative electrode: filter paper, pvdf membrane, gel, filter paper, connect power supply, V changes film.
After changeing film and finishing, TTBS washes film 5min * 3 time, removes Tris and glycine.Film is put into heat sealable plastics bag, press the filter membrane area and add confining liquid 0.1mi/cm
2, 4 ℃ of sealings are spent the night.TTBS washes film 10min * 3 time; The mouse source LySMP that adds respectively with the confining liquid dilution resists more, hatches 1h for 37 ℃; TTBS washes film 10min * 3 time, adds sheep anti mouse two anti-working fluids (1: 100), and 37 ℃ are shaken and hatch 2h; TTBS washes film 10min * 3.Drip the colour developing of TMB colour developing liquid.Obvious band (Fig. 1) appears at the 52kDa place.
Use 20mM sodium-acetate buffer pH4.2,20mM sodium-acetate buffer pH5.2,10mM phosphate buffered saline buffer pH6.2,10mM PBS pH of buffer 7.2,20mM Tris-HCl pH of buffer 8.2 resuspended SS2-H respectively, furnishing OD
600Be 1.0.The bacterial suspension of making adds in 96 orifice plates, every hole 100 μ L, each gradient 6 hole.Add lys crude extract 100 μ L in the bacterial suspension of every hole, each gradient is respectively done 3 repetitions.Respectively in control group every hole add L9000 bacterial strain that 100 μ L contain empty plasmid pNZ8148 through etc. the crude extract of condition after inducing, each gradient is respectively done 3 repetitions.Behind 37 ℃ of concussion reaction 30min, measure the maximum pH condition of bacterial turbidity decline that makes.Measuring the optimum response damping fluid is 20mM pH 7.2PBS damping fluid.
The SS2-H that will grow to logarithmic phase is resuspended with 0.01M PBS pH of buffer 7.2, transfers to OD
600Be 1.0.The bacterial suspension of making adds in 96 orifice plates, every hole 100 μ L, and every kind of bacterial strain adds 6 holes.Add lys crude extract 100 μ L in the bacterial suspension of every hole.Every strain bacterium is cooked 3 repetitions.Respectively in control group every hole add L9000 bacterial strain that 100 μ L contain empty plasmid pNZ8148 through etc. the crude extract of condition after inducing, every strain bacterium is cooked 3 repetitions.At 22 ℃, 27 ℃, 32 ℃, 37 ℃, 42 ℃ effect 30min, measure and make the maximum temperature condition of bacterial turbidity decline respectively.Measuring the lyase optimal reaction temperature is 37 ℃.
The best cracking condition of lyase is defined as pH 7.2PBS, 37 ℃.
Get swine streptococcus and make OD
600Be 1.0 bacterial suspension, mix that 37 ℃ of reaction 30min detect OD with the lyase crude extract
600, calculate the per-cent that turbidity successively decreases.The turbidity of the clinical isolating pig streptococcus bacterial strain of the surveying test-results of successively decreasing sees attached list 1.Lyase can make the bacterial turbidity of swine streptococcus, intestinal bacteria, Salmonellas etc. descend 30～77%.
Table 1 lyase is to the turbidity of each bacterial strain experimental result of successively decreasing
Listed swine streptococcus and intestinal bacteria are recorded in the following discloses document in the table:
1. Liping Wang, Lu Chengping, Tang Jiaqi have delivered the 794th～799 page of " microorganism journal " 2004 the 44th the 6th phase of volume that " 32 kinds of antibacterials are to the streptococcic antibacterial activity in vitro in clinical separation pig source.
2.Ma，Y.L.，Lu，C.P.，Isolation?and?identification?of?a?bacteriophage?capable?of?infectingStreptococcus?suis?type?2?strains.Veterinary?Microbiology，2008，132：340-3471。
3.Wang.Y，Sun.J.H.，Lu.C.P.，Purified?Recombinant?Phage?Lysin?LySMP：AnExtensive?Spectrum?of?Lytic?Activity?for?Swine?Streptococci.Current?of?Microbiology.2009，58：609-615。
4. wishing that sky is red, Gu Hongwei, Lu Chengping. the new gene trag of expression in vivo is in distribution and the immunoreactivity analysis thereof of swine streptococcus, microorganism journal, 2008,48 (12): 1642-1648.
Listed horse streptococcus zooepidemicus subspecies are recorded in the following discloses document in the table:
5. the red knot of model, Lu Chengping, Tang Jiaqi. the amalgamation and expression of streptococcus equi epizootic disease subspecies class M gene and streptococcus suis 2-type mrp gene fragment and piglet immunological test, Agricultural University Of Nanjing's journal, 2003,26 (4): 78-81
Listed streptococcus aureus is recorded in the following discloses document in the table:
6. Song's generation is flat, Xie Xiaozhen, and Xi Daoyou waits the resistance monitoring and analysis, Chinese hospital infection magazine, 2003,13 (4): 384-385 of .158 strain streptococcus aureus
Listed Salmonellas is recorded in the following discloses document in the table:
7. open river war. the classification of Salmonellas, name and Chinese Salmonellas bacterial type distribute, microbiology immunology progress, 2002,30 (2): 74-76.
Claims (9)
1. porcine streptococcus phage SMP lyase, its dna sequence dna is shown in Seq ID No.3.
2. the Lactococcus lactis expression method of a porcine streptococcus phage SMP lyase according to claim 1 is characterized in that, comprises the steps:
Step 1, purifying obtains the streptococcus suis 2-type virulent phage from streptococcus suis 2-type virulent phage host bacterium SS2-H;
Step 2 is its genomic dna of material extraction with step 1 gained phage, and is template with this DNA, uses pcr amplification and obtains lyase gene, shown in Seq ID No.3;
Step 3 is used Lactococcus lactis and is expressed step 2 amplification gained lyase gene, obtains fusion rotein.
3. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 2 is characterized in that described purifying is meant: with SS
2-H is an indicator, adopts double-deck agar method to cultivate phage.
4. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 3 is characterized in that, described double-deck agar is meant: bottom platform is a 1.5wt% agar, and top layer is the 0.7wt% agarose.
5. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 3 is characterized in that, described double-deck agar method is meant: phage is done 10 with streptococcus cultures
-2, 10
-4, 10
-6Above-mentioned phage diluent of 200 μ L and 200 μ L host bacterium SS2-H mixings are got in the constant gradient dilution, add 1mol/L CaCl again
2To final concentration 0.1mol/L, said mixture is positioned over 37 ℃ hatches behind the 15min and 50 ℃ of top-agar mixings that melted, be cast on the bottom platform, room temperature is placed to top-layer agar solidify after, be inverted in 37 ℃ of incubators and cultivate 10-12h, get the flat board that grows up to intensive continuous plaque, every plate adds 5mL SM liquid, rocks 3 hours at 4 ℃ of shaking tables, collects SM liquid and puts aseptic centrifuge tube, 4000g removed cell debris in centrifugal 10 minutes, obtained the phage of purifying.
6. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 2, it is characterized in that, described extraction is meant: add the 20mg/mL Proteinase K to final concentration 50 μ g/mL in the resuspended liquid of phage, 37 ℃ of incubations 30 minutes, add again 10%SDS to final concentration be 0.5%, behind the mixing with digestion mixture in 56 ℃ of incubations 1 hour, be cooled to room temperature, with equal-volume phenol/chloroform extracting, 3000g separated two-phase in centrifugal 5 minutes, aqueous favoring is collected in another centrifuge tube, adds the 3mol/L sodium acetate to final concentration 0.3mol/L in the aqueous favoring that reclaims, the dehydrated alcohol that adds two volumes behind the mixing washs gently, centrifugal 2 minutes of 13000g, abandon supernatant, reclaim the DNA precipitation, with the nucleic acid dissolving damping fluid dissolving phage DNA of proper volume.
7. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 2 is characterized in that, described pcr amplification specifically may further comprise the steps:
2.1) according to the EF116926 SMP dna sequence dna of Genbank, design two primer P1 and P2, at the restriction enzyme site of 5 ' end and 3 ' end insertion restriction enzyme XbalI and Hind III, wherein: primer P1 is shown in Seq ID No.1, and primer P2 is shown in SeqIDNo.2 respectively;
2.2) with primer P1, P2 by the following pcr amplification that carries out: the PCR system is the P21 μ L of P11 μ L, the 10 μ M/L of 2 * PCR mix, 25 μ L, 10 μ M/L, SM solution 5 μ L, the water 18 μ L of phage; The PCR parameter is 35 circulations, after each circulation is 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 58 ℃ of annealing 30s, 72 ℃ are extended 1min 45s; 72 ℃ are extended 10min;
2.3) after reaction finishes, get 5 μ L products and be used for 1% agarose gel electrophoresis, the PCR product reclaims test kit with the PCR product and reclaims.
8. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 2 is characterized in that, described expression may further comprise the steps:
3.1) target gene fragment is connected with the pMD18-T carrier;
3.2) evaluation of pMD18-T gene recombination plasmid;
3.3) target gene fragment is connected with the pNZ8148 carrier;
3.4) preparation of Lactococcus lactis competent cell;
3.5) electricity is transformed into Lactococcus lactis;
3.6) the LySMP induction expression of protein.
9. the Lactococcus lactis expression method of porcine streptococcus phage SMP lyase according to claim 2 is characterized in that, described expression, and concrete steps are:
I) adopt following ligation system to be connected in target gene fragment and pMD18-T carrier:
16 ℃ of reaction 30min behind the mixing, connect product and will be used to transform DH5 α competent cell, getting 2 μ L connects in the product adding DH5 α competent cell, place 30min on ice, 42 ℃ of circulator bath effect 90s, ice bath 2min adds 37 ℃ of 120 rev/mins of shaking culture 45min of 800 μ L LB substratum again, get the KanLB flat board that the coating of 100 μ L shake cultures contains 50 μ g/mL kantlex, be inverted dull and stereotyped 37 ℃ and cultivate 12-16h;
Ii) several are inoculated in the LB substratum that contains 50 μ g/mL kantlex with single bacterium colony picking of growing on the flat board, cultivate 12h for 37 ℃, extract plasmid with plasmid extraction kit, the plasmid that extracts is used XbalI, HindIII double digestion respectively, 1% agarose gel electrophoresis, if the plasmid of two bands is the pMD18-T gene recombination plasmid, cut the target gene fragment that glue reclaims 1500bp, the double digestion reaction system is as follows:
Iii) extract the pNZ8148 plasmid with plasmid extraction kit, adopt and use Xbal I, Hind III double digestion respectively, the double digestion reaction system is as follows:
Mixture places 37 ℃ of incubator effect 2h, and enzyme is cut product and dyeed with EB, carry out electrophoresis after, reclaim test kit with gel and reclaim;
The iv) target gene fragment of connecting band toughness end and Lactococcus lactis expression vector, 16 ℃ are spent the night, and connect product and are directly used in conversion, and the ligation system is as follows:
V) single bacterium colony of picking fresh culture is inoculated in 5mL GM17 liquid nutrient medium, adds in the 150mLGM17 substratum after 30 ℃ of incubated overnight, and 30 ℃ are cultured to OD
6000.5, shift nutrient solution in the 500mL centrifuge tube of precooling, place 10min on ice, 4 ℃, the centrifugal 10min of 5000r/m, electricity with the 15mL precooling transforms the damping fluid suspension, repeats 4 ℃ 3 times, the centrifugal 10min of 5000r/m, transform damping fluid with the ice-cold electricity of 1.5mL and suspend, the every pipe 40 μ L of packing ,-80 ℃ are frozen;
Vi) get the Lactococcus lactis electricity transformed competence colibacillus cell of preparation, place 5min on ice, add in every pipe competent cell and connect product 20 μ L, the soft back ice bath 1min that mixes, with competent cell be connected precooling electricity that mixture of products changes 2mm over to and transform in the cup and carry out 25 μ F, 12.5KV/cm, the electric shock of 200 Ω, recovery media SGM17MC is containing on the GM17 solid medium flat board of paraxin, 30 ℃ of insulation 24～72h, the picking transformant, be inoculated in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of overnight incubation, pcr reaction, 1% agarose gel electrophoresis, positive recombinant plasmid called after pNZ8148-lys, simultaneously, electricity changes pNZ8148 and goes into Lactococcus lactis electricity transformed competence colibacillus cell;
Vi) select clone's positive bacteria 0.8mL of incubated overnight to be inoculated in 10mL and contain in the M17 substratum of 5 μ g/mLCm, 30 ℃ of 120 rev/mins of shaking culture are to OD6000.4, and adding nisin is 1ng/mL to final concentration, 30 ℃ of shaking culture 3h.
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| CN102198265A (en) * | 2011-03-22 | 2011-09-28 | 上海交通大学 | Method for degrading streptococcus suis biofilm by applying phage lyase |
| CN103275946A (en) * | 2013-05-06 | 2013-09-04 | 上海交通大学 | Zymin ATPase-S for hydrolysis of ATP (adenosine triphosphate) and preparation method of zymin ATPase-S |
| CN103436514A (en) * | 2013-08-20 | 2013-12-11 | 昆明理工大学 | Heat-resistant lyase TSPpgh and polynucleotide coding same |
| CN104830825A (en) * | 2014-09-28 | 2015-08-12 | 中国海洋大学 | Endolysin sourced from salmonella bacteriophage and application thereof |
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| WO2016165604A1 (en) * | 2015-04-13 | 2016-10-20 | 武汉菲吉乐科生物科技有限公司 | Broad spectrum of streptococcus lyase and use thereof |
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| CN102198265A (en) * | 2011-03-22 | 2011-09-28 | 上海交通大学 | Method for degrading streptococcus suis biofilm by applying phage lyase |
| CN103275946A (en) * | 2013-05-06 | 2013-09-04 | 上海交通大学 | Zymin ATPase-S for hydrolysis of ATP (adenosine triphosphate) and preparation method of zymin ATPase-S |
| CN103436514A (en) * | 2013-08-20 | 2013-12-11 | 昆明理工大学 | Heat-resistant lyase TSPpgh and polynucleotide coding same |
| CN104830825A (en) * | 2014-09-28 | 2015-08-12 | 中国海洋大学 | Endolysin sourced from salmonella bacteriophage and application thereof |
| WO2016165604A1 (en) * | 2015-04-13 | 2016-10-20 | 武汉菲吉乐科生物科技有限公司 | Broad spectrum of streptococcus lyase and use thereof |
| US9993532B2 (en) | 2015-04-13 | 2018-06-12 | Wuhan Phagelux Bio-Tech Company Limited | Broad spectrum of Streptococcus lyase and use 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 |
| CN109371011A (en) * | 2018-11-26 | 2019-02-22 | 天津科技大学 | A method of new extraction phage genome DNA |
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