CN101831442A - Preparation method of porcine streptococcus phage perforin - Google Patents
Preparation method of porcine streptococcus phage perforin Download PDFInfo
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- CN101831442A CN101831442A CN 201010168940 CN201010168940A CN101831442A CN 101831442 A CN101831442 A CN 101831442A CN 201010168940 CN201010168940 CN 201010168940 CN 201010168940 A CN201010168940 A CN 201010168940A CN 101831442 A CN101831442 A CN 101831442A
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Abstract
The invention relates to a preparation method of porcine streptococcus phage perforin in the technical field of biological materials. The preparation method comprises the following steps of: (1) purifying to obtain a porcine streptococcus 2 type virulent phage and extracting the DNA of the phage; (2) amplifying perforin genes by applying a PCR (Polymerase Chain Reaction) through using the DNA obtained in the step (1) as a template; (3) carrying out prokaryocyte expression on the DNA obtained by amplifying in the step (2) to obtain fusion protein; (4) purifying the fusion protein and then carrying out renaturation to obtain the perforin. The purified and activated perforin can be used for cracking multiple porcine streptococcocci in vitro or improving the cracking efficiency of lyase.
Description
Technical field
The present invention relates to a kind of preparation method of technical field of biological materials, specifically be a kind of can cracking or strengthen the preparation method of the pore-forming protein (Holin) of lyase cracking swine streptococcus.
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 generation of Resistant strain further aggravates.Pore-forming protein is a kind of hydrophobic transmembrane protein by the phage gene group coding, inserts cytoplasmic membrane in specified phase and forms nonspecific hole or damage, impels lyase effectively to regulate lyase cracking bacterium near its target site.Phage perforin and lyase are used, and can improve the efficient of lyase specificity attack bacteria greatly.So pore-forming protein has certain advantage as the novel antibacterial medicine.The present invention has obtained the activated pore-forming protein of purifying by the holin gene of prokaryotic expression swine streptococcus virulent phage SMP, can splitting action be arranged to the clinical isolating swine streptococcus of many strains external.
Find by literature search, Liping Wang, Lu Chengping, Tang Jiaqi rolled up the 794th～799 page of the 6th phase in 2004 the 44th at " microorganism journal " and has delivered " 32 kinds of antibacterials are to the streptococcic antibacterial activity in vitro in clinical separation pig source ", mention in the literary composition, the isolating swine streptococcus in some pig farms shows the chemical sproof result of study of 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 objective of the invention is to overcome the deficiencies in the prior art, the preparation method of the pore-forming protein of a kind of cracking swine streptococcus is provided.The activated pore-forming protein of the purifying that the present invention obtains, it can be at the lysis efficiency of external many strains of cracking swine streptococcus or enhancing lyase.
The present invention realizes by following technical scheme, the present invention includes following steps:
Step 1, purifying obtain the streptococcus suis 2-type virulent phage, extract the DNA of phage;
Step 2 is a template with step 1 gained DNA, uses pcr amplification pore-forming protein gene;
Step 3, prokaryotic expression step 2 amplification gained DNA obtains fusion rotein;
Step 4, purified fusion protein, renaturation obtains pore-forming protein.
In the step 3, the plasmid that described prokaryotic expression adopts is pET-28a (+), the about 16kDa of pore-forming protein molecule of expression.
In the step 3, described fusion rotein is meant that a molecular weight is the fusion rotein of 16kDa.
In the step 4, described purified fusion protein is meant the column purification through Ni, obtains activated pore-forming protein.
The condition that pore-forming protein in the described step 4 is brought into play best splitting action is: adopting pH5.2 sodium-acetate buffer, optimal reaction temperature is 37 ℃.
The present invention utilizes the holin gene of pcr amplification swine streptococcus virulent phage SMP, obtain the purpose segment, again the purpose segment is connected with pET-28a (+) carrier, obtain recombinant expression plasmid, the importing e. coli bl21 is expressed, purifiedly obtain activated pore-forming protein, the pore-forming protein of acquisition is at external cleavable or strengthen the splitting action of lyase to the clinical isolating swine streptococcus of many strains, and subtilis and streptococcus aureus are had independent splitting action.
Compared with prior art, the present invention has following beneficial effect: the present invention is by the holin gene of prokaryotic expression swine streptococcus virulent phage SMP, obtain the activated pore-forming protein of purifying, it can be at the lysis efficiency of external many strains of cracking swine streptococcus or enhancing lyase.Relatively phage perforin and lyase are found the lysis efficiency of bacterium, the swine streptococcus that lyase is crossed autoclaving has splitting action, after adding pore-forming protein, itself and lyase can be worked in coordination with, the efficient undressed swine streptococcus of cracking, and lysis efficiency significantly improves.
Embodiment
The invention will be further described for following example.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example: the condition described in the laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment
One, the expression of the preparation of the purification of phage, DNA and pore-forming protein
Step 1, make even and grow up to the phage SMP (Ma of intensive continuous plaque on the plate, Y.L., Lu, C.P., Isolation andidentification of a bacteriophage capable of infecting Streptococcus suis type 2strains.Veterinary Microbiology, 2008,132:340-347, whole genome sequence have submitted GenBank, accession number EF116926. to), every plate adds 5mL SM (phage diluent, every liter contains 5.8g NaCl, 2g MgSO47H2O, 50mL 1M Tris-Cl, pH 7.5,5mL 2% gelatin), 4 ℃ of shaking tables rocked 3 hours, collected SM liquid and put aseptic centrifuge tube 4000g removal in centrifugal 10 minutes cell debris.It is 1 μ g/mL to final concentration that supernatant liquor adds pancreas DNaseI and RNaseI, 37 ℃ of incubations 30 minutes.Add NaCl to final concentration 1mol/L, ice bath is after 1 hour, and centrifugal 10 minutes of 4 ℃ of 11000g collect supernatant.Supernatant liquor adds PEG 8000 and dissolved the back ice bath 1 hour to final concentration 10% stirring at room, and phage particle is precipitated.Centrifugal 10 minutes of 4 ℃ of 12000g abandon supernatant, and sedimentary phage particle spends the night resuspended with SM liquid.
Step 2 adds the 20mg/mL Proteinase K to final concentration 50 μ g/mL in resuspended liquid, 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, 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.
Step 3, the holin Prokaryotic Expression
(1) clone of swine streptococcus virulent phage SMP holin gene
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 EcoR I restriction enzyme site, and the downstream comprises the XhoI restriction enzyme site.
(2) structure of expression vector
EcoR I and Xho I enzyme respectively 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 EcoR I and XhoI enzyme.The positive recombinant plasmid order-checking of identifying.
(3) Expression of Fusion Protein
Screening positive clone extracts plasmid, change over to e. coli bl21 (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).1 colony inoculation 5ml of picking kantlex LB substratum incubated overnight.Get 5mL incubated overnight bacterium liquid inoculation 1L kantlex LB substratum, 37 ℃ 200 rev/mins are shaken bacterium and cultivate 5h, to OD600 be 0.4～1.Adding IPTG (isopropylthio-) is 1mM to final concentration, and 27 ℃ are shaken after bacterium cultivates 4h, collect bacterium liquid.
(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 has tangible band to occur at the 16kDa place.
Two, the purifying of fusion rotein
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 the pore-forming protein of purifying.
Three, the best cracking condition of pore-forming protein determines
With swine streptococcus SS2-4 (Wang.Y, Sun.J.H., Lu.C.P., Purified Recombinant Phage LysinLySMP:An Extensive Spectrum of Lytic Activity for Swine Streptococci.Current ofMicrobiology.2009,58:609-615) centrifugal, be resuspended in the sodium-acetate buffer of pH5.2, be made into OD630 and be about 1.0 bacterial suspension, add in 96 orifice plates every hole 100 μ l.In the pore-forming protein of 20 μ g/ml purifying, add 0%, 0.5%, 0.8%, 1.0%, 3.0% and 5.0% (V/V) beta-mercaptoethanol respectively, respectively get 100 μ l and add in the bacterial suspension.Each concentration is respectively done 3 repetitions, and lysis buffer is as blank.Behind the reaction 30min, read absorbancy at the 600nm place under the room temperature, get absorbancy and reduce the optimum concn that maximum concentration adds as beta-mercaptoethanol, the optimum concn of mensuration is 0.5%.
SS2-4 is centrifugal with swine streptococcus, is resuspended in the sodium-acetate buffer of pH5.2, is made into OD
630Be about 1.0 bacterial suspension, add in 96 orifice plates every hole 100 μ l.In the pore-forming protein of 20 μ g/ml purifying, add 1mM, 2mM, 4mM, 5mM, 8mM, 10mM, 15mM halfcystine respectively, respectively get 100 μ l and add in the bacterial suspension.Each concentration is respectively done 3 repetitions, and lysis buffer is as blank.Behind the reaction 30min, read absorbancy at the 600nm place under the room temperature, get absorbancy and reduce the optimum concn that maximum concentration adds as halfcystine, the optimum concn of mensuration is 10mM.
Use 20mM pH5.2 sodium-acetate buffer, 10mM pH6.8 phosphate buffered saline buffer, 10mM pH7.2 phosphate buffered saline buffer, the resuspended suis of 20mM pH8.5 Tris-Cl damping fluid respectively, all the other steps and top same, measuring the optimum response damping fluid is 20mM pH5.2 sodium-acetate buffer.
Act on 30min at 4 ℃, 18 ℃, 25 ℃, 37 ℃ and 42 ℃ respectively, all the other steps and top same, measuring the pore-forming protein optimal reaction temperature is 37 ℃.
Four, pore-forming protein is measured the splitting action of bacterium
Get 200mL respectively and express bacterium lys-BL21 and holin-BL21 (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) induce after 4 hours centrifugal through IPTG, each is resuspended with 5mL sterilization 0.01M PBS (pH 7.2), the suspended substance ultrasonication.The lysate that obtains is done the bacterium breaking test.
The 50mL strain culture of incubated overnight grows to OD
6001.0 the back is centrifugal, and is resuspended with 1mLPBS (pH7.2), washes 3 times with PBS (pH 7.2).
Bacterium after the processing adds in 0.7% agarose that melts, and pours in the plate after the cooling, punches on flat board, drips the mixed solution of two kinds of lysates and two kinds of lysates respectively, cultivates more than the 5h, observes the transparent inhibition zone that occurs on the substratum for 37 ℃.With the BL21 that contains pET-28a (+) plasmid as blank.Observe two kinds of expression bacterium lysates and mixed solution to streptococcus suis 2-type (SS2), swine streptococcus 7 types (SS7), swine streptococcus 9 types (SS9) bacterial strain, streptococcus equi epizootic disease subspecies are with reference to strain ATCC35246, intestinal bacteria, streptococcus aureus, the cracking situation of Salmonellas and subtilis (seeing Table 1) (Wang.Y, Sun.J.H., Lu.C.P., Purified Recombinant Phage Lysin LySMP:An ExtensiveSpectrum of Lytic Activity for Swine Streptococci.Current of Microbiology.2009.58:609-615).
Table 1 pore-forming protein is to the splitting action of swine streptococcus etc.
Annotate :+lytic activity had; ++ have than the fine melt activity;-no lytic activity
The information of listed bacterial strain is open in following document in the table: Wang.Y, Sun.J.H., Lu.C.P., Purified Recombinant Phage Lysin LySMP:An Extensive Spectrum of Lytic Activity forSwine Streptococci.Current of Microbiology.2009,58:609-615.; 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; 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; Song Shiping, Xie Xiaozhen, Xi Daoyou waits the resistance monitoring and analysis, Chinese hospital infection magazine, 2003,13 (4): 384-385 of .158 strain streptococcus aureus; Li Jing, Yang Qian. the progress of biological and ecological methods to prevent plant disease, pests, and erosion subtilis, Anhui agricultural sciences, 2008,36 (1): 106-111; Zhang Hezhan. the classification of Salmonellas, name and Chinese Salmonellas bacterial type distribute, microbiology immunology progress, 2002,30 (2):
74-76。
Claims (8)
1. the preparation method of a porcine streptococcus phage perforin is characterized in that, comprises the steps:
Step 1, purifying obtain the streptococcus suis 2-type virulent phage, extract the DNA of phage;
Step 2 is a template with step 1 gained DNA, uses pcr amplification pore-forming protein gene;
Step 3, prokaryotic expression step 2 amplification gained DNA obtains fusion rotein;
Step 4, purified fusion protein, renaturation obtains pore-forming protein.
2. the preparation method of porcine streptococcus phage perforin according to claim 1 is characterized in that, in the step 3, the plasmid that described prokaryotic expression adopts is pET-28a (+), the about 16kDa of pore-forming protein molecule of expression.
3. according to the preparation method of claim 1 or 2 described porcine streptococcus phage perforins, it is characterized in that in the step 3, described prokaryotic expression is meant:
(1) the gene order design primer according to swine streptococcus virulent phage SMP carries out pcr amplification, is connected on the pMD-18T carrier and checks order, and designed primer upstream comprises EcoR I restriction enzyme site, and the downstream comprises Xho I restriction enzyme site;
(2) structure of expression vector: EcoR I and Xho I enzyme respectively cut PCR product and pET-28a (+), 10～16 ℃ of connections of spending the night of T4 dna ligase, connecting product changes in the bacillus coli DH 5 alpha, 37 ℃ of incubated overnight, extract plasmid, cut evaluation with EcoRI and Xho I enzyme, the positive recombinant plasmid order-checking of evaluation;
(3) Expression of Fusion Protein: screening positive clone extracts plasmid, and 1 colony inoculation 5ml of picking kantlex LB substratum incubated overnight is got 5mL incubated overnight bacterium liquid inoculation 1L kantlex LB substratum, and 37 ℃ 200 rev/mins are shaken bacterium cultivation 5h, to OD
600Be 0.4～1, adding isopropylthio-to final concentration is 1mM, and 27 ℃ are shaken after bacterium cultivates 4h, collect bacterium liquid;
(4) 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 has tangible band to occur at the 16kDa place.
4. the preparation method of porcine streptococcus phage perforin according to claim 1 is characterized in that, in the step 3, described fusion rotein is meant and obtains the fusion rotein that molecular weight is 16kDa.
5. the preparation method of porcine streptococcus phage perforin according to claim 1 is characterized in that, in the step 4, described purified fusion protein is meant the column purification through Ni, obtains activated pore-forming protein.
6. according to the preparation method of claim 1 or 5 described porcine streptococcus phage perforins, it is characterized in that in the step 4, described purified fusion protein comprises the steps:
1. IPTG inductive bacterium 1L is dissolved in the lysis buffer of 25mL precooling, wherein contains the 50mM sodium phosphate buffer, pH8.0, ultrasonication;
2. 8000g centrifuging and taking supernatant;
3. 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;
4. the cleaning buffer solution A that adds the 5mM imidazoles with the lysis buffer of precooling washes post, until OD
280＜0.1;
5. the cleaning buffer solution B that adds the 20mM imidazoles with the lysis buffer of precooling washes post, until OD
280＜0.1;
6. the dissolution fluid that at last adds the 250mM imidazoles with lysis buffer is washed post, collects elutriant, is the pore-forming protein of purifying.
7. according to the preparation method of claim 1 or 5 described porcine streptococcus phage perforins, it is characterized in that the condition that the pore-forming protein in the described step 4 is brought into play best splitting action is: adopting pH5.2 sodium-acetate buffer, optimal reaction temperature is 37 ℃.
8. the preparation method of porcine streptococcus phage perforin according to claim 1 is characterized in that, described step 2 is carried out in such a way:
In resuspended liquid, add the 20mg/mL Proteinase K to final concentration 50 μ g/mL, 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, 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;
With nucleic acid dissolving damping fluid dissolving phage DNA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181420A (en) * | 2011-01-31 | 2011-09-14 | 上海交通大学 | Expression method of lactococcus lactis of porcine streptococcus phage catenase |
| CN113201050A (en) * | 2020-08-06 | 2021-08-03 | 青岛诺安百特生物技术有限公司 | Staphylococcus aureus bacteriophage perforin and preparation method and application thereof |
-
2010
- 2010-05-12 CN CN 201010168940 patent/CN101831442A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 《Curr Microbiol》 20090307 Y. Wang et al. Purified Recombinant Phage Lysin LySMP: An Extensive Spectrum of Lytic Activity for Swine Streptococci 摘要，601-611页"Materials and Methods"部分 1-8 第58卷, * |
| 《veterinary microbiology》 20080518 Y.L.MA and C.P.Lu Isolation and identification of a bacteriophage capable of infecting Streptococcus suis type 2 strains 340-347 1-8 第132卷, * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181420A (en) * | 2011-01-31 | 2011-09-14 | 上海交通大学 | Expression method of lactococcus lactis of porcine streptococcus phage catenase |
| CN113201050A (en) * | 2020-08-06 | 2021-08-03 | 青岛诺安百特生物技术有限公司 | Staphylococcus aureus bacteriophage perforin and preparation method and application thereof |
| CN113201050B (en) * | 2020-08-06 | 2022-07-22 | 青岛诺安百特生物技术有限公司 | Staphylococcus aureus bacteriophage perforin and preparation method and application thereof |
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