CN1557960A - Method for separating antibiotic peptide and separated antibiotic peptide - Google Patents
Method for separating antibiotic peptide and separated antibiotic peptide Download PDFInfo
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Abstract
The invention relates to a method of isolating antibacterial peptides and the isolated peptides thereof. The method comprises: constructing a fusion DNA sequence of yeast alpha mating factor leader sequence and a random peptide, inserting the sequence into a saccharomyces cerevisiae vector which contains an inducible promotor, digesting the unlinked linear DNA with exonuclease III and mungbean sprout nuclease, electrotransforming the yeast vector after purification, culturing the transformant 24h and covering the transformant with the upper medium which contains E. coli K12D31, and detecting the bacterial inhibition ring and the bacterial inhibition rate after inducing the expression by liquid medium, selecting the positive clones and amplifying by PCR, and determining the sequence. The method of the present invention can be used easily and effectively to isolate antibacterial oligopeptides and polypeptides and can be used as an alternative way to find antibiotics.
Description
Technical field
The present invention relates to a kind of method and isolating antibacterial peptide that separates antibacterial peptide.
Background technology
Microbiotic is under lower concentration, can optionally suppress or kill and wound him and plant the microbial secondary metabolites of microorganism or tumour cell and similar compound and the structural modification thing that adopts chemistry or biological method to make.Various antibiotic antibacterial effects, majority is bacteriostatic action, germicidal action of minority tool or bacteriolysis.Antibiotic anti-microbial effect mainly is the physiology aspect that acts on mushroom, suppresses metabolic some link of microorganism cells or some enzyme system.The antibiotic mechanism of action can reduce following several types: (1) suppresses the synthetic of nucleic acid.(2) arrestin matter is synthetic.(3) permeability of change cytolemma.(4) formation of interference cell wall.(5) act on energy metabolism system or as metabolic antagonist.
Microbiotic is playing an important role aspect the guarantee human health as the important clinical drug application.Yet, along with antibiotic widespread use, cause two problems clinically: the one, bacterial drug resistance increases year by year, causes some microbiotic curative effects to reduce, even invalid, and multi-drug resistant constantly worsens.The 2nd, some non-pathogenic bacteriums become conditioned pathogen.Increasing microbiotic lost efficacy to many patients, needed the new antibiosis of exploitation usually to be prevented and treated.Traditional microbiotic finds that the resulting new antibiotic of approach is fewer and feweri, need set up some new methods and approach and develop.New development approach comprises structural modification, the new role mechanism and the screening of target site, material such as the antimicrobial enhancing agent that strengthens and protect the antibacterials performance, antibiotic enzyme inhibitor, membrane permeability toughener, efflux pump inhibitor etc. of microbiotic and synthetic antibacterial drug now.The present new target site of finding is kind surplus in the of 10 only.
Polypeptide antibiotics is one of direction of antibiotics medicine development.Traditional microbiotic is all produced by mould.Studies show that recently also has the antibiotic gene of coding in the genome of animal, plant and human body.These genes encodings mostly be some small peptides, be called antibacterial peptide.It is strong that antibacterial peptide has an anti-microbial activity, characteristics such as broad-spectrum antibacterial property.Antibacterial peptide is widespread in nature, and represents the ancient mechanism of host defense system.From genomes such as insect, batrachians, Mammals, plant, hundreds of antibacterial peptides have been separated so far.Antibacterial peptide can be used for fields such as medicine, fodder additives, food preservatives, plant genetic engineering.Because antibacterial peptide is to the particularly procaryotic toxicity of microorganism, general gene isolation method is unsuitable for separating antibacterial peptide gene.The method of traditional separation antibacterial peptide adopts repeatedly crosses post from amino acid sequencing or the mass spectrum order-checking then of various bioseparation purifying, very loaded down with trivial details.The another one method is according to known antibacterial peptide design homology fragment, carries out activity then and detects, and has significant limitation.Can also seek antibacterial peptide according to the method for combinatorial chemistry, the expense costliness, modification reaches maximum activity if antibacterial peptide needs glycosylation etc., and this method can't satisfy requirement of experiment.Be badly in need of setting up advantages of simplicity and high efficiency antibacterial peptide separation method at present.The general length of antibacterial peptide is 10 to 40 amino acid, has stronger immunogenicity, and patient is used effectively for the first time, and later result of use reduces gradually, must improve constantly dosage.The small molecules antibacterial peptide is these shortcomings of tool not.
Summary of the invention
The object of the present invention is to provide a kind of method of high efficiency separation antibacterial peptide, so that isolate oligopeptides and polypeptide with strong anti-microbial activity.The present invention also provides isolating antibiotic tripeptides and polypeptid coding sequence, to be used for the microbiotic exploitation.
The objective of the invention is to realize: make up the yeast α mating factor leading peptide and the fusion dna fragment of peptide at random, be used for the secreting, expressing of peptide at random by following technical measures; Insert the yeast saccharomyces cerevisiae carrier that contains inducible promoter with EcoR I and Xba I double digestion; Recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, it is carried out digestion process with exonuclease III and Semen Phaseoli radiati Germinatus nuclease; Electricity swashs transformed saccharomyces cerevisiae behind the purifying; Transformant is forwarded to raffinose/semi-lactosi abduction delivering flat board, covers the observation inhibition zone after the overnight growth with the upper strata substratum that contains intestinal bacteria bacterial classification K12D31; Or with surveying bacteriostasis rate behind the liquid nutrient medium abduction delivering; Positive colony is carried out pcr amplification, order-checking; Chemosynthesis part antibacterial peptide is used the agar diffusion method detection of active.
The present invention includes following concrete steps:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.With having the active Pfu archaeal dna polymerase of high-fidelity, PyrobestDNA polysaccharase or other has the active thermostable DNA polymerases amplification of high-fidelity, amplification template is that (plasmid pPICZ α A contains yeast α mating factor leading peptide dna sequence dna to plasmid pPICZ α A, the Invitrogen product), amplified production ethanol sedimentation; Hair dryer dries up; Be suspended in the sterile distilled water; With EcoR I and Xba I double digestion 6 hours to spending the night; 70 ℃ of heat inactivations.
(2) pYES2/CT (Invitrogen product) with EcoR I and Xba I double digestion 6 hours to spending the night; 70 ℃ of heat inactivations; Get the pYES2/CT of double digestion, add the PCR product and the T4 dna ligase of double digestion, 6 ℃ or 16 ℃ of connections are spent the night.
(3) cause the segmental transformant of nothing insertion too much for the linear DNA that does not connect of avoiding quantity to occupy the majority with pastoris genomic dna reorganization takes place after conversion, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company; Use H
2The O wash-out; Adding the exonuclease III of 10-20 unit handled 1 hour in 37 ℃; 70 ℃ of inactivations 15 minutes; Add 10-20 unit's Semen Phaseoli radiati Germinatus nuclease again, handled 40 minutes to 1 hour 20 minutes for 37 ℃; With the PCR purification column purifying of Qiagen company, use H
2The O wash-out; Getting 10 μ l does electric sharp;
(4) at first prepare the yeast competent cell.It is centrifugal to cultivate the S. cervisiae of OD value 1.3-1.5 at 30 ℃ of shaking tables, removes supernatant; Be resuspended in Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 20-30 ℃ is incubated 1 hour; Ice-cold sterilized water washed twice; Washing once in the ice-cold again 1mol/L sorbyl alcohol; Be resuspended in the 1mol/L sorbyl alcohol of 0.2-0.5ml ice precooling ice bath; Add 80 μ l yeast suspensions in each sterile tube and be no more than the DNA (cumulative volume is less than 10 μ l) of 100ng, mixing gently, ice bath 5 minutes; The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom; Doing electricity in 1.5 kilovolts, 25 microfarads and 200 ohm swashs; Add 450 μ l to 1ml 1mol/L sorbyl alcohols at once and swash in the cup to electricity, mixing is transferred to culture tube gently;
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days; Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped;
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day; Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth;
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed; Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant; Leave standstill and solidified in 10 minutes; Make a call to an aperture, add 0.5 μ l 50mg/ml penbritin, put super clean bench 5 minutes as over against photograph; Be inverted overnight growth at 37 ℃ of incubators; Observe inhibition zone, record is taken pictures;
(8) glucose that in addition yeast transformant is never contained sorbyl alcohol selects plating to 5-10ml raffinose/semi-lactosi abduction delivering liquid nutrient medium; Abduction delivering 7 days; Get the culture of inducing 7 days, centrifugal 1 minute of 13000rpm gets supernatant; With e. coli k12 D31 and LB substratum mixing; Put into 37 ℃ of shaking tables, 200rpm shook 3 hours, took out ice bath; The OD. value of 600nm place measure sample and contrast on spectrophotometer is a blank with the LB and the yeast raffinose/semi-lactosi abduction delivering liquid nutrient medium of equivalent; Calculate bacteriostasis rate according to formula (bacteriostasis rate=1-sample OD. value/contrast OD. value);
(9) insert fragment from the yeast transformant amplification, utilize boiling method to prepare pcr template; According to carrier pYES2/CT sequences Design upstream and downstream primer, amplification, purifying and full-automatic order-checking.Chemosynthesis part antibacterial peptide is used the agar diffusion method detection of active.
The inventive method is applicable to separates various antibacterial peptides, behind the fusion dna fragment secreting, expressing of yeast α mating factor leading peptide and peptide at random a Kex2 proteolytic enzyme restriction enzyme site is arranged, and will downcut peptide at random, secretes to born of the same parents, thereby can suppress colibacillary growth.3 antibiotic tripeptide sequences that record are:first clone is tripeptides, and dna sequence dna is AAGACCCAC, and aminoacid sequence is Lys Thr His; Second clone is tripeptides, and dna sequence dna is CCAACGCCT, and aminoacid sequence is Pro Thr Pro; The 3rd clone is tripeptides, and dna sequence dna is GCGTCTAAA, and aminoacid sequence is Ala Ser Lys; The 4th clone is 68 peptides; Dna sequence dna is ACCAACAATGATCTAGAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTC GGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCG CTGATCCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCC TCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGT, and amino acid sequence is Thr Asn Asn Asp Leu Glu Gly Pro Ser Lys Val Ser LeuSer Leu Thr Leu Ser Ser Val Ser Ile Leu Arg Val Pro Val Ile Ile Thr IleThr Ile Glu Phe Lys Pro Ala Asp Pro Arg Gly Pro His His Val Ile Ser TyrVal Thr Leu Thr Phe Thr Pro Ser Pro His Ile Arg Ser Asn Arg Lys Gly ArgSer.
Three separating obtained antibiotic 3 peptides of the inventive method can be used as the lead compound of microbiotic exploitation.
Following table 1 is the bacteriostasis rate that records with antibacterial peptide clone juice.
| First clone | Second clone | The 3rd clone | Sequence table No. 1 and No. 2 | Blank: the pYES2/CT transformant | |
| OD600nm | ?0.024 | ?0.029 | ?0.056 | ?0.037 | ?0.27 |
| Bacteriostasis rate | ?91.1％ | ?89.2％ | ?79.2％ | ?86.3％ |
Proved absolutely that from table 1 and each experiment diagram the inventive method can separate antibacterial peptide effectively.
The method that the inventive method is set up can antibiotic oligopeptides of high efficiency separation and polypeptide.The frequency of finding strong antibiotic clone is about 20%, and about 10% records sequence.A general people 1 year separable antibiotic oligopeptides and polypeptide more than 40, and the general people of traditional method just can be separated to a kind of antibacterial peptide in several years.The tripeptides that is separated to is not had an immunogenicity.Because molecular weight is little, can see through bacteria cell wall, biological accessibility is good.Owing to carry out secreting, expressing in the yeast saccharomyces cerevisiae system, be equivalent to utilize eukaryotic system to carry out a preliminary Biosafety property testing.And, can utilize Yeast gene engineering production.Because encoding sequence can produce a large amount of combinations, nearly all target position that these little peptides can directed toward bacteria.Albumen and polypeptide at yeast saccharomyces cerevisiae system secreting, expressing are subjected to glycosylation modified through the Serine of being everlasting, Threonine and N site.And all there is glycosylation site in the antibacterial peptide that the present invention is separated to, and has shown strong anti-microbial activity, and antibiotic 3 peptides can be used as the in addition chemically modified of lead compound of microbiotic exploitation, develop potent antibiotics.
Description of drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d are that separating obtained 4 antibacterial peptides clone covers formed inhibition zone behind the upper strata substratum that contains e. coli k12 D31;
Fig. 1 e is the negative contrast after empty carrier pYES2/CT yeast conversion subcovering contains the upper strata substratum of e. coli k12 D31;
Fig. 1 f for add in the upper strata substratum punching that contains e. coli k12 D31 0.5 μ l 50mg/ml penbritin over against photograph;
Fig. 2 upper left for full-automatic synthetic, purity be that 95.8% antibiotic 3 peptide Lys Thr His point sample 10ng are containing the formed inhibition zone of upper strata substratum of e. coli k12 D31;
Fig. 2 bottom right be 0.5 μ l 50mg/ml penbritin point sample the upper strata substratum that contains e. coli k12 D31 over against photograph.
Embodiment
Embodiment 1:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PfuDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 50ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 20 seconds, 15 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and 1 μ l (20 unit) exonuclease III, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 1 μ l (20 unit) Semen Phaseoli radiati Germinatus nuclease again, handles 1 hour 20 minutes for 37 ℃.With the PCR purification column purifying of Qiagen company, with 13 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 200ml YPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 100ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 25 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 200ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 100ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.2ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 doing electricity, kilovolt, 25 microfarads, 200 ohm swash.Adding 450 μ l 1mol/L sorbyl alcohols at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day.Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth.
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed.Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant.Leave standstill and solidified in 10 minutes.Make a call to an aperture in the place that does not have transformant with rifle head point, add 0.5 μ l50mg/ml penbritin, put super clean bench 5 minutes as over against photograph.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
(8) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the Pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAATACGACTCACTATAGGG;＜6〉TAGGATCAGCGGGTTTAAACTC.2 antibiotic tripeptide sequences that record are: first clone is tripeptides, and dna sequence dna is AAGACCCAC, and aminoacid sequence is Lys ThrHis.Second clone is tripeptides, and dna sequence dna is CCAACGCCT, and aminoacid sequence is Pro Thr Pro.
(9) synthesize kyrine L ys Thr His in Shanghai Bo Ya company with Peptide synthesizer.The intestinal bacteria bacterial classification K12D31 of 5 μ l overnight growth is added to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed, dull and stereotyped, leave standstill and solidified in 10 minutes.Make a call to two apertures with rifle head point, an aperture adds 10ng kyrine L ysThr His, and another aperture adds 0.5 μ l 50mg/ml penbritin as over against photograph, puts super clean bench 5 minutes.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
Embodiment 2:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PyrobestDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 100ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 49 ℃ 30 seconds, 72 ℃ 30 seconds, 18 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 40 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 40 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and the 10 exonuclease III of unit, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 20 unit Semen Phaseoli radiati Germinatus nucleases again, handles 1 hour 20 minutes for 37 ℃.With the PCR purification column purifying of Qiagen company, with 20 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 50mlYPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 50ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 25 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 50ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 30ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.2ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 kilovolt, 25 microfarads and 200 ohm do electricity and swash.Adding 1ml 1mol/L sorbyl alcohol at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) glucose that yeast transformant is never contained sorbyl alcohol selects plating to 10ml raffinose/semi-lactosi abduction delivering liquid nutrient medium.Abduction delivering 7 days.Get each 1.5ml of culture that has induced 7 days, centrifugal 1 minute of 13000rpm gets supernatant, adds 0.75ml in every sterile eppendorf tubes.Get e. coli k12 D31 overnight culture and LB substratum mixing, final concentration is 50 μ l e. coli k12 D31 overnight culture/5mlLB substratum, wherein gets 0.75ml and is added to the eppendorf pipe that sample is housed, and making cumulative volume is 1.5ml.Put into 37 ℃ of shaking tables, 200rpm shook 3 hours, took out ice bath.The OD. value of 600nm place measure sample and contrast on 752 spectrophotometers is a blank with the LB and the yeast raffinose/semi-lactosi abduction delivering liquid nutrient medium of equivalent.Calculate bacteriostasis rate according to formula (bacteriostasis rate=1-sample OD. value/contrast OD. value).
(7) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAGGATCAGCGGGTTTAAACTC;＜6〉TAGGATCAGCGGGTTTAAACTC.1 the antibiotic tripeptides dna sequence dna that records is GCGTCTAAA, and aminoacid sequence is Ala Ser Lys.
Embodiment 3
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PfuDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 50ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 51 ℃ 30 seconds, 72 ℃ 20 seconds, 20 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and the 20 exonuclease III of unit, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 10 unit Semen Phaseoli radiati Germinatus nucleases again, handles 1 hour for 37 ℃.With the PCR purification column purifying of Qiagen company, with 11 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 100ml YPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 100ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 30 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 100ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 50ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.3ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 kilovolt, 25 microfarads and 200 ohm do electricity and swash.Adding 650 μ l 1mol/L sorbyl alcohols at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day.Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth.
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed.Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant.Leave standstill and solidified in 10 minutes.Make a call to an aperture in the place that does not have transformant with rifle head point, add 0.5 μ l50mg/ml penbritin, put super clean bench 7 minutes as over against photograph.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
(8) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the Pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAATACGACTCACTATAGGG;＜6〉TAGGATCAGCGGGTTTAAACTC.1 antibiotic 68 peptide that records, its dna sequence dna and aminoacid sequence are seen sequence table.This clone's stochastic sequence is lost a base and is caused reading frame to change when primer is synthetic.
Lithium Acetate/dithiothreitol (DTT) pretreatment fluid prescription used in the foregoing description is (every 100ml): 0.1M Lithium Acetate (final concentration), 10mM Tris (final concentration), pH7.5,1mM EDTA (final concentration) adds the 10ml 0.1M dithiothreitol (DTT) of filtration sterilization to the above-mentioned solution of 90ml (final concentration is 10mM) behind the autoclaving.
Glucose/sorbyl alcohol selection plate culture medium prescription used in the foregoing description is (every a liter): 100ml10X yeast salt (containing trace element), 1M sorbyl alcohol, 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Add VITAMIN and in the time of 50-60 ℃, add penicillin, fall dull and stereotyped with bacteria growing inhibiting.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
The used glucose that does not contain sorbyl alcohol selects dull and stereotyped prescription to be (every liter) in the foregoing description: 100ml10X yeast salt (containing trace element), 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Add VITAMIN and in the time of 50-60 ℃, add penicillin, fall dull and stereotyped with bacteria growing inhibiting.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
The dull and stereotyped prescription of raffinose/semi-lactosi abduction delivering used in the foregoing description is (every liter): 100ml10X yeast salt (containing trace element), 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.The final concentration that adds filtration sterilization is 1% raffinose and 1% semi-lactosi.Add VITAMIN, fall dull and stereotyped.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
Raffinose/semi-lactosi abduction delivering liquid culture based formulas used in the foregoing description is (every a liter):: 100ml 10X yeast salt (containing trace element), 30mg leucine, 20mg Histidine, 30mg tryptophane.Autoclaving.Add 1% raffinose of filtration sterilization and 1% semi-lactosi.Add VITAMIN.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
LB prescription used in the foregoing description is (500ml): 5g peptone, 2.5g yeast extract, 5g sodium-chlor, distilled water, pH7.0.
Antibacterial peptide sequence table .seq
＜110〉Liu Qiuyun, impressively
＜120〉a kind of method and isolating antibacterial peptide that separates antibacterial peptide
<160>2
<210>1
<211>207
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(204)
<400>1
ACC?AAC?AAT?GAT?CTA?GAG?GGC?CCT?TCG?AAG?GTA?AGC?CTA?TCC?CTA?ACC???48
Thr?Asn?Ash?Asp?Leu?Glu?Gly?Pro?Ser?Lys?Val?Ser?Leu?Ser?Leu?Thr
1???????????????5???????????????????10??????????????????15
CTC?TCC?TCG?GTC?TCG?ATT?CTA?CGC?GTA?CCG?GTC?ATC?ATC?ACC?ATC?ACC???96
Leu?Ser?Ser?Val?Ser?Ile?Leu?Arg?Val?Pro?Val?Ile?Ile?Thr?Ile?Thr
20??????????????????25??????????????????30
ATT?GAG?TTT?AAA?CCC?GCT?GAT?CCT?AGA?GGG?CCG?CAT?CAT?GTA?ATT?AGT???144
Ile?Glu?Phe?Lys?Pro?Ala?Asp?Pro?Arg?Gly?Pro?His?His?Val?Ile?Ser
35??????????????????40??????????????????45
TAT?GTC?ACG?CTT?ACA?TTC?ACG?CCC?TCC?CCC?CAC?ATC?CGC?TCT?AAC?CGA???192
Tyr?Val?Thr?Leu?Thr?Phe?Thr?Pro?Ser?Pro?His?Ile?Arg?Ser?Asn?Arg
50??????????????????55??????????????????60
AAA?GGA?AGG?AGT?TAG?207
Lys?Gly?Arg?Ser?***
65
<210>2
<211>68
<212>PRT
＜213〉artificial sequence
<400>2
Thr?Asn?Asn?Asp?Leu?Glu?Gly?Pro?Ser?Lys?Val?Ser?Leu?Ser?Leu?Thr
1???????????????5???????????????????10??????????????????15
Leu?Ser?Ser?Val?Ser?Ile?Leu?Arg?Val?Pro?Val?Ile?Ile?Thr?Ile?Thr
20??????????????????25??????????????????30
Ile?Glu?Phe?Lys?Pro?Ala?Asp?Pro?Arg?Gly?Pro?His?His?Val?Ile?Ser
35??????????????????40??????????????????45
Tyr?Val?Thr?Leu?Thr?Phe?Thr?Pro?Ser?Pro?His?Ile?Arg?Ser?Asn?Arg
50??????????????????55??????????????????60
Lys?Gly?Arg?Ser
65
Claims (5)
1, a kind of method of separating antibacterial peptide is characterized in that utilizing high-fidelity PCR to make up the yeast α mating factor leading peptide and the fusion dna fragment of peptide at random, is used for the secreting, expressing of peptide at random; Insert the yeast saccharomyces cerevisiae carrier that contains inducible promoter with EcoR I and Xba I double digestion; Recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, it is carried out digestion process with exonuclease III and Semen Phaseoli radiati Germinatus nuclease; Electricity swashs transformed saccharomyces cerevisiae behind the purifying; Transformant is forwarded to raffinose/semi-lactosi abduction delivering flat board, covers the observation inhibition zone after the overnight growth with the upper strata substratum that contains intestinal bacteria bacterial classification K12D31; Or with surveying bacteriostasis rate behind the liquid nutrient medium abduction delivering; Positive colony is carried out pcr amplification, order-checking.
2, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is AAGACCCAC, aminoacid sequence is Lys Thr His.
3, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is CCAACGCCT, aminoacid sequence is Pro Thr Pro.
4, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is GCGTCTAAA, aminoacid sequence is Ala Ser Lys.
5, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is 68 peptides, and its dna sequence dna is
ACCAACAATGATCTAGAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTC
TCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGT
TTAAACCCGCTGATCCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCT
TACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGT, aminoacid sequence are Thr Asn Asn Asp Leu Glu Gly Pro Ser Lys Val Ser Leu Ser Leu Thr LeuSer Ser Val Ser Ile Leu Arg Val Pro Val Ile Ile Thr Ile Thr Ile Glu Phe Lys Pro Ala AspPro Arg Gly Pro His His Val Ile Ser Tyr Val Thr Leu Thr Phe Thr Pro Ser Pro His IleArg Ser Asn Arg Lys Gly Arg Ser.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345865C (en) * | 2005-09-16 | 2007-10-31 | 中山大学 | Antibiotic dipeptide and its chemical modifier, its preparation method and uses |
| CN102382173A (en) * | 2011-11-12 | 2012-03-21 | 山东大学 | Oligopeptide C and antibacterial application thereof |
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102093998B (en) * | 2010-12-09 | 2012-11-07 | 山东大学 | Preparation method of antibacterial peptide cecropin feed additive |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH07203975A (en) * | 1994-01-23 | 1995-08-08 | Showa Denko Kk | Gene encoding antimicrobial peptide and production thereof |
| JP3459314B2 (en) * | 1994-08-31 | 2003-10-20 | 社団法人農林水産技術情報協会 | New peptide, antimicrobial agent, new peptide gene, new recombinant DNA and novel peptide production method |
| CN1269837C (en) * | 2002-09-02 | 2006-08-16 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345865C (en) * | 2005-09-16 | 2007-10-31 | 中山大学 | Antibiotic dipeptide and its chemical modifier, its preparation method and uses |
| CN102382173A (en) * | 2011-11-12 | 2012-03-21 | 山东大学 | Oligopeptide C and antibacterial application thereof |
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
| CN103360467B (en) * | 2013-07-16 | 2015-06-03 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
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