CN106336452A - Group of NZ2114 histidine mutants, and preparation method thereof - Google Patents
Group of NZ2114 histidine mutants, and preparation method thereof Download PDFInfo
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Abstract
The invention provides a group of NZ2114 histidine mutants, and a preparation method thereof. Two histidine sites existing in an NZ2114 sequence are selectively mutated by using a protein directional transformation. The histidine mutants with the amino acid sequences represented by SEQ ID NO:1 to SEQ ID NO:5 are designed, and recombinant expression of the histidine mutants in Pichia pastoris is realized. Design of an NZ2114 histidine mutantderivative is realized for the first time, and a determination result shows that the mutants (especially the 16th histidine mutant) has substantial bacteriostatic activity on Staphylococcus aureus ATCC 25923, ATCC 43300 and ATCC 6538 and Streptococcus suis CVCC 3928 and CVCC 3309, and the MIC value is 0.0625-2 [mu]g/ml. The NZ2114 histidine mutants obtained through the method can be applied to the fields of antibacterial drugs, food additives, cosmetics and feed additives, and has wide application values and market prospect.
Description
Technical field
The present invention relates to protein engineering and genetic engineering field are and in particular to serial antibacterial peptide directional transformation and gene work
The method that journey produces.
Background technology
Plectasin plectasin is mygind seminar using the saprophytic ascus class isolated from the pine forest of Europe the north
Fungi pseudoplectania nigrella builds cdna library, by blastx and ssearchp sequence similarity search journey
Sequence, examination to there is efficiently anti-g+Bacterium active antibacterial peptide.Plectasin gene code contains the polypeptide of 95 amino acid residues
Sequence, it 1～23 is signal peptide sequence, and 24～55 is leader peptide sequences, and 56～95 is mature peptide (plectasin) sequence
Row.Plectasin theoretical molecular is 4407.9da, has six histidines (his) and five lysines (lys), in difference
In ph environment, because histidine dissociated state is different, the net charges of plectasin change between+1 to+3 (mygind etc.,
plectasin is a peptide antibiotic with therapeutic potential from a
Saprophytic fungus.nature, 2005,437 (7061): 975-980).
Plectasin has strong lethal effect to gram-positive bacterium.Mygind etc. have studied plectasin pair
The inhibitory action of more than 130 strain separate sources streptococcus pneumonias, finds for penicillin-susceptible or resistant strain, MBC
(mic) all between 0.1-8 μ g/ml, mic50For 1 μ g/ml.Gottlieb etc. (2008) research plectasin is to golden yellow Portugal
Grape coccus (staphylococcus aureus) sterilizing ability, mic value is between 1 and 32 μ g/ml, mic50For 8 μ g/ml
(gottlieb etc., antimicrobial peptides effectively kill a broad spectrum of
listeria monocytogenes and staphylococcus aureus strains independently of
Origin, sub-type, or virulence factor expression.bmc microbiol, 2008,8 (1): 205-
214).Additionally, novozymes is with plectasin as parent, build mutated library through fallibility pcr, sieve from 274 mutant
Choose the mutant nz2114 that anti-microbial property is greatly improved, bacterium is clinically separated to methicillin-resistant staphylococcus aureus (mrsa)
The average mic of strain is only 1 μ g/ml, and also has very strong inhibitory action to vancomycin-resistant enterococcus (vrsa), and mic value is only
For 2～4 μ g/ml.Additionally, sterilized dynamic experiment proves, plectasin has efficient sterilizing ability, 5 × mic
Plectasin can kill 99.9% for examination pathogen in 5h.Murine Model of Intraperitoneal Infection model shows, 10mg/kg
In plectasin 5h, germicidal efficiency is equivalent to 70mg/kg vancomycin, and in abdominal cavity, pathogen quantity reduces by 3 order of magnitude (phases
When in 99.9% germicidal efficiency).After 7 days, control group mice is all dead, by the mouse survival of 10mg/kg plectasin administration
Rate according to administration frequency different between 80～100%.Therapeutic effect in rabbit endocarditis infection model for the nz2114 also obtains phase
Like conclusion, 10mg/kg nz2114 can be more than or equal to 15mg/kg vancomycin or 12mg/kg Daptomycin curative effect, after 3 days
In related internal organs (kidney, spleen), the quantity of pathogen reduces by more than 99% (xiong etc., efficacy of nz2114, a
Novel plectasin-derived cationic antimicrobial peptide antibiotic, in
experimental endocarditis due to methicillin-resistant staphylococcus
Aureus.antimicrobial agents and chemotherapy, 2011,55 (11): 5325-5330).
Nz2114 sequence the 16th is histidine with the 18th, and because of histidine, dissociated state is different under various circumstances, makes
Nz2114 charge number in different ph environment is unstable, leads to antibacterial ability different.With charge stronger arginine and bad ammonia
It is unstable that the histidine that acid substitutes in nz2114 can alleviate the electric charge leading to because of the different dissociated states of histidine.At present, not yet
See the report being related to that histidine in nz2114 sequence is mutated.
Content of the invention
It is an object of the invention to provide a kind of utilize aox promoter inducible expression plectasin derivative nz2114 group ammonia
The method of mutant polynucleotides.
For realizing the object of the invention, the invention provides a series of antibacterial peptide, amino acid sequence comprises seq idno.1-
Any one in seq idno.5.The present invention also provides a series of antibacterial peptide genes, and nucleotide sequence comprises seq idno.6-
Any one in seq idno.10.
Additionally, the present invention comprises serial recombinant expression carrier states encoding antimicrobial peptide gene it is characterised in that carrying and being numbered with
Any one in sequence (seq idno.6-seq idno.10).
Present invention also offers the host cell containing above-mentioned expression vector.Preferably, described host cell is to finish red ferment
Female genetic engineering bacterium.
Invention further provides expression antibacterial peptide (seq idno.1-seq idno.5) in recombinant yeast pichia pastoris
It is characterised in that it is by above-mentioned recombinant yeast pichia pastoris fermented and cultured, secretion produces antibacterial peptide to method.
The present invention realizes first to histidine directional transformation in nz2114 and realizes its expression in Pichia pastoris.
Brief description
Fig. 1 is the pcr and plasmid vector ppicz α a extraction electrophoretogram of h1～h5 in the embodiment of the present invention 2, wherein, m1:
Dna molecular weight marker；M2:trans 5k marker；1-10:h1～h5；11:ppicz α a.
Fig. 2 is to linearize recombinant vector electrophoretogram, wherein, m:trans5k marker in the embodiment of the present invention 3；1-9:
Ppich1～ppich5 linearizes product；10: negative control.
Fig. 3 is that in the embodiment of the present invention 5, h1～h5 isolates and purifies rear tricine-sds-page electrophoretogram, m: ultra-low molecular
Amount albumen marker；1-8: h1～h5 after purification.
Fig. 4-8 is h1～h5 maldi-tof ms analysis after purification in the embodiment of the present invention 5.
Specific embodiment
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the modification that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, in the embodiment of the present invention, experiment material used, reagent and instrument etc. are all commercially available, if
Do not particularly point out, the conventional meanses that in embodiment, technological means used is well known to the skilled person.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as sambrook decile
Sub- Cloning: A Laboratory Manual (new york:gold spring harbor laboratory press, 1989).
Enzyme and reagent used in following examples: restriction enzyme, pfudna polymerase, t4dna ligase decile
Gou Zi not biolabs, invitrogen and promega company.Four kinds of dntp are purchased from promega company.Dna and protein molecule
Amount standard is biolabs product.Other conventional reagent adopt import packing or domestic pure analysis pure.
The culture medium being related in following examples and buffer formulation:
Lb culture medium: tryptone 10g/l, yeast soaks extract 5g/l, nacl 10g/l；Solid lb culture medium then adds
2% agarose.
Less salt lb culture medium: tryptone 10g/l, yeast soaks extract 5g/l, nacl5g/l；Solid less salt lb culture medium
Then add 2% agar powder.
Mh culture medium: casein hydrolysate 17.5g/l, beef extract powder 5g/l, starch 1.5g/l.
Mha culture medium: solid mh culture medium then adds 2% agar powder.
Ypd culture medium: peptone 20g/l, yeast soaks extract 10g/l, glucose 20g/l；Solid ypd culture medium then adds
Enter 2% agar powder.
Ypds culture medium: peptone 20g/l, yeast soaks extract 10g/l, sorbierite 182.2g/l, glucose 20g/l,
Agar powder 20g/l.
Grasp with reference to invitrogen Pichia pastoris about lb culture medium, using of the culture medium such as less salt lb, mh, ypd, ypds
Make handbook.
20mm phosphate buffer (a liquid): 3.1146g na2hpo4, 1.7628g nah2po4, plus deionized water is extremely
950ml, is placed in magnetic stirring apparatus and adjusts ph6.7 to after be completely dissolved, be settled to 1000ml.
1m nacl 20mm phosphate buffer (b liquid): 3.1146g na2hpo4, 1.7628g nah2po4, 58.44g
Nacl, plus deionized water, to 950ml, is placed in magnetic stirring apparatus and adjusts ph 6.7 to after be completely dissolved, be settled to 1000ml.
The gene magnification being related in following examples and transformant authentication method are pcr method and dna PCR sequencing PCR.
The method of protein detection being related in following examples is tricine-sds-page, with reference to (
H.tricine-sds-page.nat protoc, 2006,1 (1): 16-22).
The determination of protein concentration method being related in following examples is Coomassie Brilliant Blue.
The protein molecular method for determination of amount being related in following examples is maldi-tof ms method.
The method of the protein purification being related in following examples is based on ion chromatography.
The fermentation process being related in following examples is high density fermentation method.
The bacterial classification being related in following examples and plasmid are shown in Table 1:
Table 1 is for examination bacterial classification and plasmid
Embodiment 1nz2114 Histidine mutagenesis body MOLECULE DESIGN and gene chemical synthesis
There is histidine at 2 according to the 16th in nz2114 sequence with the 18th, with arginine and lysine, it is carried out
Mutation.Separately design single-site mutant body and double-site mutant totally 5 sequences (seq idno.1-seq idno.5).
According to Pichia pastoris codon-bias design mutant gene sequence (seq idno.6-seq idno.10).For
During ensureing to express, the integrality of sequence, holds in mutant gene sequence 5 ' and adds xhoi restriction enzyme site and kex2 cleavage
Point, adds taa, tag terminator sequence and xbai restriction enzyme site at 3 ' ends.Above sequence is had by the raw work bioengineering share in Shanghai
Limit company completes.
The structure of embodiment 2 Pichia pastoris inducible expression vector ppich1～ppich5
Respectively double digestion is carried out to the genetic fragment and carrier ppiczaa of synthesis with restriction enzyme xhoi and xbai,
Reclaim ppiczaa carrier segments and mutant gene fragment, connect, obtain carrier ppich1～ppich5；
Carrier ppich1～detailed the building process of ppich5: using restriction enzyme xhoi and xbai respectively to synthesis
Genetic fragment and ppiczaa carry out double digestion, and digestion system and condition are as follows:
Above digestion system sample-adding finishes and reacts 3 hours, 2% agarose gel electrophoresis detection, electrophoresis strip after 37 DEG C
Part: 120v, 30min.Electrophoresis finishes and is respectively cut carrier segments electrophoresis corresponding with genetic fragment using scalpel under uviol lamp
Band simultaneously carries out dna fragment recovery using Tiangeng Bioisystech Co., Ltd glue reclaim kit, provides specification by kit
Correlative detail operated.
Detected using agarose gel electrophoresis and reclaim fragment and carry out preliminary quantitation using quantitation software (genetools),
Molar ratio according to fragment/carrier (3: 1) is attached using t4dna ligase, and system and condition are as follows:
Above linked system sample-adding finishes after 22 DEG C of reactions of metal bath 2 hours, converts e.coli dh5 α.Conversion operation
Details is as follows:
1) connection product adds 100 μ l e.coli dh5 α competent cells, ice bath 30min；
2) 42 DEG C of heat shock 90s, ice bath 2～3min immediately；
3) the lb less salt culture medium of l37 DEG C of preheating of addition 900 μ, 37 DEG C, 80-100rpm renewal cultivation 1h；
4) 4000rpm centrifugation 5min, sucks 700 μ l supernatants；
5) take the 100-200 μ l lb less salt solid medium containing 25 μ g/ml zeocin for the coating after resuspended thalline；
6) it is inverted culture 12-16h for 37 DEG C.
Picking positive transformant, designs primer according to gene order, carries out bacterium solution pcr checking transformant correctness, pcr body
System, condition are as follows:
Pcr system:
Pcr condition:
2% agarose gel electrophoresis detection positive transformant bacterium solution pcr product, deposition condition: 120v, 30min.15% is sweet
Oil pipe preserves the e.coli containing recombinant expression carrier and extracts plasmid, turns p.pastoris for linearisation electricity and prepares, related real
Test details to operate according to plasmid extraction kit (Tiangeng bio tech ltd) specification.
Embodiment 3 contains the structure of ppich1～ppich5 restructuring yeast strains
The linearisation of 3.1 recombinant vector ppich1～ppich5
Using pmei, digestion, digestion system and reaction condition are carried out to composing type recombinant expression carrier ppich1～ppich5
As follows:
Above digestion system sample-adding finishes and reacts 3 hours, 2% agarose gel electrophoresis detection, electrophoresis strip after 37 DEG C
Part: 120v, 30min.Electrophoresis reclaims linearisation using dna QIAquick Gel Extraction Kit after finishing the correct linearisation of detection recombinant expression carrier
Recombinant expression carrier.
Pichia pastoris electricity conversion and the identification of 3.2 linearized vectors
1) the x-33 single bacterium colony on picking ypd flat board, is seeded to 10ml ypd fluid nutrient medium, 30 DEG C, 250rpm, overnight
Culture；
2) 50 μ l incubated overnight liquid are taken to be seeded to 100ml ypd fluid nutrient medium, 30 DEG C, 250rpm, cultivates to od600Inhale
Light value is 1.2；
3) 50ml culture, adds 50ml sterilized water resuspended after 4000rpm, 5min centrifugation by 4 DEG C；
4) 4 DEG C, after 4000rpm, 5min centrifugation, add 25ml sterilized water resuspended；
5) 4 DEG C, after 4000rpm, 5min centrifugation, add 2ml 1m sorbierite resuspended；
6) 4 DEG C, 4000rpm, 5min centrifugation after add 100 μ l1m sorbierites resuspended after be x-33 competent cell (with
Upper 6 step operations must operate on ice, and action is soft)；
7) premix 80 μ lx-33 competent cells and 5-10 μ g linearized vector, the 0.2cm electricity being transferred to precooling on ice turns
In cup, place electroporation operation (1200v, 25 μ f, 400 ω) after 5min on ice；
8) add 1ml 1m sorbierite immediately, mix；
9) 30 DEG C of incubation 1-2h；
10) the x-33 cell ypds flat board containing 100 μ g/mlzeocin for the coating after taking 100 μ l to incubate, is inverted training for 30 DEG C
Foster 2-4 days.
Single bacterium colony on picking ypds flat board is seeded in the ypd fluid nutrient medium of 100 μ g/ml zeocin, 30 DEG C,
250rpm, incubated overnight.Take 4 DEG C of 1ml overnight culture, after 12000rpm, 5min centrifugation, pbs is resuspended, boiling water bath 10min, soon
Speed is positioned over -70 DEG C of 30min, immediately boiling water bath 10min again, 4 DEG C, takes supernatant as template after 12000rpm, 5min centrifugation
Carry out pcr checking positive transformant, pcr system is as follows with condition:
Pcr system:
Pcr condition:
2% agarose gel electrophoresis detection positive transformant bacterium solution pcr product, deposition condition: 120v, 30min.By size
Correct positive transformant corresponds and is transferred on the ypd flat board containing 100 μ g/ml zeocin in case expressing further.
The expression of embodiment 4nz2114 Histidine mutagenesis body
The expression of 4.1 transformants
Picking positive transformant, is seeded to ypd fluid nutrient medium, 30 DEG C, 250rpm shaken cultivation 18-20h；0.5-1%
Inoculum concentration is forwarded to 50ml ypd fluid nutrient medium, and 30 DEG C, 250rpm shaken cultivation replaced glass with 4 layers of sterile gauze after 1 day
Paper sealed membrane wraps up shaking flask mouth, and 30 DEG C, 250rpm shaken cultivation 3 days is to fermentation ends.
4.2 recombination yeast Activities of Fermentation Broth detections
Bactericidal test is analyzed: picking s.aureusatcc 25923 single bacterium colony is inoculated in 10mlmh culture medium, 37 DEG C,
250rpm cultivates to od600nm ≈ 0.4, and 1% inoculum concentration is transferred in 50ml mh solid medium, mix, rapidly 19cm ×
In the square culture dish of 19cm, to be solidified after, carefully place Oxford cup in media surface, add 50 μ l fermented liquid supernatant.
The horizontal tricine-sds-page of 4.3 recombination yeast secretory proteins detects
Tricine-sds-page is adopted to analyze recombinant mutant further the high activity restructuring yeast strains being obtained
Expression, electrophoresis method reference (2006).
The purifying of embodiment 5nz2114 Histidine mutagenesis body
5.1 cation-exchange chromatographies purify
Bradford method measures protein concentration, 4 DEG C, takes supernatant after 12000rpm centrifugation 10min.By hiprep sp ff sun
Ion exchange column (length 16mm, internal diameter 10mm, ge healthcare) utilizes a liquid to balance loading after 5-10 column volume.Sample introduction
After finishing, first with containing 20mm, the phosphoric acid salt elution buffer (a liquid) of ph6.7 is eluted, and after peak to be penetrated has eluted, uses
The phosphoric acid salt elution buffer (b liquid) of the 20mm containing 1m nacl, ph6.7 is eluted, and collects eluting peak.Elution step is:
100%a liquid, elutes 5 column volumes, for penetrating peak；40%a, 60%b liquid, elutes 5 column volumes, is eluting peak.Using
Uv215nm monitors elution profile, and tricine-sds-page and detection target product purify situation.
5.21kda bag filter desalination
The eluting peak collected, through the dialysis of 4 DEG C of 1kd molecular cut off bag filter, every 2h changes water once, changes water 6 times.Collect
Dislysate after dialysis, (- 54 DEG C, 0.016mba) of low-temperature vacuum freeze drier is lyophilized, and is coagulated by tricine-sds page
Gel electrophoresis detect.
Embodiment 6 anti-Staphylococcus aureus Activity determination
Nz2114 Histidine mutagenesis body to the minimal inhibitory concentration (mic) of pathogen with reference to tian etc. (tian etc.,
expression of antimicrobial peptide lh multimers in escherichia coli c43
(de3) .applied microbiology and biotechnology, 2009,83 (1): 143-149) the micro meat set up
Soup dilution method, slightly changes as the case may be, and details of operation is as follows:
1) picking strains tested monoclonal is to mh culture medium, 37 DEG C, 250rpm, and shaken overnight is cultivated；
2) antibacterials are diluted in 1.5ml sterile centrifugation tube by 2 times of gradient series, concentration is the 10 of final concentration respectively
Times；
3) respectively strains tested is forwarded to 37 DEG C in mh fluid nutrient medium with 1% inoculum concentration, 250rpm shaken cultivation
To 0.5 Maxwell standard than turbid；
4) for 1000 times of bacteria culture fluid of examination, (final cell concentration is 10 for dilution5Cfu/ml about), it is transferred to steril cell
In culture plate, every hole contains bacterium solution 90 μ l after dilution；
5) the medicine 10 μ l after dilution, 3 Duplicate Samples of each concentration, and reserved no drug-negative control wells are added.Plus no
Bacterium growth plate lid, after sealing membrane closure, 37 DEG C of static gas wave refrigerator to negative controls vacate now macroscopic obvious muddiness bacterium solution.
Tissue Culture Plate is taken out, observed result, mic value is can substantially to suppress the Cmin of strain subject growth.As occurred jumping
Between hole or Duplicate Samples, the inconsistent situation of result, then retest.
H1～h5 mutant is taken to carry out Determination of Antibacterial Activity, result is as shown in table 3.
Table 2nz2114 Histidine mutagenesis body is to staphylococcus aureus activity
Table 3nz2114 Histidine mutagenesis body is to streptococcus activity
As shown in Table 2, nz2114 Histidine mutagenesis body has compared with high inhibition effect to staphylococcus aureus: to golden yellow Portugal
The mic of grape coccus atcc25923 is between 0.0625-0.125 μ g/ml；To the mic of atcc6538 between 0.25-1 μ g/ml；Right
The mic of atcc43300 is between 0.25-2 μ g/ml.
As shown in Table 3, to streptococcus, especially three plants Streptococcus suis have compared with high inhibition work nz2114 Histidine mutagenesis body
With: to the mic of Streptococcus suis cvcc3928 between 0.0312-0.25 μ g/ml；To the mic of cvcc606 between 0.0312-0.125
μg/ml；To the mic of cvcc3309 between 0.0625-0.125 μ g/ml.
Although, above used general explanation, specific embodiment and test, the present invention made retouch in detail
State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Scope.
Claims (6)
1. a series of antibacterial peptides, amino acid sequence comprises any one in seq id no.1-seq id no.5.
2. a series of antibacterial peptide genes, nucleotide sequence comprises any one in seq id no.6-seq id no.10.
3. a kind of recombinant expression carrier is it is characterised in that carry encoding antimicrobial peptide gene order described in coding claim 2
Any one in (seq id no.5-seq id no.10).
4. the host cell containing expression vector described in claim 3.
5. containing the host cell described in claim 3 it is characterised in that described host cell is pichia pastoris gene engineering
Bacterium.
6. express the method for antibacterial peptide (seq id no.1-seq id no.5) in recombinant yeast pichia pastoris it is characterised in that being
By the recombinant yeast pichia pastoris fermented and cultured described in claim 5, secretion produces antibacterial peptide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438139A (en) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | A method of antibacterial peptide T9W is prepared using Pichia yeast |
| CN110468143A (en) * | 2019-09-12 | 2019-11-19 | 中国农业科学院饲料研究所 | The preparation method and application of antibacterial peptide NZX |
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| CN101189256A (en) * | 2005-06-06 | 2008-05-28 | 诺维信公司 | Polypeptides having antimicrobial activity and polynucleotides encoding same |
| CN103923207A (en) * | 2014-04-08 | 2014-07-16 | 东北农业大学 | Preparation method of FGF-21 (fibroblast growth factor-21) mutant protein and application of FGF-21 mutant protein in treating non-alcoholic fatty liver disease |
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| CN101189256A (en) * | 2005-06-06 | 2008-05-28 | 诺维信公司 | Polypeptides having antimicrobial activity and polynucleotides encoding same |
| CN103923207A (en) * | 2014-04-08 | 2014-07-16 | 东北农业大学 | Preparation method of FGF-21 (fibroblast growth factor-21) mutant protein and application of FGF-21 mutant protein in treating non-alcoholic fatty liver disease |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438139A (en) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | A method of antibacterial peptide T9W is prepared using Pichia yeast |
| CN110468143A (en) * | 2019-09-12 | 2019-11-19 | 中国农业科学院饲料研究所 | The preparation method and application of antibacterial peptide NZX |
| CN110468143B (en) * | 2019-09-12 | 2021-06-15 | 中国农业科学院饲料研究所 | Preparation method and application of antibacterial peptide NZX |
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Application publication date: 20170118 |