CN110468143A

CN110468143A - The preparation method and application of antibacterial peptide NZX

Info

Publication number: CN110468143A
Application number: CN201910866484.0A
Authority: CN
Inventors: 王建华; 刘鹤; 毛若雨; 杨娜; 滕达; 王秀敏; 郝娅
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2019-11-19
Anticipated expiration: 2039-09-12
Also published as: CN110468143B

Abstract

The preparation method and application of present invention offer antibacterial peptide NZX.The present invention passes through optimization antibacterial peptide NZX gene order; high efficient expression of the antibacterial peptide NZX in Pichia pastoris is realized for the first time, and yield breaks through gram-grade level, overcomes the problem that yield is too low or chemical synthesis cost is excessively high in large-scale production; and perfect purification system is established, it can be achieved that large-scale production.In addition, antibacterial experiment the result shows that, antibacterial peptide NZX of the invention is to gram-positive bacteria, with significant bacteriostatic activity, while having the characteristics that low cytotoxicity, low hemolytic, can be applied to antibacterials exploitation, the fields such as feed addictive exploitation, have wide application value and market prospects.

Description

The preparation method and application of antibacterial peptide NZX

Technical field

The present invention relates to field of biotechnology, specifically, being related to the preparation method and application of antibacterial peptide NZX.

Background technique

Antibacterial peptide (AMPs) be multicellular organism be protect host from pathogenic microorganism encroach on and generate endogenous it is more Peptide, since they are played a crucial role in constituting innate immune system, antibacterial peptide (AMPs) is also defined For host defense peptide.The antibacterial peptide (AMPs) of most study is general positively charged at present, has cationic characteristic, can be negative with band The bacterial membrane of electricity combines, and leads to the disintegration (Min-Duk Seo etc., 2012) of lipid sided configuration.Most of antibacterial peptides (AMPs) there are two kinds of structural domains of hydrophily and hydrophobicity simultaneously, this structure can make antibacterial peptide (AMPs) can be with lipid group (hydrophobic region) and phospholipid fraction (hydrophilic area) is divided to combine (Jenssen etc., 2006).Antibacterial peptide (AMPs) has multiple-microorganism There are broad spectrum antibiotic activity, including gram-positive bacteria, Gram-negative bacteria, fungi and virus (Zasloff etc., 2002).Due to A possibility that antibacterial peptide has antibacterial activity good, has a broad antifungal spectrum (including drug tolerant bacteria), a variety of antibacterial mechanisms, drug resistance occurs It is lower, therefore become the strong candidate (Ali Adem Bahar etc., 2013) of new antibiotic substitute.

Plectasin is Mygid in 2005 et al. from from the saprophytic sac fungus of Northern Europe pine forest earth's surface Isolated a kind of antibacterial peptide with antibacterial functions in (Pseudoplectania nigrella), and the first fungal defense Element has efficient anti-G⁺Bacterium active antibacterial peptide.The long precursor of one 95 residue of Plectasin gene open reading frame codes Peptide, 1~23 is signal peptide sequence, and 24~55 are leader peptide sequences, and 56~95 are mature peptide (Plectasin) sequence. Plectasin mature polypeptide is made of 40 amino acid, contains 6 Cys residues and 5 Lys residues, the tetrapeptide of a difference (DEDD) mode, molecular weight 4407.99Da, net charge change between+1 to+3, depend primarily on its two histidines Ionic condition.Shape 3 is to disulfide bond (Cys4-Cys30, Cys15-Cys37, Cys19-Cys39) in the molecule by its 6 Cys, and two Level structure by two antiparallel beta sheet structures and spiral of a stable N-terminal alpha-helix of 3 pairs of disulfide bond and C-terminal and Loop district's groups between foldable structure at.Plectasin has obvious lethal effect to gram-positive bacterium.Mygind etc. is ground Plectasin has been studied carefully to the inhibiting effect of more than 130 strain separate sources streptococcus pneumonias, has been found for penicillin-susceptible or resistance bacterium Strain, minimum bactericidal concentration (MIC) is between 0.1-8 μ g/mL, MIC₅₀For 1 μ g/mL (Mygind, Fischer etc., 2005).

NZX is a kind of novel Plectasin derived peptide, it is first verified to streptococcus pneumonia and resistance to methoxy west Woods staphylococcus aureus mycophylaxin class antibacterial peptide NZX active simultaneously, can effectively kill tuberculosis point in vitro Branch bacillus, concentration are suitable with the Killing Mycobacterium Tuberculosis drug of standard.In addition, NZX is not easy to be easily degraded by proteases, work as concentration There is no cytotoxicity at up to 100 μM.In mouse mycobacterium tuberculosis infection model, the mouse lung of NZX treatment is thin after 5 days Bacterium load reduces by 46%, and almost without difference, this is further supported for therapeutic effect and Killing Mycobacterium Tuberculosis medicament benemicin NZX as future may treat drug candidate lungy.Currently, there is not yet producing antibacterial peptide using gene engineering method The relevant report of NZX.

Summary of the invention

The object of the present invention is to provide the preparation method of antibacterial peptide NZX and new applications.

In order to achieve the object of the present invention, in a first aspect, the present invention provides antibacterial peptide NZX in preparation antibacterials or composition In application, wherein the bacterium includes but is not limited to staphylococcus aureus (Staphylococcus aureus) and pig grape Coccus (Staphylococcus hyicus).

In the present invention, the amino acid sequence of the antibacterial peptide NZX is as shown in SEQ ID NO:1.

Second aspect, the present invention provide a kind of recombinant expression carrier, and the recombinant expression carrier carries following gene expression Frame:

Shown in inducible promoter-coding alpha factor signal peptide DNA sequence dna-Kex2 cleavage site-SEQ ID NO:2 DNA sequence dna-terminator codon of encoding antimicrobial peptide NZX.

Further, 5 ' ends of the DNA sequence dna of the coding alpha factor signal peptide are connected with XhoI restriction enzyme site；The volume 3 ' ends of the DNA sequence dna of code antibacterial peptide NZX are connected with XbaI enzyme cutting site.

The sequence of complete genome expression cassette is shown in SEQ ID NO:3.

The third aspect, the present invention provide the host cell (such as Pichia pastoris) for containing the recombinant expression carrier.

Fourth aspect, the present invention provide recombinant yeast pichia pastoris, are the Pichia pastoris X- containing the recombinant expression carrier 33。

5th aspect, the present invention provide the preparation method of antibacterial peptide NZX, by building containing by yeast biased codons The expression vector of the DNA sequence dna of Optimized Coding Based antibacterial peptide NZX, and Pichia pastoris is converted, the recombinant yeast pichia pastoris of acquisition is by hair Ferment culture, secretion generate antibacterial peptide NZX.

Further, the method includes cultivating in the fermentation medium above-mentioned recombinant yeast pichia pastoris, and from hair Antibacterial peptide NZX is separated in ferment product.

It the described method comprises the following steps:

1) it the preparation of seed liquor: from YPD plate picking recombinant yeast pichia pastoris single colonie, is inoculated in rich containing 80~150 μ g/ml In 10~30mL YPD fluid nutrient medium of bleomycin (Zeocin), 28~30 DEG C, 200~250rpm, shaking table culture 18~ For 24 hours, it is inoculated in 200mL YPD fluid nutrient medium with 1%v/v inoculum concentration, 28~30 DEG C, 200~250rpm, shaking table culture 16 ~18h, until OD₆₀₀For 4~6 to get seed liquor；

Preferably, the seed liquor prepare it is as follows: from YPD plate picking recombinant yeast pichia pastoris single colonie, be inoculated in and contain In the 10mL YPD fluid nutrient medium of 100 μ g/ml bleomycins, 29 DEG C, 250rpm, shaking table culture 18-24h are connect with 1%v/v Kind amount is inoculated in 200mL YPD fluid nutrient medium, and 29 DEG C, 250rpm, shaking table culture 16-18h, until OD₆₀₀=6 to get seed Liquid；

2) at 25 DEG C~29 DEG C, the salt culture of the basis 2L fermented and cultured: is added in above-mentioned seed liquor by the inoculum concentration of 10%v/v In base, pH to 5.0 is adjusted, 9.6mL Trace salts solution PMT1 (FeSO is added₄·7H₂O 65g, ZnCl₂20g, CuSO₄·5H2O 6g, MnSO₄·5H₂O 3g, CoCl₂0.5g, Na₂MoO₄0.2g, KI 0.08g, H₃BO₄0.02g, Biotin 0.2g is dense H₂SO₄5mg is dissolved in 750mL distilled water, is placed in magnetic stirring apparatus and is settled to 1L, 0.22 μm of water system filter membrane to after being completely dissolved Filtration sterilization), ventilatory capacity maintains 8vvm, revolving speed 600rpm, and dissolved oxygen maintains 20% or more；

3) feed carbon source: observation oxygen dissolving value starts to rise to 80% or more suddenly after slowly declining, and starts stream plus containing 12 ‰ 50% glucose solution of PMT1, flow acceleration are 12~24mL/L/min, and continuous flow adds 6~8h, is adjusted on revolving speed 1000rpm；

4) methanol induction: after stream plus glucose, hungry half an hour starts to add 100% methanol, by first hour flow velocity 1mL/L/min progressively increases to the 6th hour 6mL/L/min, and 1100rpm is adjusted on revolving speed, and 5.5 are adjusted on pH, controls dissolved oxygen 20% or more, until fermentation ends.

Wherein, basal salt media used in step 2) is formulated as follows: 45g glucose, 50gNH₄H₂PO₄、20g K₂SO₄、15g MgSO₄·7H₂O、6g KH₂PO4、0.4g CaSO₄With 1.5g KOH, water is added to be settled to 1L.

The present invention also provides the purification process of the recombinant protein of above-mentioned pichia pastoris X-33 genetic engineering bacterium secretion comprising Dialysis desalting, freeze-drying, redissolution and ion-exchange chromatography are carried out to fermentation liquid.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

The present invention constructs specific expression carrier, realizes antibacterial peptide NZX for the first time by optimization antibacterial peptide NZX gene order High efficient expression in Pichia pastoris, it is horizontal that yield breaks through gram-grade, overcomes that yield in large-scale production is too low or chemical synthesis The excessively high problem of cost, and perfect purification system is established, it can be achieved that large-scale production, can be applied to antibacterials exploitation, raise The fields such as feed additives exploitation, have wide application value and market prospects.

Detailed description of the invention

Fig. 1 is the product agarose gel electrophoresis testing result that PCR method expands NZX gene in the embodiment of the present invention 1；Its In, M:DNA Marker I；1: being primer PUC57-NZX as the pcr amplification product of template using F1, R1.

Fig. 2 is bacillus coli DH 5 alpha positive transformant qualification result in the embodiment of the present invention 2；Wherein, M:DNA Marker I；1-5: transformant (F1, R1) specific primer PCR product.

Fig. 3 is that pPICZ α A-NZX vector linearization electrophoresis result is recombinated in the embodiment of the present invention 3；Wherein, M:Trans5K DNA marker；1: not linearized recombinant vector pPICZ α A-NZX；2: the recombinant vector pPICZ α A-NZX of linearisation.

Fig. 4 is that 48 orifice plates induce Tricine-SDS-PAGE electrophoretogram in the embodiment of the present invention 3；Wherein, M: ultra-low molecular It measures albumen Marker, 1-4: being respectively N14, N32, N34 and N41 transformant.

Fig. 5 is the antibacterial examination of the horizontal fermentation supernatant of N14, N32, N34, N41 restructuring yeast strains shaking flask in the embodiment of the present invention 4 Test result.

Fig. 6 is that N14, N32, N34, N41 restructuring yeast strains 120h are induced in the fermentation of shaking flask level in the embodiment of the present invention 4 Clear Tricine-SDS-PAG electrophoresis detection result；Wherein, M is Ultra-low molecular weight albumen Marker, 1-4: being respectively transformant The horizontal induction fermentation liquid supernatant electrophoresis band of N14, N32, N34, N41 transformant 120h shaking flask.

In fermentation of the Fig. 7 for induction times different after N34 restructuring yeast strains induction fermentation 120h in the embodiment of the present invention 5 Clear bacteriostatic activity testing result.

Fig. 8 is N34 restructuring yeast strains induction fermentation difference induction time fermentation supernatant in the embodiment of the present invention 5 Tricine-SDS-PAGE electrophoresis detection result；Wherein, M: Ultra-low molecular weight albumen Marker；1-6: respectively indicate induction 0h, For 24 hours, 48h, 72h, 96h, 120h fermented liquid supernatant electrophoresis band.

Fig. 9 is that total protein concentration and thallus are wet in N34 restructuring yeast strains process of high-density fermentation in the embodiment of the present invention 5 Curve is changed over time again.

Figure 10 is 6 antibacterial peptide NZX Mass Spectrometric Identification result of the embodiment of the present invention.Wherein, A: fermentation liquid mass spectrogram；B: after purification RNZX mass spectrogram.

Figure 11 is 8 antibacterial peptide NZX hemolytic experimental result of the embodiment of the present invention.

Figure 12 is 9 antibacterial peptide NZX cytotoxicity experiment result of the embodiment of the present invention.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Enzyme and reagent used in the following embodiment: restriction enzyme, pfuDNA polymerase, T4DNA ligase equal part It 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 reagents are dispensed using import or domestic analysis is pure.

Culture medium involved in following embodiment and buffer formulation:

LB culture medium: tryptone 10g/L, yeast soak extract 5g/L, NaCl 10g/L；Solid LB media is then added 2% agarose.

Low salt LB medium: tryptone 10g/L, yeast soak extract 5g/L, NaCl 5g/L；Solid less salt LB culture 2% agar powder is then added in base.

MH culture medium: casein hydrolysate 17.5g/L, beef extract powder 5g/L, starch 1.5g/L.

MHA culture medium: 2% agar powder is then added in solid MH culture medium.

YPD culture medium: peptone 20g/L, yeast soak extract 10g/L, glucose 20g/L；Solid YPD culture medium then adds Enter 2% agar powder.

YPDS culture medium: peptone 20g/L, yeast soak extract 10g/L, sorbierite 182.2g/L, glucose 20g/L, Agar powder 20g/L.

BMGY culture medium (/L): yeast soaks extract 10g, and peptone 20g, glycerol 10mL, 13.4% without amino acid leaven Nitrogen source (YNB) 100m L, 0.02% biotin 2mL, 1mol/L phosphate buffer (pH6.0) 100mL.

Use in relation to the culture mediums such as LB culture medium, less salt LB, MH, YPD, YPDS is grasped referring to Invitrogen Pichia pastoris Make handbook.

20mM phosphate buffer (A liquid): 0.4654g Na₂HPO₄, 2.9172g NaH₂PO₄, add deionized water extremely 950mL is placed in magnetic stirring apparatus and adjusts pH 5.7 to after being completely dissolved, is settled to 1000mL.

1M NaCl 20mM phosphate buffer (B liquid): 0.4654g Na₂HPO₄, 2.9172g NaH₂PO₄, 58.44g NaCl adds deionized water to 950mL, is placed in magnetic stirring apparatus and adjusts pH 5.7 to after being completely dissolved, be settled to 1000mL.

Gene magnification involved in following embodiment and transformant identification method are PCR method and DNA sequencing method.

Method of protein detection involved in following embodiment is Tricine-SDS-PAGE, referring to ( H.Tricine–SDS-PAGE.Nat protoc,2006,1(1):16-22)。

Determination of protein concentration method involved in following embodiment is Coomassie Brilliant Blue.

Protein molecular method for determination of amount involved in following embodiment is MALDI-TOF MS method.

The method of protein purification involved in following embodiment is based on ion chromatography.

Fermentation process involved in following embodiment is high density fermentation method.

Strain involved in following embodiment and plasmid are shown in Table 1:

Table 1 is for examination strain and plasmid

The acquisition of 1 antibacterial peptide NZX genetic fragment of embodiment

The optimization and design of 1.1 antibacterial peptide NZX expressed sequences

According to the amino acid sequence of antibacterial peptide NZX: GFGCNGPWSEDDIQCHNHCKSIKGYKGGYCARGGFVCKCY (SEQ ID NO:1) designs the encoding gene of antibacterial peptide NZX, passes through codon optimization according to Pichia pastoris codon-bias DNA sequence dna (rNZX) is as follows afterwards: GGTTTTGGTTGTAACGGTCCATGGTCTGAAGATGATATTCAATGTCATAACCATTG TAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAGAGGTGGTTTTGTTTGTAAGTGTTAC(SEQ ID NO: 2)。

In order to effectively terminate rNZX accurate translation, the C-terminal of two terminator codon TAA, TGA insertion rNZX coded sequences. Realize that natural secretion is expressed to be able to achieve rNZX in Pichia pastoris, the N of rNZX is inserted into the site signal peptide cutting site Kex2 End.In order to be able to achieve rNZX gene cloning and expression, XhoI, XbaI endonuclease site are designed at NZX gene both ends；In order to have Effect improves the cutting efficiency of XhoI, XbaI endonuclease, is inserted into protection respectively at XhoI, XbaI endonuclease site both ends Base.Gene expression cassette sequence is following (SEQ ID NO:3):

tctCTCGAGaaaagaGGTTTTGGTTGTAACGGTCCATGGTCTGAAGATGATATTCAATGTCATAACCA TTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAGAGGTGGTTTTGTTTGTAAGTGTTACtaatgaTCT AGAgc

Wherein, italic is protection base, is restriction enzyme site at underscore, and the italic place of underlining is kex2 gene expression production Object cutting identification sequence, runic is two terminator codons.

1.2PCR expands NZX gene expression frame

Designed NZX gene order is integrated on pUC57 plasmid vector, in order to obtain a large amount of NZX genes, design one PCR amplification is carried out to PCR amplification primer:

F1:5 '-TCTCTCGAGAAAAGAGGTTTTGGTT-3 '

R1:5 '-GCTCTAGATCATTAGTAACAC-3 '

Using the pUC57 plasmid of recombination as template, PCR reaction system is as follows:

Reaction condition is as follows: 94 DEG C of 5min of denaturation；94 DEG C of 30s are denaturalized, anneal 58 DEG C of 30s, extend 72 DEG C of 40s, 30 A circulation；It terminates and extends 72 DEG C of 10min.

PCR product is recycled through PCR product kits, obtains the NZX genetic fragment of high-purity, -20 DEG C save backup. Fig. 1 show testing result of the PCR product through 2% agarose gel electrophoresis.

The building of 2 yeast recombinant expression carrier of embodiment

The 2.1 NZX genetic fragments for obtaining embodiment 1 recovery purifying piece after XhoI and XbaI endonuclease double digestion Section.Meanwhile with XhoI and XbaI double digestion pPICZ α A carrier (be purchased from Invitrogen).

Double digestion system is as follows:

The above digestion system sample-adding, which finishes, is placed on 37 DEG C of reaction 4h of PCR instrument, the detection of 2% agarose gel electrophoresis, electrophoresis Condition: 120V, 30min.Digestion products are recycled with DNA product QIAquick Gel Extraction Kit, and -20 DEG C save backup.NZX gene and pPICZ α A carrier is after XbaI and XhoI are double digested, with T4 DNA ligase by the pPICZ α A carrier of NZX gene and linearisation It is attached.Linked system is as follows:

Condition of contact: in 16 DEG C of connections overnight of PCR instrument after the above linked system sample-adding.

2.2 are transformed into the recombinant vector of acquisition in bacillus coli DH 5 alpha, and step of converting is as follows:

1) 50 μ L E.coli DH5 α competent cells, ice bath 30min is added in 10 μ L connection products；

2) 42 DEG C of heat shock 45s, immediately 2~3min of ice bath；

3) the LB less salt culture mediums of 37 DEG C of 450 μ L preheatings of addition, 37 DEG C, 100rpm renewal cultivation 1h；

4) 100-200 μ L is taken to be coated with the LB less salt solid medium containing 25 μ g/mL Zeocin after thallus is resuspended；

5) 12-16h is cultivated in 37 DEG C of inversions.

The identification of 2.3 bacillus coli DH 5 alpha positive transformants

The single colonie that picking is grown on less salt LB plate is inoculated in 10mL LB liquid medium (containing 25 μ g/mL Zeocin), 37 DEG C, 250rpm is incubated overnight, and identifies positive transformant by bacterium colony PCR.The sun that picking is verified through special primer Property transformant be inoculated in 10mL less salt LB liquid medium (containing 25 μ g/mL Zeocin), 37 DEG C, 250rpm is incubated overnight, and is taken 500 μ g sequencing, is compared with design gene order, and it is whether completely correct that foreign gene insertion is verified from DNA level.

Picking positive transformant carries out bacterium solution PCR and verifies transformant correctness, PCR body according to gene order design primer System, condition are as follows:

PCR system:

PCR condition: 94 DEG C of 5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 28 circulations；72℃10min.

PCR product is through 2% agarose gel electrophoresis testing goal band (Fig. 2).Sequencing result shows NZX genetic fragment Insertion point, direction and sequence are completely correct, are consistent with design.15% glycerol tube saves the E.coli containing recombinant expression carrier And plasmid is extracted, it is denoted as pPICZ α A-NZX, turns Pichia pastoris (P.pastoris) for linearisation electricity and prepares.Embodiment 3 contains NZX The building of recombinant yeast

The linearisation of 3.1 recombinant vector pPICZ α A-NZX

Digestion is carried out to composing type recombinant expression carrier pPICZ α A-NZX using PmeI, digestion system and reaction condition are such as Under:

The above digestion system sample-adding, which finishes, is placed on 37 DEG C of reaction 4h of PCR instrument, the detection of 2% agarose gel electrophoresis, electrophoresis Condition: 120V, 30min.Electrophoresis result (Fig. 3) display: pPICZ α A-NZX recombinant vector total Linearization.

The preparation of 3.2 pichia pastoris X-33 competence

1) the X-33 single colonie on picking YPD plate is seeded to 10mL YPD fluid nutrient medium, 29 DEG C, 250rpm, stays overnight Culture；

2) it takes pichia pastoris X-33 to be incubated overnight liquid with 1% inoculum concentration, is seeded to 100mL YPD fluid nutrient medium, 29 DEG C, 250rpm, culture to OD₆₀₀Light absorption value is 1.1-1.3；

3) 50mL culture, is added 50mL sterile water after 4000rpm, 5min centrifugation and is resuspended by 4 DEG C；

4) 4 DEG C, after 4000rpm, 5min centrifugation, supernatant is removed, 25mL sterile water is added and is resuspended；

5) 4 DEG C, after 4000rpm, 5min centrifugation, supernatant is removed, 2mL 1M sorbierite is added and is resuspended；

6) 4 DEG C, after 4000rpm, 5min centrifugation, supernatant is removed, being added after 100 μ L 1M sorbierites are resuspended is X-33 impression State cell；

3.3 electrotransformation

It takes 100 μ L competent yeast cells that linearisation recombinant plasmid freeze-dried powder is added, mixes gently, go to the electricity of ice pre-cooling In revolving cup, 5min is placed on ice, and electricity turns, parameter 1200V, 25 μ F, 400 Ω.The mountain 1M of 1ml ice pre-cooling is added in electricity immediately after turning Pears alcoholic solution, mixing are transferred in 2mL centrifuge tube, and 29 DEG C, recovery 2h, the bacterium solution after taking 100 μ L to recover is coated on containing 100 μ g/ On the YPDS plate of mL Zeocin antibiotic, 29 DEG C of inversion cultures, until growing single colonie.

3.4 48 orifice plates induction screening positive transformant

500 μ L BMGY culture mediums are added in every hole in 48 orifice plates, and half of the single colonie grown in picking step 3.3 is put into In 48 orifice plates.The blank control wells that bacterium is not added are respectively set, pPICZ α A empty plasmid negative control hole can induce out with determining Positive control wells.29 DEG C, 250rpm shaken cultivation for 24 hours after, 2.5 μ L methanol (final concentration of 0.5%) of methanol, note is added in every hole It is denoted as 0h respectively every adding a methanol for 24 hours for 0h, for 24 hours, 48h, 72h collect the hair in 48 orifice plates after inducing 72h respectively Zymotic fluid is in 1.5mL centrifuge tube, centrifuging and taking supernatant, carries out antibacterial activity detection and Tricine-SDS-PAGE electrophoresis.

48 orifice plates induction Tricine-SDS-PAGE electrophoretogram is shown in Fig. 4.

The screening of the horizontal high efficient expression NZX restructuring yeast strains of 4 shaking flask of embodiment

The 4.1 horizontal inducing expressions of positive transformant shaking flask

The positive transformant (being denoted as Pichia pastorisX-33NZX) screened in picking step 3.4, is seeded to 10mL YPD fluid nutrient medium, 29 DEG C, 250rpm shaken cultivation 18-20h；1% inoculum concentration is forwarded to the training of 200mL BMGY liquid Base is supported, glassine paper sealed membranes is replaced after 29 DEG C, 250rpm shaken cultivation 1 day with 4 layers of sterile gauze and wraps up shaking flask mouths, each shaking flask Adding 1mL methanol induction, (final concentration of 0.5%) of methanol is denoted as 0h, and 29 DEG C, 250rpm shaken cultivation is collected every for 24 hours later 2mL fermentation liquid is in 2mL centrifuge tube, until induction 120h terminates.

The detection of 4.2 recombination yeast Activities of Fermentation Broth

Bactericidal test analysis: picking S.aureus ATCC43300 single colonie is inoculated in 10mL MH culture medium, and 37 DEG C, 250rpm is cultivated to OD₆₀₀About 0.4,1% inoculum concentration is transferred in 50mL MH solid medium, is mixed, rapidly 19cm In the rectangular culture dish of × 19cm, after to be solidified, the fermented liquid supernatant of 30 μ L induction 0-120h is added.

The horizontal Tricine-SDS-PAGE detection of 4.3 recombination yeast secretory proteins

Tricine-SDS-PAGE is further used to analyze recombinant mutant high activity restructuring yeast strains obtained Expression, electrophoresis method reference (2006)。

The present invention successfully optimizes encoding antimicrobial peptide NZX gene, and constructs pPICZ α A-NZX recombinant expression carrier, warp Successful conversion obtains tetra- plants of higher recombinations of expression of N14, N32, N34, N41 into pichia pastoris X-33 after PmeI linearisation Yeast strain, antibacterial peptide NZX realize high-caliber secreting, expressing in pichia pastoris X-33.

The horizontal fermentation supernatant bacteriostatic test result of N14, N32, N34, N41 restructuring yeast strains shaking flask is shown in Fig. 5, N14, N32, N34, N41 restructuring yeast strains 120h induction horizontal fermentation supernatant Tricine-SDS-PAG electrophoresis detection result of shaking flask are shown in Fig. 6.

The high density fermentation of 5 restructuring yeast strains N34 of embodiment

The horizontal fermented liquid supernatant protein concentration of transformant N14, N32, N34, N41 shaking flask is respectively 161mg/L, 124mg/ L, 253mg/L and 208mg/L selects the highest N34 transformant of expression to carry out the hydraulic test of subsequent fermentation tank.It is flat from YPD Plate picking N34 transformant single colonie, being inoculated in liquid amount is 10ml YPD fluid nutrient medium (containing 100 μ g/ml Zeocin) In 50mL shaking flask, 29 DEG C, 250rpm, 18-24h, liquid amount is inoculated in as 200ml YPD seed liquid culture medium with 1% inoculum concentration 1L shaking flask in, 29 DEG C, 250rpm, 16-18h, OD₆₀₀About 6, it is spare as high density fermentation seed liquor.

High density fermentation is carried out using 5L fermentor, fermentation process is divided into three phases: (1) the thalli growth stage: is added 2L basal salt media, 121 DEG C of sterilizing 20min are cooled to 29 DEG C, adjust pH to 5.0, and 9.6mL PMT1 is added, and access 200mL Bacterium solution (1:10), ventilatory capacity maintain 8vvm, revolving speed 600rpm, and dissolved oxygen maintains 20% or more；(2) stream plus Glucose-grown Stage: observation oxygen dissolving value starts to fly up after slowly declining, and starts stream plus 50% glucose solution (12 ‰ PMT1), and stream accelerates Degree is 24mL/L/min, and continuous flow adds 6h, 1000rpm is adjusted on revolving speed, and other fermentation conditions are constant；(3) methanol transition induces Stage: after fermentation condition variation, stream plus glucose 6h, hungry half an hour starts to add 100% methanol, by first hour stream Fast 1mL/L/min progressively increases to the 6th hour 6mL/L/min, and 1100rpm is adjusted on revolving speed, and 5.5 are adjusted on pH, controls molten For oxygen 20% or more, other fermentation conditions are constant, until fermentation ends.

Since transition induction, sampled every for 24 hours, it is living for detecting thallus weight in wet base and protein expression situation analysis and antibacterial Property analysis.The fermentation supernatant bacteriostatic activity testing result of different induction times is shown in after N34 restructuring yeast strains induction fermentation 120h Fig. 7.N34 restructuring yeast strains induction fermentation difference induction time fermentation supernatant Tricine-SDS-PAGE electrophoresis detection result is shown in Fig. 8.Total protein concentration and thallus weight in wet base change over time curve in 29 DEG C of induction restructuring yeast strains N34 process of high-density fermentation See Fig. 9.The result shows that by after the high density fermentation of the highest recombination yeast N34 progress fermentation tank level of expression quantity, to its total egg White concentration, antibacterial peptide NZX concentration and biomass are detected, and 29 DEG C, total protein concentration reaches 2820mg/L after 120h fermentation, raw Object amount reaches 270g/L.And the higher antibacterial peptide NZX product of purity is obtained by single step purification, the rate of recovery is about 68%, is produced Amount is about 742mg/L.

The purifying of 6 antibacterial peptide NZX of embodiment

1, cation-exchange chromatography purifies

HiPrep SP FF cation exchange column (length 16mm, internal diameter 10mm, supplier GE Healthcare) is utilized A liquid balances loading after 3-5 column volume.After sample introduction, first with containing 20mM, the phosphoric acid salt elution buffer (A liquid) of pH5.7 It is eluted, after peak to be penetrated has eluted, with the 20mM containing 0.6M NaCl, the phosphoric acid salt elution buffer (B liquid) of pH5.7 It is eluted, collects eluting peak, UV280nm monitors elution profile, and Tricine-SDS-PAGE and detection target product purify feelings Condition.

2,1KDa bag filter desalination

The eluting peak being collected into is dialysed through 4 DEG C of 1kD molecular cut off bag filter, and it is primary that every 2h changes water, is changed water 6 times.It collects Dialyzate after dialysis, (- 54 DEG C, 0.016MPa) of low-temperature vacuum freeze drier freeze-dryings obtain antibacterial peptide NZX freeze-dried powder and produce Product, mass spectrogram after purification show that rNZX molecular weight is consistent (Figure 10) with theoretical molecular weight.

The detection of 7 antibacterial peptide NZX antibacterial activity of embodiment

It is the anti-of 1280 μ g/mL with sterile physiological water compound concentration according to antibacterial peptide NZX freeze-dried powder is obtained in embodiment 6 Bacterium peptide NZX, antibacterial peptide Plectasin and ceftriaxone sodium solution, 2 times of doubling dilutions to 1.25 μ g/mL of final concentration will be different dense Antibacterial peptide NZX, antibacterial peptide Plectasin and the ceftriaxone sodium solution of degree are added separately in sterile 96 porocyte culture plates, often 10 μ L of hole, 3, each sample parallel, and equal amount (10 μ L) sterile saline prepares MIC plate as negative control.Bacterial strain is adopted With MH fluid nutrient medium culture, 37 DEG C of shaken cultivations to OD₆₀₀=0.4, bacterium solution is prepared into concentration and is equivalent to 0.5 Maxwell than turbid The bacteria suspension of standard, the sterile MH fluid nutrient medium after 37 DEG C are incubated for are diluted to 10⁵After CFU/mL, to the MIC plate prepared Every hole adds 90 μ L bacteria suspensions in sample well, and 37 DEG C of constant-temperature incubation 16-18h observe and record experimental result.NZX to pathogen most The micro broth dilution method of the foundation such as small Mlc (MIC) reference Tian, slightly change as the case may be (Tian etc., 2009).NZX is shown in Table 2 to the antibacterial activity of gram-positive bacteria, to staphylococcus aureus and Staphylococcus hyicus antibacterial activity It is significantly better than 6-26 times of antibiotic Ceftriaxone Sodium and parent peptide Plectasin 2-8 times.

Antibacterial activity of 2 NZX of table to gram-positive bacteria

The hemolytic of 8 antibacterial peptide NZX of embodiment is tested

According to the antibacterial peptide NZX freeze-dried powder obtained in embodiment 6, antibacterial peptide NZX is dissolved in sterile saline, is matched It is set to the mother liquor that concentration is 512 μ g/mL, the final concentration of 2 μ g/mL of 2 times of doubling dilutions.Take the ICR female mice of SPF grades of 6 week old, eyeball Blood is taken, is collected using heparin sodium anticoagulant tube.The blood of acquisition is centrifuged 10min, with 10mM PBS (pH7.3) in 4 DEG C, 1500rpm Repeated washing red blood cell 3 times, until supernatant it is colorless and transparent be made 8% red blood cell suspension.Respectively take 100 μ L red blood cell suspensions and Antibacterial peptide NZX solution, is added 96 orifice plates, and 37 DEG C of incubations 1h, 1500rpm are centrifuged 5min, draw supernatant to ELISA ELISA Plate and detect UV absorption under 540nm.Physiological saline and 0.1%Triton X-100 are respectively 0% and 100% haemolysis control experiment.It is molten Blood degree calculation formula is following (referring to Jung, Park etc., 2007):

Haemolysis degree (%)=[(Abs_{540nm antibacterial peptide NZX}-Abs_{540nm physiological saline})/(Abs_540nm _0.1%Triton _X-100- Abs_{540nm physiological saline})] × 100%

Experimental result is as shown in figure 11.

The cytotoxicity experiment of 9 antibacterial peptide NZX of embodiment

Mtt assay detects NZX and Ceftriaxone Sodium to Hacat cytotoxicity.MTT can be by the succinic acid in living cells mitochondria Dehydrogenase is reduced to insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation, is deposited on into the cell, and this reaction does not occur for dead cell.DMSO can dissolve Bluish violet in cell crystallizes first a ceremonial jade-ladle, used in libation, can reflect indirectly living cells quantity by solution light absorption value after dissolution.Certain cell quantity In range, crystallization forming amount is directly proportional to cell number.

37 DEG C, 5%CO₂And under the conditions of saturated humidity, Hacat cell culture is in MEM complete medium.Liquid-transfering gun piping and druming Cell, cell is resuspended in MEM complete medium, with 2.5 × l0⁵Cells/mL density is inoculated in 96 orifice plates, every 100 μ L of hole, if Set 3 repetitions.Moved back for 24 hours except culture medium, every hole by concentration gradient be added 100 μ L concentration be 1,2,4,8,16,32,64,128, 256 μ g/mL sample peptides, control wells add the PBS solution of equivalent.Continue after being incubated for for 24 hours, remove culture medium, PBS is washed twice, every hole It is middle that the MTT (being protected from light operation) that 100 μ L concentration are 5mg/mL is added.96 orifice plates are moved to incubator to continue to cultivate 4h.After abandoning MTT liquid 150 μ L DMSO are added in every hole, and oscillator vibrates 10min, are completely dissolved to bottom hole crystallization, 570nm wavelength surveys each hole absorbance. Cell survival rate (with reference to Jiao etc., 2015) is calculated according to the following formula:

Survival rate (%)=processing group OD value/control group OD value × 100%

Experimental result is as shown in figure 12.

The present invention is by carrying out antibacterial activity detection to antibacterial peptide NZX after purification, the results show that the anti-gram sun of NZX Property bacterium activity be significantly better than antibiotic Ceftriaxone Sodium, the minimal inhibitory concentration of NZX anti-Staphylococcus aureus ATCC43300 (MIC) value is 0.46 μM, and minimal inhibitory concentration (MIC) value of anti-Staphylococcus hyicus NCTC10350 is 0.91 μM, and ceftriaxone Minimal inhibitory concentration (MIC) value of sodium anti-Staphylococcus aureus ATCC43300 and Staphylococcus hyicus NCTC10350 is 12.09μM.The hemolytic of NZX the experimental results showed that, NZX of the concentration within the scope of 1-256 μ g/mL hardly generates red blood cell Haemolysis.The cytotoxicity experiment of NZX the result shows that, under 1-128 μ g/mL concentration, the survival rate of Hacat cell exists NZX 90% or more, illustrate that it does not have toxic action to intact animal histocyte.NZX cell in 256 μ g/mL of higher concentration is deposited Motility rate is declined slightly (72%), but toxicity is not strong.Antibacterial peptide NZX is prepared according to the method provided by the invention, and there is expression quantity Height, antibacterial activity is good, and the low feature of toxicity has a extensive future.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Bibliography:

1.Seo M D,Won H S,Kim J H,et al.Antimicrobial peptides for therapeutic applications:A review[J].Molecules,2012,17(12):12276-12286

2.Jenssen,Hamill P,Hancock R E W.Peptide antimicrobial agents [J].Clinical Microbiology Reviews,2006,19(3):491-511.

3.Zasloff,Michael.Antimicrobial peptides of multicellular organisms [J].Nature,2002,415(6870):389-395.

4.Bahar A,Ren D.Antimicrobial Peptides[J].Pharmaceuticals,2013,6(12): 1543-1575.

5.Mygind P H,Fischer R L,Schnorr K M,et al.Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus[J].Nature (London),2005,437(7061):975-980.

6.Tenland E,Krishnan N,Anna,et al.A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis[J].Tuberculosis,2018,113:231-238.

7.H.Tricine-SDS-PAGE.[J].Nature Protocols,2006,1(1):16-22.

8.Tian Z G,Dong T T,Yang Y L,et al.Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3)[J].Applied Microbiology and Biotechnology,2009,83(1):143-149.

9.Jung H J,Park Y,Sung W S,et al.Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance[J].Biochimica et Biophysica Acta,2007,1768(6):0-1405.

Jiao J,Mao R,Wang X,et al.GAP-initiated constitutive expression of a novel plectasin-derived peptide MP1106 by Pichia pastoris and its activity against Streptococcus suis[J].Process Biochemistry,2015,50(2):253-261.

Sequence table

<110>Institute of Feeds,China Academy of Agriculture Sciences

<120>preparation method and application of antibacterial peptide NZX

<130> KHP191113661.9

<160>3

<170>SIPOSequenceListing1.0

<210> 1

<211> 40

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Gly Phe Gly Cys Asn Gly Pro Trp Ser Glu Asp Asp Ile Gln Cys His

1 5 10 15

Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Arg

20 25 30

Gly Gly Phe Val Cys Lys Cys Tyr

35 40

<210> 2

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ggttttggtt gtaacggtcc atggtctgaa gatgatattc aatgtcataa ccattgtaag 60

tctattaagg gttacaaggg tggttactgt gctagaggtg gttttgtttg taagtgttac 120

<210> 3

<211> 149

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tctctcgaga aaagaggttt tggttgtaac ggtccatggt ctgaagatga tattcaatgt 60

cataaccatt gtaagtctat taagggttac aagggtggtt actgtgctag aggtggtttt 120

gtttgtaagt gttactaatg atctagagc 149

Claims

1. application of the antibacterial peptide NZX in preparation antibacterials or composition, which is characterized in that the bacterium bag includes golden yellow grape Coccus (Staphylococcus aureus) and Staphylococcus hyicus (Staphylococcus hyicus)；

Wherein, the amino acid sequence of the antibacterial peptide NZX is as shown in SEQ ID NO:1.

2. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier carries following gene expression frame:

It is encoded shown in inducible promoter-coding alpha factor signal peptide DNA sequence dna-Kex2 cleavage site-SEQ ID NO:2 DNA sequence dna-terminator codon of antibacterial peptide NZX.

3. recombinant expression carrier according to claim 2, which is characterized in that the DNA sequence dna of the coding alpha factor signal peptide 5 ' end be connected with XhoI restriction enzyme site；3 ' ends of the DNA sequence dna of the encoding antimicrobial peptide NZX are connected with XbaI enzyme cutting site.

4. the host cell containing recombinant expression carrier described in Claims 2 or 3.

5. host cell according to claim 4, which is characterized in that it is Pichia pastoris.

6. recombinant yeast pichia pastoris, which is characterized in that it is the Pichia pastoris containing recombinant expression carrier described in Claims 2 or 3 X-33。

7. the preparation method of antibacterial peptide NZX, which is characterized in that the method includes in the fermentation medium to claim 6 institute The recombinant yeast pichia pastoris stated is cultivated, and antibacterial peptide NZX is separated from tunning；

8. the method according to the description of claim 7 is characterized in that the following steps are included:

1) it the preparation of seed liquor: from YPD plate picking recombinant yeast pichia pastoris single colonie, is inoculated in rich next mould containing 80~150 μ g/ml In 10~30mL YPD fluid nutrient medium of element, 28~30 DEG C, 200~250rpm, shaking table culture 18~for 24 hours, it is connect with 1%v/v Kind amount is inoculated in 200mL YPD fluid nutrient medium, and 28~30 DEG C, 200~250rpm, 16~18h of shaking table culture, until OD₆₀₀For 4~6 to get seed liquor；

2) at 25 DEG C~29 DEG C, 2L basal salt media fermented and cultured: is added in above-mentioned seed liquor by the inoculum concentration of 10%v/v In, pH to 5.0 is adjusted, 9.6mL Trace salts solution PMT1 is added, ventilatory capacity maintains 8vvm, revolving speed 600rpm, and dissolved oxygen maintains 20% or more；

3) feed carbon source: observation oxygen dissolving value starts to rise to 80% or more suddenly after slowly declining, and starts stream plus containing 12 ‰ PMT1 50% glucose solution, flow acceleration is 12~24mL/L/min, and continuous flow adds 6~8h, be adjusted to 1000rpm on revolving speed；

4) methanol induction: after stream plus glucose, hungry half an hour started to add 100% methanol, by first hour flow velocity 1mL/ L/min progressively increases to the 6th hour 6mL/L/min, and 1100rpm is adjusted on revolving speed, 5.5 is adjusted on pH, control dissolved oxygen exists 20% or more, until fermentation ends；

Wherein, basal salt media used in step 2) is formulated as follows: 45g glucose, 50gNH₄H₂PO₄、20g K₂SO₄、 15g MgSO₄·7H₂O、6g KH₂PO4、0.4g CaSO₄With 1.5g KOH, water is added to be settled to 1L.