CN110468143B - Preparation method and application of antibacterial peptide NZX - Google Patents

Abstract

The invention provides a preparation method and application of antibacterial peptide NZX. According to the invention, by optimizing the gene sequence of the antimicrobial peptide NZX, the efficient expression of the antimicrobial peptide NZX in pichia pastoris is realized for the first time, the yield is in the way of overcoming the gram level, the problems of too low yield or too high chemical synthesis cost in large-scale production are solved, a perfect purification system is established, and the large-scale production can be realized. In addition, an antibacterial experiment result shows that the antibacterial peptide NZX has obvious antibacterial activity on gram-positive bacteria, has the characteristics of low cytotoxicity and low hemolysis, can be applied to the fields of antibacterial drug development, feed additive development and the like, and has wide application value and market prospect.

Description

Preparation method and application of antibacterial peptide NZX

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of antibacterial peptide NZX.

Background

Antimicrobial peptides (AMPs) are endogenous polypeptides produced by multicellular organisms to protect hosts from pathogenic microorganisms, and are also defined as host defense peptides because they play a crucial role in constituting the innate immune system. Currently the most studied antimicrobial peptides (AMPs) are generally positively charged, have cationic properties and can bind to negatively charged bacterial membranes, leading to the disassembly of the lipid bilateral structure (Min-Duk sio et al, 2012). Most antimicrobial peptides (AMPs) have both hydrophilic and hydrophobic domains, which may enable the antimicrobial peptides (AMPs) to bind to lipid components (hydrophobic regions) and phospholipid components (hydrophilic regions) (jensen et al, 2006). Antimicrobial peptides (AMPs) have a broad spectrum of antimicrobial activity against a variety of microorganisms, including gram-positive bacteria, gram-negative bacteria, fungi, and viruses (Zasloff et al, 2002). The antibacterial peptide has good antibacterial activity, wide antibacterial spectrum (including drug-resistant bacteria), multiple antibacterial mechanisms and low possibility of drug resistance, so the antibacterial peptide becomes a powerful candidate of a novel antibiotic substitute (Ali Adem Bahar and the like, 2013).

Plectasin was a 2005 antibiotic peptide isolated by Mygid et al from Psilocystis putrescentia (Pseudoplectania nigrella) in Pinus martensii and having an antibacterial function, and is also a first fungal defensin having a high G-resistance⁺An antibacterial peptide with antibacterial activity. The Plectasin gene open reading frame encodes a precursor peptide with the length of 95 residues, wherein 1-23 parts of the Plectasin gene open reading frame are signal peptide sequences, 24-55 parts of the Plectasin gene open reading frame are leader peptide sequences, and 56-95 parts of the Plectasin gene open reading frame are mature peptide (Plectasin) sequences. Plectasin mature polypeptide is composed of 40 amino acidsContaining 6 Cys residues and 5 Lys residues, a distinctive tetrapeptide (DEDD) pattern with a molecular weight of 4407.99Da and a net charge varying between +1 and +3, depending mainly on the ionic state of its two histidines. The 6 Cys of the polypeptide have 3 pairs of disulfide bonds (Cys4-Cys30, Cys15-Cys37 and Cys19-Cys39) in the molecule, and the secondary structure consists of an N-terminal alpha-helix and two anti-parallel beta-folded structures at the C terminal which are stabilized by the 3 pairs of disulfide bonds and a Loop region between the helix and the folded structures. Plectasin has an obvious killing effect on gram-positive bacteria. Mygind et al studied the inhibitory effect of Plectasin on over 130 strains of Streptococcus pneumoniae from different sources and found that the minimum bactericidal concentration (MIC) was between 0.1 and 8. mu.g/mL for penicillin-sensitive or resistant strains, with MIC being between₅₀At 1. mu.g/mL (Mygind, Fischer et al, 2005).

NZX is a novel Plectasin derived peptide, the first fungal defensin antimicrobial peptide NZX that has been shown to be active against both Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus, and is effective in killing Mycobacterium tuberculosis in vitro at concentrations comparable to standard anti-Mycobacterium tuberculosis drugs. Furthermore, NZX is not readily degraded by proteases and is not cytotoxic at concentrations as high as 100. mu.M. In a mouse mycobacterium tuberculosis infection model, the bacterial load of NZX-treated mouse lung is reduced by 46% after 5 days, and the treatment effect is almost not different from that of the anti-mycobacterium tuberculosis drug rifampicin, which further supports NZX as a candidate drug for treating tuberculosis in the future. At present, no report related to the production of the antibacterial peptide NZX by using a genetic engineering method is found.

Disclosure of Invention

The invention aims to provide a preparation method and a new application of antibacterial peptide NZX.

To achieve the objects of the present invention, in a first aspect, the present invention provides the use of antimicrobial peptide NZX in the preparation of an antimicrobial medicament or composition, wherein the bacteria include but are not limited to Staphylococcus aureus (Staphylococcus aureus) and Staphylococcus suis (Staphylococcus hyicus).

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

In a second aspect, the present invention provides a recombinant expression vector carrying the following gene expression cassettes:

inducible promoter-DNA sequence encoding alpha factor signal peptide-Kex 2 cleavage site-DNA sequence encoding antibacterial peptide NZX shown in SEQ ID NO. 2-stop codon.

Further, the 5' end of the DNA sequence for coding the alpha factor signal peptide is connected with an XhoI enzyme cutting site; the XbaI cleavage site is connected to the 3' end of the DNA sequence encoding the antimicrobial peptide NZX.

The sequence of the complete gene expression cassette is shown in SEQ ID NO. 3.

In a third aspect, the invention provides a host cell (e.g., pichia pastoris) containing the recombinant expression vector.

In a fourth aspect, the invention provides recombinant pichia pastoris, which is pichia pastoris X-33 containing the recombinant expression vector.

In a fifth aspect, the invention provides a preparation method of the antimicrobial peptide NZX, wherein an expression vector containing a DNA sequence which is optimized by yeast preferred codons and encodes the antimicrobial peptide NZX is constructed, pichia pastoris is transformed, and the obtained recombinant pichia pastoris is subjected to fermentation culture and secretion to generate the antimicrobial peptide NZX.

Further, the method comprises culturing the recombinant pichia pastoris in a fermentation medium, and separating the antibacterial peptide NZX from the fermentation product.

The method comprises the following steps:

1) preparing a seed solution: selecting a recombinant Pichia pastoris single colony from an YPD plate, inoculating the recombinant Pichia pastoris single colony in 10-30 mL YPD liquid culture medium containing 80-150 mu g/mL bleomycin (Zeocin), carrying out shake culture at 28-30 ℃ and 200-250 rpm for 18-24h, inoculating the recombinant Pichia pastoris single colony in 200mL YPD liquid culture medium at 1% v/v inoculum size, carrying out shake culture at 28-30 ℃ and 200-250 rpm for 16-18h until the recombinant Pichia pastoris single colony reaches OD₆₀₀4-6, and obtaining a seed solution;

preferably, the seed liquid is prepared as follows: picking recombinant Pichia pastoris single colony from YPD plate, inoculating in 10mL YPD liquid culture medium containing 100 ug/mL bleomycin, shake culturing at 29 deg.C and 250rpm for 18-24h, inoculating in 200mL YPD liquid culture medium at 1% v/v inoculum sizeCulturing at 29 deg.C and 250rpm for 16-18h to OD₆₀₀Obtaining seed liquid as 6;

2) fermentation culture: adding the seed solution into 2L of basic salt culture medium at 25-29 ℃ according to the inoculation amount of 10% v/v, adjusting the pH to 5.0, and adding 9.6mL of trace salt solution PMT1 (FeSO)₄·7H₂O 65g，ZnCl₂ 20g，CuSO₄·5H2O 6g，MnSO₄·5H₂O 3g，CoCl₂ 0.5g，Na₂MoO₄ 0.2g，KI 0.08g，H₃BO₄0.02g, Biotin 0.2g, concentrated H₂SO₄5mg, dissolving in 750mL of distilled water, placing in a magnetic stirrer until the solution is completely dissolved, fixing the volume to 1L, filtering and sterilizing by a 0.22-micron water-based filter membrane, and maintaining the ventilation volume at 8vvm, the rotating speed at 600rpm and the dissolved oxygen at more than 20%;

3) feeding a carbon source: observing that the dissolved oxygen value is slowly reduced and then suddenly rises to more than 80 percent, feeding a 50 percent glucose solution containing 12 per mill PMT1 at a feeding speed of 12-24 mL/L/min for 6-8 h continuously, and adjusting the rotating speed to 1000 rpm;

4) methanol induction: after glucose is fed, starving for half an hour, beginning to supplement 100% methanol, gradually increasing from the flow rate of 1mL/L/min in the first hour to 6mL/L/min in the sixth hour, increasing the rotation speed to 1100rpm, increasing the pH to 5.5, and controlling the dissolved oxygen to be more than 20% until the fermentation is finished.

Wherein, the basic salt culture medium used in the step 2) is prepared as follows: 45g glucose, 50gNH₄H₂PO₄、20g K₂SO₄、15g MgSO₄·7H₂O、6g KH₂PO4、0.4g CaSO₄And 1.5g KOH, and water was added to make a volume of 1L.

The invention also provides a purification method of the recombinant protein secreted by the pichia pastoris X-33 genetic engineering bacteria, which comprises the steps of carrying out dialysis desalination, freeze drying, redissolution, ion exchange chromatography and the like on fermentation liquor.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

according to the invention, the antibacterial peptide NZX gene sequence is optimized, a specific expression vector is constructed, the efficient expression of the antibacterial peptide NZX in pichia pastoris is realized for the first time, the yield breaks through gram-level, the problems of too low yield or too high chemical synthesis cost in large-scale production are solved, a complete purification system is established, the large-scale production can be realized, the antibacterial peptide NZX gene sequence can be applied to the fields of antibacterial drug development, feed additive development and the like, and the antibacterial peptide NZX gene has wide application value and market prospect.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection of the product of NZX gene amplified by PCR in example 1 of the present invention; wherein M is DNA Marker I; 1: PCR amplification products using F1 and R1 as primers and PUC57-NZX as templates.

FIG. 2 is the result of identifying E.coli DH5 alpha positive transformants in example 2 of the present invention; wherein M is DNA Marker I; 1-5: transformant (F1, R1) specific primer PCR product.

FIG. 3 shows the results of linearized electrophoresis of the recombinant pPICZ α A-NZX vector of example 3 of the present invention; wherein, M: trans5K DNA marker; 1: the non-linearized recombinant vector pPICZ alpha A-NZX; 2: linearized recombinant vector pPICZ alpha A-NZX.

FIG. 4 is an electrophoresis chart of 48-well plate induced Tricine-SDS-PAGE in example 3 of the present invention; wherein, M: ultra-low molecular weight protein Marker, 1-4: transformants were N14, N32, N34 and N41, respectively.

FIG. 5 shows the results of shake flask horizontal fermentation supernatant bacteriostasis tests of recombinant yeast strains N14, N32, N34 and N41 in example 4 of the present invention.

FIG. 6 shows the results of electrophoresis detection of Tricine-SDS-PAG in the supernatant of 120 h-induced shake flask horizontal fermentation of recombinant yeast strains N14, N32, N34 and N41 in example 4 of the present invention; wherein, M is an ultra-low molecular weight protein Marker, 1-4: the supernatant of the fermentation liquor is subjected to electrophoresis by a 120h shake flask horizontal induction method, wherein the supernatant is a transformant N14, a transformant N32, a transformant N34 and a transformant N41.

FIG. 7 shows the results of the bacteriostatic activity of the fermentation supernatant obtained by different induction times after 120h of induction and fermentation of the recombinant N34 yeast strain in example 5.

FIG. 8 shows the results of SDS-PAGE electrophoresis of fermented supernatant obtained by inducing fermentation with recombinant yeast strain N34 at different induction times in example 5; wherein, M: an ultra-low molecular weight protein Marker; 1-6: respectively representing the bands of fermentation liquor supernatant by electrophoresis method for inducing 0h, 24h, 48h, 72h, 96h and 120 h.

FIG. 9 is the total protein concentration and wet weight of the strain of recombinant N34 yeast in example 5 of the present invention during high density fermentation.

FIG. 10 shows the mass spectrometric identification of antimicrobial peptide NZX of example 6 of the present invention. Wherein, A: a mass spectrum of the fermentation liquid; b: purified rNZX mass spectrum.

FIG. 11 shows the results of the hemolytic test of antimicrobial peptide NZX in example 8 of the present invention.

FIG. 12 shows the results of the cytotoxicity test of the antibacterial peptide NZX in example 9 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Enzymes and reagents used in the following examples: restriction enzymes, pfu DNA polymerase, T4DNA ligase, etc. were purchased from Biolabs, Invitrogen and Promega, respectively. Four dNTPs were purchased from Promega. DNA and protein molecular weight standards were products of Biolabs. Other conventional reagents are imported and subpackaged or domestic analytically pure.

Media and buffer formulations referred to in the following examples:

LB culture medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl; solid LB medium was supplemented with 2% agarose.

Low-salt LB medium: 10g/L of tryptone, 5g/L of yeast extract and 5g/L of NaCl; 2% agar powder is added into the solid low-salt LB culture medium.

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

MHA medium: 2% agar powder was added to the solid MH medium.

YPD medium: peptone 20g/L, yeast extract 10g/L, glucose 20 g/L; 2% agar powder was added to the solid YPD medium.

YPDS medium: 20g/L of peptone, 10g/L of yeast extract, 182.2g/L of sorbitol, 20g/L of glucose and 20g/L of agar powder.

BMGY medium (/ L): 10g of yeast extract, 20g of peptone, 10mL of glycerol, 13.4% amino acid-free yeast nitrogen source (YNB)100m L, 2mL of 0.02% biotin, and 100mL of 1mol/L phosphate buffer (pH 6.0).

For the use of LB medium, low-salt LB, MH, YPD, YPDS and other media, the Invitrogen Pichia pastoris instruction manual was referred to.

20mM phosphate buffer (liquid a): 0.4654g Na₂HPO₄，2.9172g NaH₂PO₄Adding deionized water to 950mL, placing in a magnetic stirrer until the deionized water is completely dissolved, adjusting the pH value to 5.7, and metering to 1000 mL.

1M NaCl 20mM phosphate buffer (liquid B): 0.4654g Na₂HPO₄，2.9172g NaH₂PO₄58.44g of NaCl, adding deionized water to 950mL, placing in a magnetic stirrer until the NaCl is completely dissolved, adjusting the pH value to 5.7, and metering to 1000 mL.

The gene amplification and transformant identification methods referred to in the following examples were the PCR method and the DNA sequencing method.

The protein detection method referred to in the following examples is Tricine-SDS-PAGE, reference (see: (C)

H.Tricine–SDS-PAGE.Nat protoc,2006,1(1):16-22)。

The protein concentration measurement method described in the following examples is the Coomassie Brilliant blue method.

The molecular weight of the protein referred to in the following examples was determined by MALDI-TOF MS.

The method of protein purification referred to in the following examples is based on ion chromatography.

The fermentation process referred to in the following examples is a high density fermentation process.

The species and plasmids referred to in the following examples are shown in Table 1:

TABLE 1 test strains and plasmids

EXAMPLE 1 acquisition of antimicrobial peptide NZX Gene fragment

1.1 optimization and design of antibacterial peptide NZX Gene expression sequence

According to the amino acid sequence of antimicrobial peptide NZX: GFGCNGPWSEDDIQCHNHCKSIKGYKGGYCARGGFVCKCY (SEQ ID NO:1), designing the coding gene of antimicrobial peptide NZX according to the codon preference of pichia pastoris, and obtaining the following DNA sequence (rNZX) after codon optimization: GGTTTTGGTTGTAACGGTCCATGGTCTGAAGATGATATTCAATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAGAGGTGGTTTTGTTTGTAAGTGTTAC (SEQ ID NO: 2).

To effectively terminate translation expression of rNZX, two stop codons TAA, TGA were inserted into the C-terminus of the rNZX coding sequence. In order to realize natural secretion expression of rNZX in Pichia pastoris, a signal peptide cleavage site Kex2 site is inserted into the N end of rNZX. In order to realize the clone expression of rNZX gene, XhoI and XbaI endonuclease sites are designed at both ends of NZX gene; in order to effectively improve the cutting efficiency of XhoI and XbaI endonucleases, protective bases are respectively inserted into two ends of the XhoI and XbaI endonucleases. The expression cassette gene sequence is as follows (SEQ ID NO: 3):

tctCTCGAGaaaagaGGTTTTGGTTGTAACGGTCCATGGTCTGAAGATGATATTCAATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAGAGGTGGTTTTGTTTGTAAGTGTTACtaatgaTCT AGAgc

wherein, the italic is a protective base, the underlined part is a restriction enzyme site, the italic underlined part is a kex2 gene expression product cutting recognition sequence, and the bold is two stop codons.

1.2PCR amplification of NZX Gene expression cassette

The designed NZX gene sequence is integrated into a pUC57 plasmid vector, and in order to obtain a large amount of NZX genes, a pair of PCR amplification primers are designed for PCR amplification:

F1：5′-TCTCTCGAGAAAAGAGGTTTTGGTT-3′

R1：5′-GCTCTAGATCATTAGTAACAC-3′

using the recombinant pUC57 plasmid as a template, the PCR reaction system was as follows:

the reaction conditions were as follows: initial denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 40s, and 30 cycles; the extension was terminated at 72 ℃ for 10 min.

The PCR product is purified and recovered by a PCR product kit to obtain a high-purity NZX gene fragment, and the high-purity NZX gene fragment is stored at the temperature of-20 ℃ for later use. FIG. 1 shows the result of detection of PCR products by 2% agarose gel electrophoresis.

EXAMPLE 2 construction of recombinant expression vectors for Yeast

2.1 the NZX gene fragment obtained in example 1 was digested simultaneously with XhoI and XbaI endonucleases, and the purified fragment was recovered. At the same time, pPICZ α A vector (from Invitrogen) was double-digested with XhoI and XbaI.

The double enzyme digestion system is as follows:

after the enzyme digestion system is added with sample, the mixture is placed in a PCR instrument for reaction at 37 ℃ for 4h, and 2% agarose gel electrophoresis detection is carried out, wherein the electrophoresis conditions are as follows: 120V, 30 min. The enzyme digestion product is recovered by a DNA product recovery kit and stored at-20 ℃ for later use. NZX gene and pPICZ alpha A vector are digested by XbaI and XhoI, then NZX gene is connected with linearized pPICZ alpha A vector by T4DNA ligase. The linking system is as follows:

connection conditions are as follows: after the sample is added, the connection system is connected with a PCR instrument at 16 ℃ overnight.

2.2 transformation of the obtained recombinant vector into E.coli DH 5. alpha. the transformation procedure was as follows:

1) add 10. mu.L ligation into 50. mu.L E.coli DH 5. alpha. competent cells, ice-bath for 30 min;

2) performing heat shock at 42 ℃ for 45s, and immediately performing ice bath for 2-3 min;

3) adding 450 μ L LB low salt culture medium preheated at 37 deg.C, restoring culture at 37 deg.C and 100rpm for 1 h;

4) after the thalli is resuspended, 100. mu.L of the thalli is taken and coated with LB low-salt solid culture medium containing 25. mu.g/mL Zeocin;

5) culturing at 37 deg.C for 12-16 h.

2.3 identification of E.coli DH5 alpha Positive transformants

Single colonies grown on low-salt LB plates were picked and inoculated into 10mL of LB liquid medium (containing 25. mu.g/mL Zeocin), cultured overnight at 37 ℃ and 250rpm, and positive transformants were identified by colony PCR. The positive transformant verified by the specific primer is selected and inoculated in 10mL of low-salt LB liquid medium (containing 25 mug/mL Zeocin), cultured overnight at 37 ℃ and 250rpm, 500 mug of sequence is selected for sequencing, compared with the designed gene sequence, and whether the insertion of the exogenous gene is completely correct or not is verified on the DNA level.

Selecting positive transformants, designing primers according to gene sequences, and carrying out bacteria liquid PCR to verify the correctness of the transformants, wherein the PCR system and conditions are as follows:

and (3) PCR system:

PCR conditions were as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 40s at 72 ℃ and 28 cycles; 10min at 72 ℃.

The PCR product was subjected to 2% agarose gel electrophoresis to detect the band of interest (FIG. 2). Sequencing results show that the NZX gene fragment has completely correct insertion site, direction and sequence and is consistent with the design. E.coli containing recombinant expression vector was stored in 15% glycerol tube and plasmid was extracted, designated pPICZ α A-NZX, prepared for linearized pichia pastoris (P.pastoris). EXAMPLE 3 construction of NZX-containing recombinant Yeast

3.1 linearization of the recombinant vector pPICZ. alpha.A-NZX

The recombinant expression vector pPICZ alpha A-NZX is subjected to enzyme digestion by PmeI, and the enzyme digestion system and the reaction conditions are as follows:

after the enzyme digestion system is added with sample, the mixture is placed in a PCR instrument for reaction at 37 ℃ for 4h, and 2% agarose gel electrophoresis detection is carried out, wherein the electrophoresis conditions are as follows: 120V, 30 min. The electrophoresis results (fig. 3) show that: the pPICZ alpha A-NZX recombinant vector was completely linearized.

3.2 preparation of Pichia pastoris X-33 competence

1) Selecting an X-33 single colony on the YPD plate, inoculating the single colony to 10mL of YPD liquid culture medium, culturing at 29 ℃ and 250rpm overnight;

2) inoculating 1% of the overnight Pichia pastoris X-33 culture medium to 100mL YPD liquid medium, culturing at 29 deg.C and 250rpm to OD₆₀₀The light absorption value is 1.1-1.3;

3) centrifuging 50mL of culture at 4 ℃ and 4000rpm for 5min, and adding 50mL of sterile water for resuspension;

4) centrifuging at 4 ℃ and 4000rpm for 5min, removing supernatant, and adding 25mL of sterile water for resuspension;

5) centrifuging at 4 ℃ and 4000rpm for 5min, removing supernatant, and adding 2mL of 1M sorbitol for resuspension;

6) centrifuging at 4 deg.C and 4000rpm for 5min, removing supernatant, adding 100 μ L1M sorbitol, and resuspending to obtain X-33 competent cells;

3.3 electrotransformation

100 mu L of yeast competent cells are added into the linearized recombinant plasmid freeze-dried powder, the mixture is gently mixed, the mixture is transferred into an electric rotor cup precooled by ice, and the electric rotor cup is placed on the ice for 5min, wherein the parameters are 1200V, 25 mu F and 400 omega. Immediately adding 1mL of ice-precooled 1M sorbitol solution after electrotransfer, uniformly mixing, transferring into a 2mL centrifuge tube, recovering for 2h at 29 ℃, taking 100 mu L of recovered bacteria liquid, coating on a YPDS plate containing 100 mu g/mL Zeocin antibiotic, and carrying out inverted culture at 29 ℃ until a single colony grows out.

3.448 well plate induction screening positive transformant

Each well of the 48-well plate was filled with 500. mu.L of BMGY medium, and half of the single colonies grown in step 3.3 were picked and placed in the 48-well plate. Blank control holes without bacteria, pPICZ alpha A empty plasmid negative control holes and positive control holes which can be induced are respectively arranged. After shaking culture at 29 ℃ and 250rpm for 24h, 2.5 mul of methanol (the final concentration of methanol is 0.5%) is added into each well and recorded as 0h, methanol is added every 24h and recorded as 0h, 24h, 48h and 72h respectively, after induction for 72h, fermentation liquor in 48-well plates is collected into a 1.5mL centrifuge tube respectively, and the supernatant is centrifuged to carry out antibacterial activity detection and Tricine-SDS-PAGE electrophoresis.

The 48-well plate induced Tricine-SDS-PAGE is shown in FIG. 4.

Example 4 screening of recombinant Yeast strains highly expressing NZX at Shake flask level

4.1 Positive transformants Induction of expression at Shake flask level

Selecting the positive transformant (marked as Pichia pastoris X-33NZX) screened in the step 3.4, inoculating the positive transformant into 10mL YPD liquid culture medium, and carrying out shaking culture at 29 ℃ and 250rpm for 18-20 h; transferring 1% inoculum size to 200mL BMGY liquid medium, culturing at 29 ℃ under shaking at 250rpm for 1 day, replacing cellophane sealing membrane with 4 layers of sterilized gauze, wrapping shaking bottle mouth, adding 1mL of methanol to each shaking bottle for induction (final concentration of methanol is 0.5%), recording for 0h, culturing at 29 ℃ under shaking at 250rpm, and collecting 2mL of fermentation liquid in 2mL centrifuge tubes every 24h until 120h of induction is finished.

4.2 detection of antibacterial Activity of recombinant Yeast fermentation broth

Experimental analysis of zone of inhibition: s. aureus ATCC43300 single colony was picked and inoculated into 10mL MH medium, cultured at 37 ℃ and 250rpm to OD₆₀₀About 0.4, 1% inoculum size was transferred to 50mL MH solid medium, mixed well, poured quickly into a square petri dish of 19cm × 19cm, after coagulation, 30 μ L of fermentation broth supernatant was added for induction for 0-120 h.

4.3 recombinant Yeast secretory protein level Tricine-SDS-PAGE detection

The obtained high-activity recombinant yeast strain is further analyzed for the expression level of the recombinant mutant by Tricine-SDS-PAGE (electrophoresis method reference) ((S))

2006)。

The invention successfully optimizes the gene for coding the antimicrobial peptide NZX, constructs a pPICZ alpha A-NZX recombinant expression vector, successfully converts the pPICZ alpha A-NZX recombinant expression vector into pichia pastoris X-33 after PmeI linearization to obtain four recombinant yeast strains with higher expression levels of N14, N32, N34 and N41, and the antimicrobial peptide NZX realizes high-level secretion expression in the pichia pastoris X-33.

The results of the shake flask horizontal fermentation supernatant bacteriostasis test of the recombinant yeast strains N14, N32, N34 and N41 are shown in figure 5, and the results of the shake flask horizontal fermentation supernatant Tricine-SDS-PAG electrophoresis test induced by the recombinant yeast strains N14, N32, N34 and N41 for 120h are shown in figure 6.

Example 5 high Density fermentation of recombinant Yeast Strain N34

Transformants N14, N32, N34 and N41, the supernatant protein concentrations of the shake flask horizontal fermentation broth were 161mg/L, 124mg/L, 253mg/L and 208mg/L, respectively, and the N34 transformant with the highest expression level was selected for the subsequent fermenter level test. Single colonies of the N34 transformant were picked from YPD plates, inoculated into 50mL flasks containing 10mL YPD liquid medium (containing 100. mu.g/mL Zeocin) at 29 ℃ for 18-24h, inoculated into 1L flasks containing 200mL YPD seed liquid medium at 29 ℃ for 16-18h at 250rpm and OD₆₀₀About 6, and is used as high-density fermentation seed liquid for standby.

The high-density fermentation is carried out by adopting a 5L fermentation tank, and the fermentation process is divided into three stages: (1) and (3) a thallus growth stage: adding 2L of basal salt culture medium, sterilizing at 121 deg.C for 20min, cooling to 29 deg.C, adjusting pH to 5.0, adding 9.6mL PMT1, inoculating 200mL bacterial liquid (1: 10), maintaining ventilation at 8vvm, rotating speed at 600rpm, and maintaining dissolved oxygen at above 20%; (2) and (3) a fed-batch glucose growth stage: observing that the dissolved oxygen value is suddenly increased after slowly decreasing, and feeding 50% glucose solution (12 ‰ PMT1) at a speed of 24mL/L/min for 6h continuously, wherein the rotation speed is increased to 1000rpm, and other fermentation conditions are unchanged; (3) methanol transition induction stage: and (3) changing fermentation conditions, starving for half an hour after glucose is fed for 6 hours, starting to supplement 100% methanol, gradually increasing the flow rate from 1mL/L/min in the first hour to 6mL/L/min in the sixth hour, increasing the rotation speed to 1100rpm, increasing the pH to 5.5, controlling the dissolved oxygen to be more than 20%, and keeping other fermentation conditions unchanged until the fermentation is finished.

Samples are taken every 24h from the beginning of transition induction and are used for detecting the wet weight of thalli and analyzing the protein expression condition and the antibacterial activity. The results of the bacteriostatic activity detection of the fermentation supernatant of the N34 recombinant yeast strain at different induction times after 120h of induced fermentation are shown in FIG. 7. The results of electrophoresis detection of Tricine-SDS-PAGE in the fermented supernatant obtained by inducing fermentation with the recombinant yeast strain of N34 at different induction times are shown in FIG. 8. The total protein concentration and wet weight of the cells during high density fermentation of recombinant yeast strain N34 induced at 29 ℃ are shown in FIG. 9. The results show that after the recombinant yeast N34 with the highest expression level is subjected to high-density fermentation at a fermentation tank level, the total protein concentration, the antimicrobial peptide NZX concentration and the biomass are detected, the total protein concentration reaches 2820mg/L and the biomass reaches 270g/L after fermentation at 29 ℃ for 120 h. And the antibacterial peptide NZX product with higher purity is obtained by a one-step purification method, the recovery rate is about 68 percent, and the yield is about 742 mg/L.

EXAMPLE 6 purification of antimicrobial peptide NZX

1. Purification by cation exchange chromatography

A HiPrep SP FF cation exchange column (16 mm in length and 10mm in inner diameter, supplier GE Healthcare) was loaded after 3-5 column volumes were equilibrated with solution A. After the sample injection is finished, a phosphate elution buffer solution (solution A) containing 20mM and pH5.7 is used for elution, after the elution of a penetration peak is finished, a phosphate elution buffer solution (solution B) containing 20mM and pH5.7 of 0.6M NaCl is used for elution, the elution peak is collected, the elution condition is monitored by UV280nm, Tricine-SDS-PAGE is carried out, and the purification condition of a target product is detected.

2. Desalination of 1KDa dialysis bag

The collected elution peak was dialyzed at 4 ℃ in a dialysis bag with a cut-off of 1kD, and water was changed once every 2 hours and 6 times. Collecting dialyzed dialysate, and lyophilizing with vacuum freeze dryer at low temperature (-54 deg.C, 0.016MPa) to obtain antibacterial peptide NZX lyophilized powder, wherein the purified mass spectrogram shows that the molecular weight of rNZX is consistent with the theoretical molecular weight (FIG. 10).

Example 7 antimicrobial peptide NZX antimicrobial Activity assay

Antibacterial agent obtained according to example 6Peptide NZX lyophilized powder, antibacterial peptide NZX, antibacterial peptide Plectasin and ceftriaxone sodium solutions with the concentration of 1280 mu g/mL are prepared by sterile physiological water, the solutions are diluted by 2 times to the final concentration of 1.25 mu g/mL, antibacterial peptide NZX, antibacterial peptide Plectasin and ceftriaxone sodium solutions with different concentrations are respectively added into a sterile 96-well cell culture plate, 10 mu L of each well is obtained, 3 samples are in parallel, and the same amount (10 mu L) of sterile physiological saline is used as a negative control to prepare a MIC plate. Culturing the strain with MH liquid culture medium, and shake culturing at 37 deg.C to OD₆₀₀(0.4), the bacterial suspension was prepared to a concentration corresponding to the 0.5M turbidimetric standard and diluted to 10 ℃ in sterile MH liquid medium incubated at 37 ℃⁵After CFU/mL, 90. mu.L of bacterial suspension was added to each well of the prepared MIC plate sample well, incubated at 37 ℃ for 16-18h for observation and recording of experimental results. NZX Minimum Inhibitory Concentration (MIC) against pathogenic bacteria was slightly modified as the case may be, with reference to the broth dilution method established by Tian et al (Tian et al, 2009). NZX the antibacterial activity to gram-positive bacteria is shown in table 2, the antibacterial activity to staphylococcus aureus and staphylococcus suis is obviously superior to 6-26 times of antibiotic ceftriaxone sodium and 2-8 times of parent peptide Plectasin.

TABLE 2 NZX antibacterial Activity against gram-Positive bacteria

Example 8 hemolytic assay of antimicrobial peptide NZX

Antimicrobial peptide NZX was dissolved in sterile physiological saline to prepare a mother solution having a concentration of 512 μ g/mL, and the final concentration of 2 μ g/mL was diluted 2-fold in accordance with the lyophilized powder of antimicrobial peptide NZX obtained in example 6. Taking a 6-week-old SPF-grade ICR female mouse, taking blood from eyeballs, and collecting by using a heparin sodium anticoagulation tube. The collected blood was centrifuged at 1500rpm at 4 ℃ for 10min, and the red blood cells were washed repeatedly 3 times with 10mM PBS (pH7.3) until the supernatant was colorless and transparent to prepare an 8% red blood cell suspension. Adding 100 mu L of erythrocyte suspension and antimicrobial peptide NZX solution into a 96-well plate, incubating for 1h at 37 ℃, centrifuging for 5min at 1500rpm, and absorbing supernatant until an ELISA plate detects the ultraviolet absorbance at 540 nm. Saline and 0.1% Triton X-100 were 0% and 100% hemolysis control experiments, respectively. The degree of hemolysis is calculated as follows (cf. Jung, Park et al, 2007):

degree of hemolysis (%) - (Abs)_{540nm antibacterial peptide NZX}-Abs_{540nm physiological saline})/(Abs_{540nm 0.1％Triton X-100}-Abs_{540nm physiological saline})]×100％

The results of the experiment are shown in FIG. 11.

Example 9 cytotoxicity assay of antimicrobial peptide NZX

The MTT method detects NZX and ceftriaxone sodium cytotoxicity to Hacat. MTT can be reduced to insoluble blue-violet crystalline formazan by succinate dehydrogenase in mitochondria of living cells, deposited in cells, while dead cells do not. DMSO can dissolve blue-purple formazan crystal in cells, and the number of living cells can be indirectly reflected by the absorbance of the solution after the dissolution. Within a certain range of cell number, the amount of crystal formation is directly proportional to the number of cells.

37℃，5％CO₂And saturated humidity conditions, Hacat cells were cultured in MEM complete medium. Cells were blown on a pipette, suspended in MEM in complete medium, and cultured at 2.5 × l0⁵cells/mL density were seeded in 96-well plates at 100. mu.L per well in 3 replicates. After 24h the medium was removed and 100. mu.L of sample peptide at 1, 2, 4, 8, 16, 32, 64, 128, 256. mu.g/mL was added to each well in a concentration gradient and an equal amount of PBS solution was added to the control wells. After a further 24h incubation, the medium was removed, washed twice with PBS and 100. mu.L of MTT at a concentration of 5mg/mL was added to each well (protected from light). The 96-well plate was moved to an incubator for further 4 h. And (3) discarding the MTT solution, adding 150 mu L DMSO into each well, oscillating for 10min by an oscillator, and measuring the absorbance of each well at the wavelength of 570nm after the crystals at the bottom of the well are completely dissolved. Cell viability was calculated according to the following formula (see Jiao et al, 2015):

survival (%). ratio of OD value of treated group/OD value of control group X100%

The results of the experiment are shown in FIG. 12.

The result of the antibacterial activity detection of purified antibacterial peptide NZX shows that NZX antibacterial activity against gram-positive bacteria is obviously better than that of antibiotic ceftriaxone sodium, the Minimum Inhibitory Concentration (MIC) value of NZX antibacterial staphylococcus aureus ATCC43300 is 0.46 mu M, the Minimum Inhibitory Concentration (MIC) value of staphylococcus suis NCTC10350 is 0.91 mu M, and the Minimum Inhibitory Concentration (MIC) values of ceftriaxone sodium antibacterial staphylococcus aureus ATCC43300 and staphylococcus suis NCTC10350 are both 12.09 mu M. NZX, it was found that NZX at a concentration in the range of 1-256. mu.g/mL hardly hemolyzes erythrocytes. NZX shows that the survival rate of Hacat cells is above 90% at NZX concentration of 1-128 mug/mL, which indicates that it has no toxic action on normal animal tissue cells. NZX cell viability decreased slightly (72%) at higher concentrations of 256. mu.g/mL, but the toxicity was not strong. The antibacterial peptide NZX prepared by the method provided by the invention has the characteristics of high expression level, good antibacterial activity, low toxicity and wide application prospect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference documents:

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.

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sequence listing

<110> institute of feed of Chinese academy of agricultural sciences

Preparation method and application of <120> 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)

1. The application of the antibacterial peptide NZX in preparing antibacterial drugs or compositions is characterized in that the bacteria are Staphylococcus suis (Staphylococcus hyicus);

wherein the amino acid sequence of the antibacterial peptide NZX is shown as SEQ ID NO. 1.

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