CN116196393A - Application of antibacterial peptide AP138 in preparation of antibacterial drugs - Google Patents
Application of antibacterial peptide AP138 in preparation of antibacterial drugs Download PDFInfo
- Publication number
- CN116196393A CN116196393A CN202211668258.XA CN202211668258A CN116196393A CN 116196393 A CN116196393 A CN 116196393A CN 202211668258 A CN202211668258 A CN 202211668258A CN 116196393 A CN116196393 A CN 116196393A
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- Prior art keywords
- antibacterial
- peptide
- antibacterial peptide
- fermentation
- pichia pastoris
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses application of an antibacterial peptide AP138 in preparation of antibacterial drugs. The invention uses genetic engineering technology to make the gene coding the antimicrobial peptide AP138 be recombined and expressed in Pichia pastoris by codon optimization. The invention realizes the high-yield expression of the antibacterial peptide AP138 in the Pichia pastoris genetically engineered bacteria for the first time, the concentration of the protein in the supernatant of the fermentation liquor reaches 3.1g/L, the fermentation liquor is purified by an ion exchange column to obtain a high-purity sample, the high-purity sample has better antibacterial activity on gram-positive bacteria staphylococcus aureus, streptococcus, staphylococcus epidermidis and the like, and the MIC is 4-16 mug/mL (0.9-3.6 mu M); meanwhile, the antibacterial peptide AP138 has the characteristics of low cytotoxicity and low hemolysis, can be applied to the fields of antibacterial drugs, food antistaling agents, food additives, cosmetics, feed additives and the like, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of an antibacterial peptide AP138 in preparation of antibacterial drugs.
Background
Antibacterial peptides (AMPs) are endogenous polypeptides produced by multicellular organisms to protect the host from pathogenic microorganisms, and are also defined as host defenses since they play a critical role in constructing the innate immune system. The composition has the characteristics of cation, amphipathy, short amino acid sequence and the like, and can inhibit and kill bacteria, fungi, viruses and protozoa (Zaseoff et al, 2002); low drug resistance and multiple bactericidal target sites, the bactericidal mechanism is different from existing antibiotics and the broad activity as immunomodulators, AMPs are therefore considered one of the most powerful candidates for future antibacterial drugs (Ali Adem Bahar et al, 2013). However, natural antibacterial peptide has the defects of low antibacterial activity, difficult synthesis, high cost, low yield, difficult purification and the like. It is therefore a difficulty and hope to develop a process for reducing the cost and obtaining a high purity and yield of the antibacterial peptide drug. Therefore, the problems of low yield and low purity are solved by combining the fermentation expression and purification processes.
Plectasin is isolated from surface saprophytic ascomycetes of northern European pine in 2005 by Mygid et al, and has good activity on gram-positive bacteria. It includes a precursor peptide, which is generally inactive, and a mature peptide, consisting of 40 amino acids, containing 6 Cys residues and 5 Lys residues, a distinct tetrapeptide (ddd) pattern, with a molecular weight of 4407.99Da, with a net charge varying between +1 and +3, depending mainly on the ionic state of its two histidines. Its 6 Cys form a 3-to-disulfide bond (Cys 4-Cys30, cys15-Cys37, cys19-Cys 39) in the molecule, and its secondary structure is stabilized by an N-terminal of the 3-to-disulfide bondTwo antiparallel beta-sheet structures at the alpha-helix and the C-terminus, and a Loop region between the helix and the sheet structure. Mygind et al studied the inhibition of Plectasin on 130 more strains of Streptococcus pneumoniae of different origins, and found that the minimum bactericidal concentration (MIC) was between 0.1-8. Mu.g/mL for penicillin-sensitive or resistant strains 50 1 μg/mL (Mygind, fischer et al 2005).
Disclosure of Invention
The invention aims to provide an application of an antibacterial peptide AP138 in preparation of antibacterial drugs.
To achieve the object of the present invention, in a first aspect, the present invention provides the use of an antimicrobial peptide AP138 for the preparation of an antimicrobial medicament, said antimicrobial peptide AP138 comprising or consisting of the amino acid sequence:
i) An amino acid sequence shown as SEQ ID NO. 1; or (b)
ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)
iii) A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).
In the present invention, the bacteria are gram positive bacteria including, but not limited to, staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), streptococcus agalactiae (Streptococcus dysgalactiae), streptococcus agalactiae (Streptococcus agalactiae).
The invention also provides any one of the following applications of the antimicrobial peptide AP 138:
1) The method is used for food preservation;
2) Is used for preparing food additives or feed additives;
3) Is used in the field of cosmetics;
4) Used for preparing disinfectant or antiseptic.
In a second aspect, the present invention provides a method for preparing the antimicrobial peptide AP138, comprising the steps of:
(1) Design of a gene expression cassette: the DNA sequence encoding the antibacterial peptide AP138 is optimized according to codons favored by pichia pastoris, an XhoI cleavage site and a Kex2 cleavage site are added at the 5 'end of the optimized gene sequence (SEQ ID NO: 2), and TAA and TAG terminator sequences and an XbaI cleavage site are added at the 3' end, so that the DNA sequence has a nucleotide sequence shown as SEQ ID NO: 3.
(2) Construction of recombinant yeast expression vectors: the DNA sequence shown in SEQ ID NO. 3 and the vector pPICZalpha A are connected after double digestion by XhoI and XbaI, so as to construct the recombinant yeast expression vector.
(3) Preparing genetically engineered bacteria: and (3) linearizing the recombinant yeast expression vector, then converting the recombinant yeast into Pichia pastoris X-33, and screening out the recombinant yeast with high-level expression of the antibacterial peptide AP138.
(4) Preparation of antibacterial peptide AP 138: and (3) fermenting and culturing the recombinant saccharomycete obtained in the step (3), adding 50% glucose and methanol for induction to obtain fermentation liquor, collecting supernatant, purifying and desalting to obtain the antibacterial peptide AP138.
The step (4) is specifically as follows:
(1) preparing seed liquid: selecting single colony of the screened high-expression yeast transformant, shaking culture at 30 deg.C and 250rpm for 18-24 hr in 10mL YPD medium containing zeocin (bleomycin) at 100 μg/mL, inoculating at 0.5% -1% into 200mL YPD liquid medium, shaking culture at 30 deg.C and 250rpm for 16-18 hr to OD 600nm The value is 5, and the seed liquid is obtained;
(2) fermentation culture initial stage: 200mL of the seed solution was inoculated in a proportion of 10% into 2L of a basal salt medium (45 g of glucose, 50g of NH) 4 H 2 PO 4 、20g K 2 SO 4 、15g MgSO 4 ·7H 2 O、6g KH 2 PO 4 、0.4g CaSO 4 And 1.5g KOH, adding water to a constant volume of 2L), controlling the temperature at 29 ℃, adjusting the pH value to 5.0, maintaining the ventilation rate at 8-10vvm, and maintaining the rotating speed at 600rpm for 6 hours, then adjusting the rotating speed to 800rpm, and maintaining the dissolved oxygen at more than 20%;
(3) and (3) a sugar supplementing growth stage: observing that the dissolved oxygen value starts to slowly decrease and then suddenly increases, the rotating speed is increased to 1000rpm, the dissolved oxygen is increased to more than 70%, and the PMT1 (FeSO) with 12% 4 ·7H 2 O 65g，ZnCl 2 20g，CuSO 4 ·5H 2 O 6g，MnSO 4 ·5H 2 O 3g，CoCl 2 0.5g，Na 2 MoO 4 0.2g，KI 0.08g，H 3 BO 4 0.02g of biotin, 0.2g of H with a concentration of 98.3% 2 SO 4 5mL of 50% glucose solution with the volume fixed to 1L) by adding water, the flow acceleration is 12-36 mL/L/min, the continuous flow is carried out for 6-8 h, and the dissolved oxygen is maintained above 20%; (4) methanol induction: after glucose is fed in, starving for 0.5h-1h, starting to supplement 100% methanol, gradually increasing the flow rate from 1mL/L/min in the first hour to 10mL/L/min in the tenth hour, adjusting the rotating speed to 1100rpm, adjusting the pH to 5.5, and controlling the dissolved oxygen to be more than 20% until the fermentation is finished.
The invention also provides a purification method of the recombinant protein secreted by the Pichia pastoris X-33 genetically engineered bacterium, which comprises the steps of ion exchange chromatography, desalination, freeze vacuum drying, redissolution and the like of fermentation liquor.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention uses genetic engineering technology to make the gene coding the antimicrobial peptide AP138 be recombined and expressed in Pichia pastoris by codon optimization. The invention realizes the high-yield expression of the antibacterial peptide AP138 in the genetically engineered bacteria of pichia pastoris for the first time, the concentration of the protein in the supernatant of the fermentation liquor reaches 2.98g/L, the fermentation liquor is purified by an ion exchange column to obtain a high-purity sample, and the high-purity antibacterial peptide AP has better antibacterial activity on gram positive bacteria staphylococcus aureus ATCC25923, ATCC43300, CVCC546, clinical isolate E48, streptococcus ATCC13813, ATCC3399, CAU-FRI2 and staphylococcus epidermidis ATCC35984 and ATCC12228, and MIC is 4-16 mug/mL (0.9-3.6 mu M); meanwhile, the antibacterial peptide AP138 has the characteristics of low cytotoxicity and low hemolysis, can be applied to the fields of antibacterial drugs, food antistaling agents, food additives, cosmetics, feed additives and the like, and has wide application prospect.
Drawings
FIG. 1 is an agarose gel electrophoresis of a recombinant plasmid pPICZαA-AP138 according to a preferred embodiment of the present invention; wherein M is DNA Marker I;1-2: pPICZαA-AP138.
FIG. 2 shows the result of linearization electrophoresis of recombinant pPICZαA-AP138 vector in accordance with the preferred embodiment of the present invention; wherein M: trans5K DNA Marker;1-2: the linearized recombinant vector pPICZαA-AP138.
FIG. 3 shows the results of antibacterial activity detection of fermentation supernatants at different induction times after induction fermentation 96 by 48-well plate AP138 recombinant yeast strains in the preferred embodiment of the invention.
FIG. 4 shows the detection results of shake flask level induction fermentation supernatant Tricine-SDS-PAGE of different transformants of the AP138 recombinant yeast strain in the preferred embodiment of the present invention; wherein M: ultra-low molecular weight protein markers; 1-4: the bands of the supernatant of the fermentation broths of the AP138-8, the AP138-28, the AP138-68 and the AP138-94 are respectively induced.
FIG. 5 shows the results of antibacterial activity detection of shake flask level induced fermentation supernatants of recombinant yeast strains of antibacterial peptides A-8, A-28, A-68, A-94 (AP 138-8, AP138-28, AP138-68, AP 138-94) according to the preferred embodiment of the invention.
FIG. 6 shows the results of antibacterial activity detection of fermentation supernatants at different induction times after the A-94 recombinant yeast strains were induced to ferment for 0h, 24h, 48h, 72h, 96h, 120h in the preferred embodiment of the invention.
FIG. 7 shows the results of detecting the fermentation supernatant Tricine-SDS-PAGE electrophoresis at different induction times after the A-94 recombinant yeast strain is induced to ferment for 120 hours in the preferred embodiment of the present invention; wherein M: ultra-low molecular weight protein markers; 1-6: respectively representing the electrophoresis bands of the supernatants of fermentation liquor induced for 0h, 24h, 48h, 72h, 96h and 120 h.
FIG. 8 shows the time-varying curves of the wet weight and total protein of the cells during the induction of 0h, 24h, 48h, 72h, 96h, 120h, respectively, in the high-density fermentation process of the recombinant A-94 yeast strain according to the preferred embodiment of the present invention.
FIG. 9 shows the result of Tricine-SDS-PAGE electrophoresis after purification of the antibacterial peptide AP138 according to the preferred embodiment of the present invention; wherein M: ultra-low molecular weight protein markers; 1-6: the peak was the breakthrough, 45.5% of the B-solution elution peak, 70% of the B-solution elution peak, and 100% of the B-solution elution peak, respectively.
FIG. 10 shows the mass spectrum identification result after the purification of the antibacterial peptide in the preferred embodiment of the present invention.
FIG. 11 shows the results of an experiment for hemolysis of the antimicrobial peptide AP138 in the preferred embodiment of the present invention.
FIG. 12 shows the cytotoxicity test results of the antimicrobial peptide AP138 according to the preferred embodiment of the present invention.
Detailed Description
The invention provides a novel antimicrobial peptide AP138, which comprises or consists of the following amino acid sequences:
i) An amino acid sequence shown as SEQ ID NO. 1; or (b)
ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)
iii) A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).
The invention also provides any one of the following applications of the antimicrobial peptide AP 138:
A. is used for preparing antibacterial drugs;
B. is used for preparing disinfectant;
C. for the preparation of preservatives;
the bacteria are gram positive bacteria.
The gram positive bacteria include staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), streptococcus agalactiae (Streptococcus dysgalactiae) and streptococcus agalactiae (Streptococcus agalactiae).
The invention also provides a recombinant expression vector carrying the following gene expression cassette: inducible promoter-DNA sequence encoding alpha factor Signal peptide-Kex 2 cleavage site-DNA sequence encoding antibacterial peptide AP 138-stop codon shown in SEQ ID NO: 2.
Further, the 5 'end of the DNA sequence encoding the antimicrobial peptide AP138 is connected with an XhoI restriction enzyme site, and the 3' end is connected with an XbaI restriction enzyme site.
The invention also provides a host cell containing the recombinant expression vector, preferably Pichia pastoris cell X-33.
The invention also provides a genetic engineering bacterium, the recombinant vector is introduced into the pichia pastoris cell in a linearization way, and high-expression transformants are screened.
The invention also provides a method for culturing the genetically engineered bacteria, which optimizes the original fermentation process, mainly reflected in that the temperature is increased from 29 ℃ to 30 ℃, the primary fermentation stage is continued for 16-18 hours from 800rpm, and is optimized by firstly continuing for 8 hours at 600rpm, then continuing for 8-10 hours at 800rpm, and the methanol flow acceleration. The specific implementation comprises the following steps:
1) Preparing seed liquid: single colonies of yeast transformants were picked from YPD plates, inoculated into 10mL of YPD liquid medium containing 100. Mu.g/mL zeocin, shake-cultured at 30℃and 250rpm for 18-24 hours, inoculated into 200mL of YPD liquid medium at 1% (v/v) inoculum size, shake-cultured at 30℃and 250rpm for 16-18 hours to OD 600nm The value is 5, and the seed liquid is obtained;
2) Fermentation culture: adding the seed solution into 2L of basic salt culture medium according to the inoculation amount of 10% (v/v) at the temperature of 25-29 ℃, adjusting the pH value to 5.0, adding 9.6ml of PMT1, maintaining the ventilation rate at 8vvm, maintaining the rotating speed at 600rpm, and maintaining the dissolved oxygen at more than 20%;
3) Feeding a carbon source: observing that the dissolved oxygen value starts to slowly decrease and then suddenly rises to more than 80%, beginning to flow the 50% glucose solution containing 12%PMT 1, the flow acceleration is 12-24 mL/L/min, the continuous flow is carried out for 6-8 h, and the rotating speed is adjusted to 1000rpm;
4) Methanol induction: after glucose is fed in, starving is carried out for half an hour, 100% methanol is added, the flow rate of 1mL/L/min in the first hour is gradually increased to 6mL/L/min in the sixth hour, the rotating speed is adjusted to 1100rpm, the pH is adjusted to 5.5, and dissolved oxygen is controlled to be more than 20% until the fermentation is finished.
Wherein, the basic salt culture medium formula used in the step 2) is as follows: 45g glucose, 50g NH 4 H 2 PO 4 、20gK 2 SO 4 、15g MgSO 4 ·7H 2 O、6g KH 2 PO 4 、0.4g CaSO 4 And 1.5g KOH, water was added to a constant volume of 2L.
The invention also provides a method for expressing the antibacterial peptide AP138 in the recombinant pichia pastoris, and the obtained recombinant pichia pastoris is subjected to fermentation culture and secretion to produce the antibacterial peptide AP138.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
The medium and buffer formulations referred to in the examples below:
LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl; solid LB medium was added with 2% agarose.
Low salt LB medium: 10g/L tryptone, 5g/L yeast extract and 5g/L NaCl; 2% of agar powder is added into the solid low-salt LB culture medium.
MHB medium: 17.5g/L casein hydrolysate, 5g/L beef extract powder and 1.5g/L starch.
MHA medium: 2% of agar powder is added into the solid MHB culture medium.
YPD medium: 20g/L of peptone, 10g/L of yeast extract and 20g/L of glucose; the solid YPD medium was supplemented with 2% agar powder.
YPDS medium: 20g/L peptone, 10g/L yeast extract, 182.2g/L sorbitol, 20g/L glucose and 20g/L agar powder.
BMGY medium (per L): 10g of yeast extract, 20g of peptone, 10mL of glycerol, 100mL of 13.4% amino acid-free yeast nitrogen source (YNB), 2mL of 0.02% biotin, 1mol/L phosphate buffer, pH6.0 and 100mL.
For the use of LB medium, low-salt LB, MHB, MHA, YPD, YPDS and the like, reference is made to the Invitrogen Pichia instruction manual.
50mM phosphate buffer (solution A): 7.786g Na 2 HPO 4 ，4.407g NaH 2 PO 4 Deionized water was added to 950mL, and after complete dissolution in a magnetic stirrer, pH7 was adjusted to 1000mL.
1M NaCl 50mM phosphate buffer (solution B): 70786g Na 2 HPO 4 ，4.407g NaH 2 PO 4 58.44g NaCl, deionized water was added to 950mL, and after complete dissolution in a magnetic stirrer, pH7 was adjusted to 1000mL.
The methods for identifying the gene amplification and transformant in the following examples are a PCR method and a DNA sequencing method.
The protein detection method in the following examples was Tricine-SDS-PAGE, reference [ (]
H.,et al,2006)。
The protein concentration measurement method in the following examples was the coomassie brilliant blue method.
The method for determining the molecular weight of the protein referred to in the following examples was MALDI-TOF MS method.
The methods of protein purification referred to in the examples below are based on ion chromatography.
The fermentation process referred to in the examples below is a high density fermentation process.
The sequence of the antibacterial peptide NZ2114 is: GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGG FVCKCY.
The sequence of the antibacterial peptide MP1102 is: GFGCNGPWQEDDVKCHNHCKSIKGYKGGYCAKGG FVCKCY.
The sequence of the antibacterial peptide MP1106 is: GFGCNGPWSEKDMHCHNHCKSIKGYKGGYCAKGG FICKCY.
The strains and plasmids referred to in the following examples are shown in Table 1:
TABLE 1 test strains and plasmids
Example 1 acquisition of the sequence of the antibacterial peptide AP138 Gene
According to an antibacterial peptide database DBAASP (https:// www.dbaasp.org/home), screening and obtaining a Plectasin derivative peptide AP138 amino acid sequence GFGANGPWSEDDLRAHRHAKSIKGYRGGYCAKGGFVA KCY (SEQ ID NO: 1), predicting and analyzing physicochemical properties of the Plectasin derivative peptide, namely, the Plectasin derivative peptide NZ2114 with better AP138 specific activity, has higher positive charges, amphipathy and possibility of antibacterial peptide, selecting an AP138 sequence, designing a coding gene of the antibacterial peptide AP138 according to the codon preference of pichia pastoris, and obtaining a DNA sequence through codon optimization: ggttttggttgtaacggtccatggtctgaagatgatttgagatgtcatagacattgtaagtctattaagggttacaga ggtggtta ctgtgctaagggtggttttgtttgtaagtgttactaataa (SEQ ID NO: 2).
To effectively terminate rAP138 translational expression, two stop codons TAA, TGA were added 3' of the coding AP138 gene sequence. To enable native secretory expression of rAP138 in pichia pastoris, a signal peptide cleavage site Kex2 site was inserted 5' to rAP. To realize construction of recombinant expression vector pPICZaA-AP138, xhoI and XbaI endonuclease sites are designed at two ends of the AP138 gene. The expression cassette gene sequence is as follows:
The physicochemical property prediction analysis of the antimicrobial peptide AP138 and other derivative peptides is as follows:
the secondary structure prediction and antibacterial peptide index analysis of the antibacterial peptide AP138 and other derivative peptides are as follows:
EXAMPLE 2 construction of Yeast recombinant expression vectors
1. The AP138 gene fragment obtained in example 1 was digested with XhoI and XbaI endonucleases, and the purified fragment was recovered. At the same time, pPICZ. Alpha.A vector (purchased from Invitrogen) was double digested with XhoI and XbaI.
The double enzyme digestion system is as follows:
after the enzyme digestion system is added, the sample is placed in a water bath kettle at 37 ℃ for reaction for 4 hours, 2% agarose gel electrophoresis detection is carried out, and electrophoresis conditions are as follows: 120V,30min. The digested product is recovered using a DNA product recovery kit. After double digestion of the AP138 gene and the pPICZ alpha A vector with XbaI and XhoI, the AP138 gene was ligated with the linearized pPICZ alpha A vector using T4DNA ligase. The connection system is as follows:
connection conditions the connection system is connected at 16 ℃ overnight in a PCR instrument after the sample addition.
2. The obtained recombinant vector is transformed into escherichia coli DH5 alpha, and the transformation steps are as follows:
1) 10. Mu.L of ligation product was incubated in 50. Mu.L of E.coli DH 5. Alpha. Competent cells for 30min on ice;
2) Heat shock is carried out for 45s at 42 ℃, and ice bath is carried out immediately for 2-3 min;
3) 450. Mu.L of LB low-salt medium preheated at 37 ℃ is added, and the culture is resumed at 100rpm at 37 ℃ for 1h;
4) After re-suspending the cells, 100. Mu.L of LB low-salt solid medium containing 25. Mu.g/mL Zeocin was applied;
5) Inverted culturing at 37deg.C for 12-16 hr.
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), incubated overnight at 37℃at 250rpm, and positive transformants were identified by colony PCR. Positive transformants verified by specific primers were picked and inoculated into 10mL of low-salt LB liquid medium (containing 25. Mu.g/mL zeocin), cultured overnight at 37℃at 250rpm, and 500. Mu.L of the transformants were sequenced and verified.
Selecting positive transformants, and carrying out bacterial liquid PCR to verify the correctness of the transformants, wherein the PCR system and the conditions are as follows:
PCR system:
primer F: 5' -GGCAAATGGCATTCTGACAT-3
Primer R: 5' -GACTGGTTCCAATTGACAAGC-3
PCR conditions: 94 ℃ for 5min;94℃for 30s,58℃for 30s,72℃for 40s,28 cycles; and at 72℃for 10min.
E.coli plasmids of recombinant expression vectors were extracted and identified by 1% agarose gel electrophoresis (FIG. 1) and prepared for linearization of electric pichia pastoris (P.pastoris).
EXAMPLE 3 construction of recombinant Yeast containing AP138 Gene
1. Linearization of recombinant vector pPICZαA-AP138
The endonuclease PmeI is utilized to carry out enzyme digestion on the constitutive recombinant expression vector pPICZalpha A-AP138, and the enzyme digestion system and the reaction conditions are as follows:
after the enzyme digestion system is added, the sample is placed in a water bath kettle at 37 ℃ for reaction for 4 hours, 1% agarose gel electrophoresis detection is carried out, and electrophoresis conditions are as follows: 155V,30min. The electrophoresis result shows that the pPICZαA-AP138 recombinant vector is completely linearized (FIG. 2).
2. Linearized plasmid concentration
And (3) putting the linearized plasmid into a vacuum freeze dryer, taking out for standby after the sample is powdery.
3. Preparation of Pichia pastoris X-33 competence
1) Picking X-33 single colony on YPD plate, inoculating to 10mL YPD liquid culture medium, and culturing at 30deg.C and 250rpm overnight;
2) Taking Pichia pastoris X-33 overnight culture solution, inoculating to 200mL YPD liquid culture medium at 30deg.C under shaking culture at 250rpm to OD 600nm The light absorption value is 1.1-1.3;
3) Subpackaging into 50mL sterile centrifuge tube, centrifuging at 4000rpm at 4deg.C for 5min, discarding supernatant, adding 50mL sterile ddH 2 O water resuspension;
4) After centrifugation at 4000rpm at 4℃for 5min, the supernatant was removed and resuspended in 50mL of sterile water;
5) After centrifugation at 4000rpm at 4℃for 5min, the supernatant was removed and resuspended in 2mL of 1M pre-chilled sorbitol;
6) Finally, after centrifugation at 4000rpm at 4 ℃ for 5min, the supernatant is removed, and 100 mu L of 1M pre-chilled sorbitol is added for resuspension, thus obtaining pichia pastoris X-33 competent cells.
4. Electric conversion
100 mu L of yeast competent cells are added into the linearized recombinant plasmid freeze-dried powder, the mixture is gently mixed and transferred into an ice-precooled sterile electric rotating cup, and the mixture is placed on ice for 5min, and the parameters are 1200V,25 mu F and 400 omega. Immediately after electrotransformation, 1mL of ice-precooled 1M sorbitol solution is added, the mixture is evenly mixed and transferred into a 2mL centrifuge tube, the temperature is 29-30 ℃, the mixture is recovered for 2 hours, 100 mu L and 150 mu L of recovered bacterial liquid are coated on a YPDS plate containing 100 mu g/mL zeocin antibiotics, and the mixture is inversely cultured at the temperature of 29-30 ℃ until single colony is grown.
5. Positive transformants were selected by 48-well plate induction
Adding 500 mu L of YPD culture medium into each hole of a 48-hole plate, picking half of single colonies growing in the step 4, inoculating into the 48-hole plate, and carrying out shaking culture at 30 ℃ and 250rpm for 24 hours; inoculating to BMGY culture medium containing 500 μl at 1%, shaking culturing at 30deg.C and 250rpm for 24 hr; 2.5 mu L of methanol (the final concentration of the methanol is 0.5%) is added into each hole, the induction is recorded as 0h, the methanol is added every 24h, the induction is respectively recorded as 0h, 24h, 48h and 72h, after the induction is carried out for 72h, fermentation liquor in a 48-hole plate is respectively collected in a 1.5mL centrifuge tube, and the supernatant is centrifugally taken for antibacterial activity detection. The supernatant was induced by the 48-well plate, and the results of the inhibition zone test are shown in FIG. 3.
Example 4 screening of recombinant Yeast strains expressing AP138 at high levels in shake flasks
1. Positive transformant shake flask level induced expression
The positive transformants selected in example 3 (designated as Pichia pastoris X-33AP138, inoculated into 10mL YPD liquid medium, shaking culture at 30℃and 250rpm for 16-18h, 1% inoculum size was transferred to 200mL BMGY liquid medium, shaking culture at 30℃and 250rpm for 24h, 1mL methanol was added to each flask for induction (0.5% final concentration of methanol), 4 layers of sterilized gauze were used to replace the sealing film to wrap the flask mouth, shaking culture at 30℃and 250rpm was performed, 1mL methanol was added every 24h until the induction was completed.
2. Antibacterial activity detection bacteriostasis circle experimental analysis of recombinant yeast fermentation liquor
Single colonies of S.aureus ATCC43300 were picked up and inoculated into 10mL of MHB medium, cultured at 37℃and 250rpm overnight, then transferred to fresh 10mL of MHB medium at 1% inoculum size, and then transferred to 50mL of MHB solid medium to give a bacterial count of 1X 10 6 CFU/mL, mix well, pour into square 19cm X19 cm Petri dish rapidly, after solidification, add 30. Mu.L of fermentation broth supernatant induced for 96 h.
3. Tricine-SDS-PAGE detection of recombinant yeast secreted protein level
The recombinant mutant expression level of the obtained high-activity recombinant yeast strain is further analyzed by Tricine-SDS-PAGE, and the electrophoresis method is referred to
2006。
The invention successfully optimizes the gene for encoding the antibacterial peptide AP138, constructs a pPICZ alpha A-AP38 recombinant expression vector, and successfully converts the pPICZ alpha A-AP38 recombinant expression vector into Pichia pastoris X-33 after PmeI linearization to obtain four recombinant yeast strains with higher expression levels, namely A-8, A-28, A-68 and A-94, wherein the antibacterial peptide AP138 realizes high-level secretory expression in the Pichia pastoris X-33. The results of antibacterial tests on shake-flask horizontal fermentation supernatants of the recombinant yeast strains A-8, A-28, A-68 and A-94 are shown in FIG. 4, and the results of electrophoresis detection on shake-flask horizontal fermentation supernatant Tricine-SDS-PAG induced by the recombinant yeast strains A-8, A-28, A-68 and A-94 for 120 hours are shown in FIG. 5.
EXAMPLE 5 high Density fermentation of recombinant Yeast Strain A-33
According to the selection of positive transformants, the A-94 shake flask level protein expression level was highest, and therefore, the A-94 recombinant yeast strain was selected for subsequent high-density fermentation. Single colonies of transformants were picked from YPD plates, inoculated into 50mL shake flasks of 10mL YPD liquid medium (containing 100. Mu.g/mL zeocin), shake-cultured at 30℃and 250rpm for 16-18h, inoculated into 1L shake flasks of 200mL YPD seed liquid medium at 0.5-1% inoculum size, shake-cultured at 30℃and 250rpm for 16-18h, OD 600nm About 4-6, as a seed liquid for high density fermentation.
The high-density fermentation is carried out by adopting a 5L fermentation tank, and the fermentation process is divided into three stages: (1) a bacterial growth stage: adding 2L of basic salt culture medium, sterilizing at 121deg.C for 20min, cooling to 29 deg.C, regulating pH to 5.0, adding 9.6mL of PMT1, inoculating 200mL of bacterial liquid (1:10, v/v), maintaining ventilation at 8vvm, maintaining rotation speed at 600rpm for about 6 hr, regulating to 800rpm, and maintaining dissolved oxygen at above 20%; (2) a fed-batch glucose growth stage: observing that the dissolved oxygen value starts to slowly decrease and then suddenly rises, and regulating the rotating speed to 1000rpm to enable the dissolved oxygen to reach about 70%, enabling the dissolved oxygen to reach an equilibrium state, starting to feed 50% glucose solution (12%PMT 1), enabling the flow acceleration to be 24mL/L/min, and continuously feeding for 6 hours; (3) methanol transition induction phase: after glucose is fed for 6 hours under the condition of changing fermentation conditions, feeding 100% methanol is started after the glucose is fed for 6 hours, the flow rate of 1mL/L/min in the first hour is gradually increased to 6mL/L/min in the sixth hour, the rotating speed is adjusted to 1100rpm, the pH is adjusted to 5.5, dissolved oxygen is controlled to be more than 20%, and other fermentation conditions are unchanged until the fermentation is finished.
Samples were taken every 24h from the start of transient induction for protein expression profile analysis and antibacterial activity analysis. FIG. 6 shows the bacteriostatic effect of the recombinant yeast strain high-density fermentation supernatant, FIG. 7 shows the electrophoresis chart of the fermentation supernatant protein, and FIG. 8 shows the variation curve of the wet weight of the thallus and the total protein time in the fermentation process. The expression yield is higher than that of other plectasin derivative peptides NZ2114, MP1102 and MP1106. The high-density fermentation total protein yield of each Plectasin-derived peptide is shown in table 2:
TABLE 2 high Density fermentation Total protein yield of Plactasin derived peptides
EXAMPLE 6 purification of the antibacterial peptide AP138
1. Cation exchange chromatography purification
The conditions for purifying the antibacterial peptide AP138 are searched and optimized, 20mM phosphate elution buffer (A solution) with pH value of 6.7 and 20mM phosphate elution buffer (B solution) with pH value of 0.6M NaCl are firstly selected, and then the concentration of the phosphate elution buffer is respectively adjusted from 20mM to 50mM and the pH value is respectively adjusted from 6.7 to 7; then different solutions A and B are arranged for eluting the impurity band and the eluting target band, and the specific steps are as follows:
HiPrep SP FF cation exchange column (length 16mm, inner diameter 10mm,GE Healthcare) was loaded after 3-5 column volumes were equilibrated with solution A. After the sample injection, eluting the impurity band with a phosphate elution buffer (solution A) containing 50mM, pH7, and after the breakthrough peak is eluted, eluting with a phosphate elution buffer (solution B) containing 50mM, pH7, 0.6M NaCl, collecting the elution peak, and monitoring the elution condition according to UV 280 nm. The purified AP138Tricine-SDS-PAGE gel is shown in FIG. 9, and the mass spectrum detection result of the purified AP138 is shown in FIG. 10.
2. Tangential flow filtration salt rejection
The collected peak sample was filtered through 0.22 μm for further use. First using ddH 2 O is cleaned and flows through a filter membrane packaging machine tangentially, when the pH value of the flowing sample is neutral, sample injection is started, 1L of sample is concentrated to 200mL, and 600mL of ddH is added 2 O, continuing filtering; the above steps were performed three times. The final sample was concentrated to 200mL and the sample was collected.
3. Sample lyophilization
And (3) putting the collected sample into a vacuum freeze dryer, and freeze-drying the sample through freezing, vacuumizing and freeze-drying to obtain an antibacterial peptide AP138 freeze-dried powder product.
Example 7 antibacterial peptide AP138 antibacterial Activity assay
According to example 6, an antibacterial peptide AP138 lyophilized powder was obtained, an antibacterial peptide AP138 and vancomycin solution having a concentration of 128. Mu.g/mL was prepared with sterilized physiological water, diluted 2-fold to a final concentration of 1.25. Mu.g/mL, and the antibacterial peptide AP138 solution and vancomycin (Van) solution having different concentrations were added to a sterile 96-well cell culture plate, 10 mL/well, three parallel, the same amount (10. Mu.L) of sterile physiological saline was used as a negative control, and MIC plates were prepared. The strain is cultured by MHB liquid culture medium, and shake culture is carried out at 37 ℃ until OD 600nm Preparation of bacterial solution into bacterial suspension with concentration equivalent to 0.5 Mirabilitum standard, dilution to 10 by sterile MH liquid culture medium after incubation at 37deg.C 5 After CFU/mL, 90 mu L of bacterial suspension is added to each well of the prepared MIC plate sample hole, the temperature is kept constant at 37 ℃ for 16-18 hours, experimental MIC results are observed and recorded, meanwhile, 10 mu L of 2 XMIC, 4 XMIC and 8 XMIC bacterial solutions are taken out for tearing on an MHA plate, and no bacterial growth is observed and recorded as the minimum sterilization concentration. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of AP138 against pathogenic bacteria were determined by reference to a micro broth dilution method and the results are shown in table 3.
TABLE 3 antibacterial Activity of AP138 against gram-Positive bacteria
Example 8 test of antibacterial peptide AP138 hemolysis
According to the lyophilized powder of the antimicrobial peptide AP138 obtained in example 6, the antimicrobial peptide AP38 was dissolved in a sterile physiological saline to prepare a mother solution having a concentration of 512. Mu.g/mL, and the final concentration was diluted by a 2-fold ratio to 2. Mu.g/mL. ICR mice of SPF grade of 6-8 weeks old were taken, blood was collected from eyeballs, and collected into heparin sodium anticoagulation tubes. The collected blood was centrifuged at 1500rpm at 4℃for 10min, and the red blood cells were repeatedly washed 3 to 5 times with 10mM PBS (pH 7.3) to prepare an 8% red blood cell suspension from the supernatant which was colorless and transparent. 100 μl of each of the erythrocyte suspension and the antimicrobial peptide AP138 was taken at 1:1 (v/v), added to a 96-well plate, incubated at 37deg.C for 1h, collected, centrifuged at 1500rpm for 5min, and the supernatant was aspirated to an ELISA plate for detection of UV absorbance at 540 nm. While saline and 0.1% Triton X-100 were 0% and 100% hemolysis control experiments, respectively. The calculation formula of the degree of hemolysis is as follows (refer to Jung, park et al, 2007), and the result is shown in FIG. 11.
Degree of hemolysis (%) = [ (Abs) 540nm antimicrobial peptide AP138 －Abs 540nm physiological saline )/(Abs 540nm 0.1％Triton X-100 －Abs 540nm physiological saline )]×100％
Example 9 cytotoxicity experiments on the antimicrobial peptide AP138
37℃，5％CO 2 And culturing RAW264.7 cells in a DMEM complete medium under saturated humidity conditions. The pipette gun blows the cells, and the DMEM complete medium resuspension of the cells, 1×l0 5 cells/mL were seeded at a density of 100. Mu.L per well in 96-well plates and 3 replicates were set. 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 per well in a concentration gradient, and an equal amount of PBS solution was added to the control wells. After further incubation for 24h, the medium was removed, washed twice with PBS, 100. Mu.L fresh DMEM medium was added to each well, followed by MTT reagent, and the 96-well plates were moved to an incubator for further incubation for 4h. The absorbance of each well was measured at 490 nm. Cell viability was calculated according to the following formula (see Li-Zhi Wu et al, 2021) and the results are shown in FIG. 12.
Survival (%) = treatment/control OD x 100%
Example 10 stability experiment of antibacterial peptide AP138
First, a stable salt ion assay was performed, and after incubation of the antimicrobial peptide AP138 in 50mM, 100mM, 150mM, 200mM, 300mM NaCl solution at 37℃for 2 hours, MIC assay was performed, and the procedure was the same as in example 7.
Then, a heat stability test was performed, and after incubation of the antimicrobial peptide AP138 at 20℃at 40℃at 60℃at 80℃at 100℃for 1 hour, MIC test was performed, and the procedure was the same as in example 7. The results show that the antimicrobial peptide AP138 remained active at high salt concentration of 300mM NaCl (Table 4) and better antimicrobial activity at 80 ℃ (Table 5). Has better high-salt ion resistance stability and higher heat resistance stability.
TABLE 4 salt ion stability
TABLE 5 thermal stability
The invention successfully optimizes the encoding antibacterial peptide AP138 gene, constructs a pPICZ alpha A-AP138 recombinant expression vector, and successfully converts the pPICZ alpha A-AP138 recombinant expression vector into pichia pastoris X-33 after PmeI linearization to obtain a recombinant yeast strain. The antibacterial activity of the purified antibacterial peptide AP138 is detected, and the result shows that the antibacterial activity (0.95-1.91 mu M) of the AP138 against gram-positive bacteria is better than that of plectasin, and the hemolytic experiment result of the AP138 shows that the concentration is in the range of 1-256 mu g/mL and almost does not hemolyze erythrocytes. The cytotoxicity test result of the AP138 shows that the survival rate of the AP138 at the concentration of 256 mu g/mL is higher than 66%, and the toxicity is smaller. The antibacterial peptide AP138 has the characteristics of good antibacterial activity and low toxicity, and has good application prospect in production practice.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Reference is made to:
1.Seo MD,Won HS,Kim JH,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.[0147].
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.[0149].
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.
.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.
10.Wu LZ,Huang ML,Qi CL,Shen LJ,Zou Y,Yang R,Sheng JF,Chen SM.Overexpression of Notch2 enhances radiosensitivity via inhibition of the AKT/mTOR signaling pathway in nasopharyngeal carcinoma.Bioengineered.2021 Dec；12(1):3398-3409.
11.Zhang,Y.,Teng,D.,Mao,R.et al.High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics,postantibiotic and synergy against Staphylococcus aureus.Appl Microbiol Biotechnol 98,681–694(2014).https://doi.org/10.1007/s00253-013-4881-2.
12 J.Jiao,R.Mao,X.Wang,Y.Zhang,D.Teng,X.Feng,J.Wang,GAP-initiated constitutive expression of a novel plectasin-derived peptide MP1106by Pichia pastoris and its activity against Streptococcus suis,Process Biochem.50(2015)253–261.
Claims (4)
1. use of an antimicrobial peptide AP138 in the preparation of an antimicrobial medicament, said antimicrobial peptide AP138 comprising or consisting of the amino acid sequence:
i) An amino acid sequence shown as SEQ ID NO. 1; or (b)
ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)
iii) A polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).
2. The use according to claim 1, wherein the bacteria are gram positive bacteria.
3. The use according to claim 2, wherein the gram-positive bacteria comprise staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), streptococcus agalactiae (Streptococcus dysgalactiae), streptococcus agalactiae (Streptococcus agalactiae).
4. Any of the following applications of the antimicrobial peptide AP 138:
1) The method is used for food preservation;
2) Is used for preparing food additives or feed additives;
3) Is used in the field of cosmetics;
4) For preparing disinfectants or preservatives;
the antimicrobial peptide AP138 is as described in claim 1.
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| ANITA UMERSKA等: "Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus", INT J NANOMEDICINE., 8 August 2017 (2017-08-08), pages 5687 - 5699 * |
| ANNE-CLAIRE GROO等: "Reverse micelle-lipid nanocapsules: a novel strategy for drug delivery of the plectasin derivate AP138 antimicrobial peptide", INT J NANOMEDICINE., 15 November 2018 (2018-11-15), pages 7565 * |
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