CN106146629B - Trypsin resistant antibacterial peptides and preparation method thereof - Google Patents

Abstract

The invention provides a group of antibacterial peptides with trypsin resistance and a preparation method thereof. Selective mutations were made to the six trypsin active sites present in the NZ2114 sequence using protein directed engineering techniques. Designing an amino acid sequence as SEQ ID NO: 1-SEQ ID NO: 33 and to achieve recombinant expression thereof in pichia pastoris. The invention realizes the design of the NZ2114 antitrypsin derivative for the first time, and the degradation rate of trypsin by measuring partial mutants (especially double mutants and all triple mutants) is obviously improved and is 16.67-25%. The trypsin resistant antibacterial peptide has remarkable antibacterial activity on staphylococcus aureus ATCC25923, ATCC43300 and ATCC6538, and the MIC is between 0.25 and 16 mu g/ml. The trypsin resistant antibacterial peptide obtained by the method can be applied to the fields of antibacterial drugs, food additives, cosmetics, feed additives and the like, and has wide application value and market prospect.

Description

Trypsin resistant antibacterial peptides and preparation method thereof

Technical Field

The invention relates to the field of protein engineering and genetic engineering, in particular to a method for directional modification and genetic engineering production of a series of antibacterial peptides.

Background

Plectasin is a Mygind research group which constructs a cDNA library by using saprophytic ascomycete pseudomaculalla isolated from northern pine of Europe and screens the cDNA library to have high-efficiency G resistance through BLASTX and SSEARCHp sequence similarity search programs⁺An antibacterial peptide with antibacterial activity. Plectasin baseThe coding sequence of the polypeptide containing 95 amino acid residues is a signal peptide sequence from 1 to 23, a leader peptide sequence from 24 to 55 and a mature peptide (Plectasin) sequence from 56 to 95. The theoretical molecular weight of Plectasin is 4407.9Da, has six histidines (His) and five lysines (Lys), and the net charge number of Plectasin varies from +1 to +3 due to different histidine dissociation states in different pH environments (Mygind et al, Plectasin a peptide antibody with a therapeutic potential from an aspartic fusion gene from Nature, 2005, 437 (7061): 975) -980).

Plectasin has a strong killing effect on gram-positive bacteria. Mygind et al studied the inhibitory effect of Plectasin on over 130 strains of Streptococcus pneumoniae of different origins and found that the minimum bactericidal concentration (MIC) was between 0.1 and 8. mu.g/ml for penicillin sensitive or resistant strains, and that MIC was₅₀At 1. mu.g/ml. Gottlieb et al (2008) studied the bactericidal activity of Plectasin on Staphylococcus aureus (Staphylococcus aureus), with MIC values between 1 and 32. mu.g/ml, MIC₅₀At 8. mu.g/ml (Gottlieb et al, antibodies against microorganisms against Listeria monocytogenes and Staphylococcus aureus antibiotic against orientation, sub-type, or viral factor expression. BMC Microbiol, 2008, 8 (1): 205-. In addition, the Novozymes uses Plectasin as a parent body, constructs a mutation library through error-prone PCR, screens the mutant NZ2114 with greatly improved antibacterial performance from 274 mutants, has an average MIC of only 1 mug/ml for clinical isolates of methicillin-resistant staphylococcus aureus (MRSA), has a strong inhibiting effect on vancomycin-resistant enterococcus (VRSA), and has an MIC value of only 2-4 mug/ml. In addition, sterilization kinetics experiments prove that Plectasin has high sterilization capacity, and 99.9 percent of tested pathogenic bacteria can be killed by 5 times MICplectasin within 5 hours. The mouse abdominal cavity infection model shows that the sterilization efficiency is equivalent to 70mg/kg vancomycin within 5h of 10mg/kg plectasin, and the number of pathogenic bacteria in the abdominal cavity is reduced by 3 orders of magnitude (equivalent to 99.9 percent sterilization efficiency). After 7 days, all mice in the control group die, and the survival rate of the mice dosed with 10mg/kg Plectasin is 80-100% according to the dosing frequency. The treatment effect of NZ2114 in the rabbit endocarditis infection model is also similar, and the NZ2114 with the concentration of 10mg/kg can be larger than that of NZ2114Or 15mg/kg vancomycin or 12mg/kg daptomycin, the number of pathogenic bacteria in the relevant organs (kidney, spleen) decreases by more than 99% after 3 days (Xiong et al, effective of NZ2114, anovel plectasin-derived cationic peptide antibody, experimental endicular product to metallic-resistant specific Staphylococcus aureus, antibiotic biological Agents and Chemotherapy, 2011, 55 (11): 5325-.

Among the major digestive enzymes in the gastrointestinal tract, plectasin and NZ2114 have good stability to pepsin, and hardly generate degradation after being incubated for 4h in pepsin. However, it is very sensitive to trypsin and almost completely loses activity after incubation with trypsin. Analysis shows that 6 trypsin cleavage sites, namely R14, K20, K23, K26, K32 and K38 exist in the NZ2114 sequence, and the existence of the sites seriously influences the stability of the NZ2114 trypsin, thereby restricting the oral treatment and the use effect as an additive. At present, no relevant report about the stability modification of NZ2114 trypsin is found.

Disclosure of Invention

The invention aims to provide a method for improving the resistance of NZ2114 trypsin by utilizing a protein oriented design and modification technology.

To achieve the object of the present invention, the present invention provides a series of antibacterial peptides, the amino acid sequence of which comprises any one of SEQ id No.1 to SEQ id No. 33. The invention also provides a series of antibacterial peptide genes, and the nucleotide sequence of the antibacterial peptide genes comprises any one of SEQ ID No.34-SEQ ID No. 66.

In addition, the invention comprises a series of recombinant expression vectors, which are characterized in that any one of the gene sequences (SEQ ID No.34-SEQ ID No.66) encoding the antibacterial peptide is carried.

The invention also provides a host cell containing the expression vector. Preferably, the host cell is pichia pastoris gene engineering bacteria.

The invention further provides a method for expressing the antibacterial peptide (SEQ ID No.1-SEQ ID No.33) in the recombinant pichia pastoris, which is characterized in that the recombinant pichia pastoris is fermented and cultured, and is secreted to generate the antibacterial peptide.

The invention realizes the directional modification of the trypsin resistant antibacterial peptide NZ2114 series derivatives for the first time and realizes the expression of the derivatives in pichia pastoris.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available, and unless otherwise specified, technical means used in the examples are conventional means well known to those skilled in the art.

The experimental procedures for the following examples, in which specific conditions are not specified, are generally performed according to conventional conditions, such as the molecular cloning Laboratory Manual (New York: Gold Spring Harbor Laboratory Press, 1989), for example, Sambrook, et al.

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.

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): 0.465g of Na₂HPO₄，2.917g 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): 0.465g of Na₂HPO₄，2.917g 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 Trypsin-resistant antimicrobial peptide molecular design and Gene Synthesis

The active site and key structural sites were bound by the presence of 6 trypsin cleavage sites (R14, K20, K23, K26, K32, K38) in the NZ2114 sequence. 33 sequences (SEQ ID No.1-SEQ ID No.33) of the single-site mutant, the double-site mutant and the triple-site mutant are respectively designed.

The mutant gene sequence (SEQ ID No.34-SEQ ID No.66) was designed according to the codon preference of Pichia pastoris. In order to ensure the integrity of the sequence in the expression process, an XhoI cleavage site and a Kex2 cleavage site are added at the 5 'end of the mutant gene sequence, and a TAA, a TAG terminator sequence and an XbaI cleavage site are added at the 3' end. The above sequence was completed by Shanghai Bioengineering Co., Ltd.

Example 2 construction of Pichia constitutive expression vectors pGAPNZTYR 1-pGAPNZTYR 33

Carrying out double enzyme digestion on the synthesized gene fragment and the vector pGAPZaA by using restriction endonucleases XhoI and XbaI respectively, recovering a pGAPZaA vector fragment and a mutant gene fragment, and connecting to obtain a vector pGAPNZTYR 1-pGAPNZTYR 33;

the detailed construction process of the vector pGAPNZTYR 1-pGAPNZTYR 33 is as follows: the synthesized gene fragment and pGAPZaA were double digested with restriction enzymes XhoI and XbaI, respectively, under the following conditions:

after the enzyme digestion system is added with sample, the reaction is carried out for 3 hours at 37 ℃, and 2% agarose gel electrophoresis detection is carried out, wherein the electrophoresis conditions are as follows: 120V, 30 min. After electrophoresis, the carrier segments and the electrophoresis strips corresponding to the gene segments are respectively cut by a scalpel under an ultraviolet lamp, DNA segments are recovered by a gel recovery kit of Tiangen biotechnology limited company, and the operation is carried out according to the relevant details of the instruction provided by the kit.

The recovered fragments were detected by agarose gel electrophoresis and preliminary quantitated using quantitation software (GeneTools) using T4DNA ligase at a fragment/vector (3: 1) molar ratio, under the following system and conditions:

after the sample of the ligation system is completely loaded, the reaction is carried out for 2 hours at 22 ℃ in a metal bath, and E.coli DH5 α is transformed, wherein the transformation operation details are as follows:

1) the ligation product was added to 100. mu.L E.coli DH5 α competent cells and ice-cooled for 30 min;

2) thermally shocking for 90s at 42 ℃, and immediately carrying out ice bath for 2-3 min;

3) adding 900 μ L LB low-salt culture medium preheated at 37 deg.C, and restoring culture at 37 deg.C and 80-100rpm for 1 h;

4) centrifuging at 5000rpm for 2min, and sucking 700 μ L of supernatant;

5) 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;

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

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:

detecting a PCR product of the positive transformant bacterium liquid by 2% agarose gel electrophoresis, wherein the electrophoresis conditions are as follows: 120V, 30 min. The E.coli containing the recombinant expression vector is stored in a 15% glycerol tube and the plasmid is extracted, preparation is carried out for linear transformation of P.pastoris, and relevant experimental details are operated according to the specification of a plasmid extraction kit (Tiangen biotechnology limited).

Example 3 construction of recombinant Yeast strains containing pGAPNZTYR 1-pGAPNZTYR 33

3.1 linearization of recombinant vectors pGAPNZTYR 1-pGAPNZTYR 33

Carrying out enzyme digestion on the constitutive recombinant expression vectors pGAPNZTYR 1-pGAPNZTYR 33 by using AvrII, wherein the enzyme digestion system and the reaction conditions are as follows:

after the enzyme digestion system is added with sample, the reaction is carried out for 3 hours at 37 ℃, and 2% agarose gel electrophoresis detection is carried out, wherein the electrophoresis conditions are as follows: 120V, 30 min. And after electrophoresis is finished, detecting the correct linearization of the recombinant expression vector, and recovering the linearized recombinant expression vector by using a DNA recovery kit.

3.2 Pichia pastoris electrotransformation and identification of linearized vectors

1) Selecting an X-33 single colony on the YPD plate, inoculating the single colony to 10mL of YPD liquid culture medium, culturing at 30 ℃ and 250rpm overnight;

2) inoculating 50 μ L of overnight culture into 100mL YPD liquid medium, culturing at 30 deg.C and 250rpm to OD₆₀₀The light absorption value is 1.2;

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, and adding 25mL of sterile water for resuspension;

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

6) centrifuging at 4 deg.C and 4000rpm for 5min, adding 100 μ L of 1M sorbitol, and resuspending to obtain X-33 competent cells (the above 6 steps are performed on ice with gentle motion);

7) premixing 80 mu L X-33 competent cells and 5-10 mu g of linearized vector, transferring to a 0.2cm electric rotor pre-cooled on ice, and standing on ice for 5min before operation of the electric rotor (1200V, 25 mu F, 400 omega);

8) 1mL of 1M sorbitol is immediately added and mixed evenly;

9) incubating at 30 ℃ for 1-2 h;

10) 100 μ L of incubated X-33 cells were plated on YPDS plates containing 100 μ g/mL Zeocin and cultured at 30 ℃ for 2-4 days with inversion.

Single colonies on YPDS plates were picked and inoculated into 100. mu.g/mL YPD liquid medium of Zeocin, and cultured overnight at 30 ℃ and 250 rpm. Taking 1mL of overnight culture, centrifuging at 4 ℃, 12000rpm for 5min, then re-suspending by PBS, quickly placing in a boiling water bath for 10min at-70 ℃ for 30min, immediately boiling water bath again for 10min, centrifuging at 4 ℃, 12000rpm for 5min, taking the supernatant as a template to carry out PCR verification on positive transformants, wherein a PCR system and conditions are as follows:

and (3) PCR system:

PCR conditions were as follows:

detecting a PCR product of the positive transformant bacterium liquid by 2% agarose gel electrophoresis, wherein the electrophoresis conditions are as follows: 120V, 30 min. Positive transformants of the correct size were transferred in one-to-one correspondence to YPD plates containing 100. mu.g/mL Zeocin for further expression.

Example 4 expression of Trypsin-resistant antimicrobial peptides

4.1 expression of transformants

Selecting positive transformants, inoculating the positive transformants to a YPD liquid culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 18-20 h; inoculating 0.5-1% of inoculum size to 50mL YPD liquid culture medium, performing shake culture at 30 deg.C and 250rpm for 1 day, replacing cellophane sealing membrane with 4 layers of sterilized gauze, wrapping shake flask mouth, and performing shake culture at 30 deg.C and 250rpm for 3 days until fermentation is finished.

4.2 detection of antibacterial Activity of recombinant Yeast fermentation broth

Experimental analysis of zone of inhibition: s. aureus ATCC25923 single colony is selected and inoculated in 10mLMH culture medium, cultured at 37 ℃ and 250rpm until OD600nm is approximately equal to 0.4, 1 percent of inoculum size is transferred to 50mL MH solid culture medium, evenly mixed, quickly poured into a square culture dish of 19cm multiplied by 19cm, after solidification, an oxford cup is carefully placed on the surface of the culture medium, and 50 mu L of fermentation broth supernatant is added.

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 recombinant AgPlectasin by Tricine-SDS-PAGE (electrophoresis method reference) ((S))

2006)。

Example 5 purification of Trypsin-resistant antimicrobial peptides

5.1G 25 desalination

Collecting the fermentation supernatant, freeze-drying, re-dissolving with deionized water, measuring the protein concentration by a Bradford method, centrifuging at 13000rpm for 5min at 4 ℃, and taking the supernatant. Desalting was performed on AKTA using a desalting column Sephadex G-25(Bed volume, 53 mL; GE Healthcare) with a loading of 4mL and a flow rate of 8mL/min, and freeze-drying was performed after desalting.

5.2 cation exchange chromatography purification

The protein concentration was determined by the Bradford method, and the supernatant was collected after centrifugation at 13000rpm for 5min at 4 ℃. A HiTrap SP FF cation exchange column (length 25mm, inner diameter 7mm, GE Healthcare) was loaded after 5-10 column volumes were equilibrated with solution A. After the completion of the sample injection, the elution was carried out in a gradient using 50mM phosphate elution buffer (solution B) containing 1M NaCl, pH 5.7. The elution step is as follows: eluting 5 column volumes with 70% solution A and 30% solution B; eluting 5 column volumes with 40% solution A and 60% solution B; 100% solution B, 5 column volumes eluted. And (3) monitoring the elution condition by using UV215nm, collecting an elution peak, and detecting the purification condition of the target product by Tricine-SDS-PAGE and a zone of inhibition experiment.

Example 6 antimicrobial peptide Trypsin degradation Rate testing

Weighing the purified freeze-dried powder, dissolving with Tris-HCl (pH 8.0), and determining the protein concentration by a Bradford method. And mixing 100 mu L of sample with trypsin with equal concentration and equal amount, incubating for 3h at 37 ℃, and determining the bacteriostatic potency of the mixture on staphylococcus aureus ATCC25923 by a double dilution method. The results (Table 2) show that the mutants show different degrees of trypsin tolerance, wherein the trypsin tolerance of the double mutant SEQ ID No.17 and the triple mutant SEQ ID No. 26-SEQ ID No.33 is greatly improved, and the degradation rate is below 25%.

TABLE 2 degradation rate of antimicrobial peptides with trypsin

Example 7 Activity assay against Staphylococcus aureus

The Minimum Inhibitory Concentration (MIC) of trypsin-resistant antimicrobial peptides against pathogenic bacteria was determined by the microbialdilution method established by Tian et al (Tian et al, Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3), Applied Microbiology and Biotechnology, 2009, 83 (1): 143-:

1) selecting a test strain to be monoclonal to MH culture medium, culturing at 37 ℃ and 250rpm, and shaking overnight;

2) serially diluting the antibacterial drug into 1.5mL sterile centrifuge tubes according to a gradient of 2 times, wherein the concentration is 10 times of the final concentration;

3) transferring the test strains into MH liquid culture medium at 37 deg.C with 1% inoculum size, and performing shake culture at 250rpm to 0.5 McLeod standard turbidimetry;

4) diluting the test bacteria culture solution 1000 times (final thallus concentration of 10)⁵CFU/mL) and transferring the diluted bacterial solution to a sterile cell culture plate, wherein each hole contains 90 mu L of diluted bacterial solution;

5) diluted drug 10 μ L, 3 replicates per concentration, was added and no drug negative control wells were reserved. Adding a sterile culture plate cover, sealing by a sealing film, and then statically culturing at 37 ℃ until a negative control is empty to show visible obvious turbid bacterial liquid. The cell culture plate is taken out, and the MIC value is the minimum concentration which can obviously inhibit the growth of the tested strain according to the observation result. And if the hole jumping or the result inconsistency among the parallel samples occurs, retesting.

The results of the antibacterial activity assay using mutants having a trypsin degradation rate of 50% or less are shown in Table 3.

TABLE 3 Trypsin-resistant antimicrobial peptides Activity against Staphylococcus aureus

As can be seen from Table 2, the antitrypsin antibacterial peptide has a strong inhibitory effect on Staphylococcus aureus: MIC for Staphylococcus aureus ATCC25923 is between 0.25-1 μ g/ml; MIC for ATCC6538 was between 1-4. mu.g/ml; the MIC for ATCC43300 was between 4-16. mu.g/ml, and the effect was superior to vancomycin and ampicillin.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (6)

1. An antibacterial peptide, the amino acid sequence of which is any item of SEQ ID NO.17 and SEQ ID NO. 26-33.

2. A gene expressing the antibacterial peptide of claim 1, the nucleotide sequence of which is any one of SEQ ID No.50 and SEQ ID No. 59-66.

3. A recombinant expression vector comprising the antimicrobial peptide gene sequence of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. The host cell of claim 4, wherein the host cell is a Pichia pastoris genetically engineered bacterium.

6. A method for expressing antibacterial peptide in recombinant Pichia pastoris, which is characterized in that the recombinant Pichia pastoris of claim 5 is fermented and cultured, and the antibacterial peptide is secreted and produced.