CN115960187A - Recombinant antibacterial peptide PmHis3, recombinant expression vector, engineering bacterium and application thereof - Google Patents

Recombinant antibacterial peptide PmHis3, recombinant expression vector, engineering bacterium and application thereof Download PDF

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CN115960187A
CN115960187A CN202310032181.5A CN202310032181A CN115960187A CN 115960187 A CN115960187 A CN 115960187A CN 202310032181 A CN202310032181 A CN 202310032181A CN 115960187 A CN115960187 A CN 115960187A
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pmhis3
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antibacterial peptide
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牟海津
梁青平
赵迎军
朱常亮
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Ocean University of China
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Abstract

The invention relates to a recombinant antibacterial peptide PmHis3, a recombinant expression vector, an engineering bacterium and application thereof, belonging to the field of genetic engineering and biotechnology, wherein the amino acid sequence of the recombinant antibacterial peptide PmHis3 is shown in SEQ ID NO. 1. The nucleotide sequence of the amino acid shown in the code SEQ ID NO.1 is shown in SEQ ID NO.2, the invention provides a preparation method of heterologous expression of recombinant antibacterial peptide PmHis3 in Trichoderma reesei, wherein the recombinant antibacterial peptide PmHis3 is generated by plectasin through site-directed mutagenesis, has an inhibiting effect on microorganisms such as staphylococcus aureus, has good thermal stability, digestive enzyme stability and pH stability, and is beneficial to the application of the recombinant antibacterial peptide PmHis3 in the preparation of bacteriostatic agents or bactericides.

Description

Recombinant antibacterial peptide PmHis3, recombinant expression vector, engineering bacterium and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering and biology, and particularly relates to a recombinant antibacterial peptide PmHis3, a recombinant expression vector, an engineering bacterium and application thereof.
Background
Staphylococcus aureus is a common pathogenic bacterium, widely distributed in air, water, human bodies and animal bodies, and can cause wound infection, skin inflammation and food-borne diseases. Staphylococcus aureus is easy to generate drug resistance, and a drug-resistant strain MRSA has drug resistance to various antibiotics, so that the staphylococcus aureus is the key point of prevention and control in the medical field. China has made a limit on staphylococcus aureus in 8 types of foods such as meat products, aquatic products, food products, instant bean products, instant fruit and vegetable products, beverages, frozen drinks, instant seasonings and the like. In addition, relevant standards in catering, hairdressing, cosmetics, medical treatment, animal feed, etc. have made a limit on staphylococcus aureus. Therefore, it is necessary to develop bacteriostatic agents or bactericides mainly targeting staphylococcus aureus.
The antibacterial peptide has the advantages of safety, environmental protection, high efficiency, difficult generation of drug resistance and the like, and can effectively replace antibiotics. Plectasin is a fungal antibacterial peptide derived from Pseudoplectania nigrella, has 3 disulfide bonds and a unique CS alpha beta structure, has a strong inhibition effect on gram-positive bacteria, and has good stability and no hemolytic activity. Plectasin can be combined with a cell wall precursor substance Lipid-II of gram-positive bacteria such as staphylococcus aureus and the like, so that the formation of a cell wall is inhibited, and the plectasin is similar to vancomycin; however, the binding site is mainly phosphate group of Lipid-II, which is less prone to cause drug resistance of bacteria. Plectasin is researched and applied in the fields of feed, cosmetics, medical treatment and the like, and is one of antibacterial peptides with research prospects and research values.
At present, myceliomycin plectasin is applied to bacteriostatic agents or bactericides and has several main problems. One is that the antibacterial activity of plectasin in the prior art is still in a large difference compared with the traditional antibiotics, the MIC of plectasin to staphylococcus aureus is 16-32 mug/mL, and the MIC of vancomycin is 1-2 mug/mL; the other is the safety problem in the production process, and the host engineering strain for producing plectasin such as pichia pastoris needs to be added with inducers such as methanol and the like before fermentation production.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a recombinant antibacterial peptide PmHis3, a recombinant expression vector, an engineering bacterium and application thereof. In order to improve the antibacterial activity of plectasin, the invention designs a plectasin mutant PmHis3 by increasing the positive charge of antibacterial peptide, and finds that the inhibitory activity of PmHis3 on staphylococcus aureus is further improved compared with plectasin.
The invention is realized based on the following technical scheme:
a recombinant antibacterial peptide PmHis3, the amino acid sequence of the recombinant antibacterial peptide PmHis3 is shown in SEQ ID NO. 1. The SEQ ID NO.1 is a mutant obtained by site-specific mutagenesis of the amino acid sequence fragment shown in SEQ ID NO. 3. The nucleic acid of the prosequence plectasin codes for the mutation of the aspartic acid at position 11 of SEQ ID NO.3 to the asparagine coding for SEQ ID NO. 1.
The nucleotide sequence of the amino acid sequence coded by the SEQ ID NO.1 is shown as SEQ ID NO.2, and the SEQ ID NO.2 is translated into the coded nucleotide sequence on the basis of the antibacterial peptide PmHis3 of the original amino acid sequence SEQ ID NO. 1.
SEQ ID NO.1:GFGCNGPWDENDMQCHNHCKSIKGYKGGYCAKGGF VCKCY；
SEQ ID NO.2:gaattcggatttggatgtaacggtccgtgggatgaaaacgatatgcaatgtcataaccat tgtaagtctattaagggatacaagggaggttactgtgctaagggtggttttgtttgtaagtgttactaataagcggccgc；
SEQ ID NO.3:GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGF VCKCY.
A recombinant expression vector comprises the antibacterial peptide gene.
A recombinant engineering bacterium comprises the recombinant expression vector, and a host strain is Trichoderma reesei Tu6.
The invention comprises a preparation method of recombinant antibacterial peptide PmHis3, which comprises the following steps: translating an amino acid sequence of plectasin into a nucleic acid sequence according to the preference of trichoderma reesei codons, amplifying the nucleic acid sequence by using PCR, verifying a PCR product, inserting the verified PCR product into a trichoderma reesei expression vector through in vitro homologous recombination, and constructing a recombinant antibacterial peptide trichoderma reesei expression vector plectasin-pCBHG; constructing a recombinant expression vector PmHis3-pCBHG of the recombinant antibacterial peptide PmHis3 by PCR site-directed mutagenesis by taking plectasin-pCBHG as a template, detecting the PCR fragment by using agarose gel electrophoresis, carrying out sequencing verification on the PCR fragment, and transforming the PCR fragment into escherichia coli DH5 alpha; and transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, and obtaining the recombinant antibacterial peptide PmHis3 through fermentation expression after screening a positive transformant and verification.
The invention relates to an application of recombinant antibacterial peptide PmHis3 in preparing feed and feed additives.
The invention relates to an application of a recombinant antibacterial peptide PmHis3 in preparation of a bacteriostatic agent or a bactericide, wherein the bacterium is staphylococcus aureus, candida albicans and pseudomonas aeruginosa.
Compared with the prior art, the invention has the beneficial effects that:
the recombinant antibacterial peptide PmHis3 has good thermal stability and pH stability, the stability of trypsin and pepsin is improved, and the recombinant antibacterial peptide PmHis3 has stronger inhibiting effect on staphylococcus aureus, candida albicans and pseudomonas aeruginosa than plectasin.
The trichoderma reesei used in the invention is safe and harmless, can be directly added into feed according to national standards, has the advantages of strong promoter, efficient synthetic secretion mechanism and the like, has the advantages of independent advantages in large-scale fermentation, and can further improve the yield.
Drawings
FIG. 1 is a schematic diagram of the electrophoresis of PCR amplified nucleic acids of a recombinant expression vector;
FIG. 2 is a schematic diagram of the construction of a recombinant expression vector PmHis 3-pCBHG;
FIG. 3 is a schematic diagram of the thermal stability of the recombinant antibacterial peptide PmHis 3;
FIG. 4 is a schematic representation of pepsin stability of the recombinant antimicrobial peptide PmHis 3;
FIG. 5 is a schematic representation of trypsin stability of the recombinant antimicrobial peptide PmHis 3;
FIG. 6 is a pH stability diagram of the recombinant antibacterial peptide PmHis3.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments with reference to the attached drawings, but the scope of the present invention is not limited in any way by the embodiments.
Example 1 construction of recombinant expression vector of Plectasin for Trichoderma reesei, plectasin-PCBHG
The amino acid sequence of plectasin, which is published in UQI49964.1 in NCBI, is used as an original amino acid sequence, and the plectasin is translated into a nucleotide sequence by using SnapGene 3.2.1 software for artificial synthesis. And constructing a Trichoderma reesei recombinant expression vector plectasin-PCBHG of the plectasin by using the synthesized nucleotide sequence as a template.
Linearized primer pairs using trichoderma reesei Tu6 vector backbone PCBHG-f:5'-GCTCCGTGGCGAAAGCCT-3' and PCBHG-r:5'-AGCACGAGCTGTGGCCAAG-3' was linearized by PCR using the enzyme
Figure BDA0004047629540000041
Super-Fidelity DNA Polymerase, PCR reaction system (50. Mu.L) was: 5 × Phanta Buffer 25 μ L; dNTP 1 u L; />
Figure BDA0004047629540000042
0.5 mu L; pf 2. Mu.L; pr 2. Mu.L; 1 μ L of vector PCBHG; ddH 2 O18.5. Mu.L. The Trichoderma reesei Tu6 carrier framework PCR adopts gradient annealing temperature, and the reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for 7min, and 5 cycles; denaturation at 94 ℃ for 30sec,Annealing at 60 ℃ for 30sec, stretching at 72 ℃ for 7min, and 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 7min, 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 7min, and 25 cycles; total extension was 10min at 72 ℃.
The PCR product verified by agarose gel electrophoresis was digested, 1. Mu.L of DpnI enzyme was used, and 5. Mu.L of rCutSmart Buffer was added. The reaction conditions were 37 ℃ for 2h. And (3) recovering and purifying by using a Cycle Pure Kit PCR purification Kit to obtain a linear fragment of the PCBHG carrier. Carrying out in-vitro homologous recombination and connection on the artificially synthesized plectasin nucleic acid fragment and a linearized vector, wherein the used homologous recombination ligase is Exnase II, and the 5 mu L connector system is as follows: exnase II 0.5. Mu.L, CE II Buffer 2. Mu.L, linear vector backbone 0.5. Mu.L, ddH 2 O1. Mu.L, plectasin gene 1. Mu.L.
The reaction was performed at 37 ℃ for 30min to ligate into a loop, and then the product was transformed into E.coli DH 5. Alpha. And cultured in LB solid medium supplemented with bleomycin (zeocin) at 37 ℃ for 16h. And after a single colony grows out, selecting the single colony for colony PCR and sequencing, and completing construction of the plectasin-PCBHG trichoderma reesei recombinant expression vector plectasin-PCBHG after comparison and verification.
Example 2 site-directed mutagenesis of the recombinant antimicrobial peptide PmHis3 Gene
Using plectasin-PCBHG prepared in example 1 as a template, a PmHis3 nucleotide fragment was prepared by site-directed mutagenesis PCR. The PCR amplification enzyme used was
Figure BDA0004047629540000051
High fidelity amplification enzyme, PCR reaction system (50 μ L) was:
Figure BDA0004047629540000052
Buffer 25μL；dNTP 1μL；/>
Figure BDA0004047629540000053
0.5 mu L; pf 2. Mu.L; pr 2. Mu.L, synthesizing a template 1. Mu.L; ddH 2 O18.5. Mu.L. The PCR amplification reaction conditions of the PmHis3 gene are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30sec, annealing at 57 ℃ for 30sec, extension at 72 ℃ for 7min, and 35 cycles; 72 ℃ for 10minA total extension; 4 ℃ is prepared. Wherein the sequence of a forward primer PmHis3-f of the PmHis3 gene is 5'-GGGATGAAAACGATATGCAATG-3', and the sequence of a reverse primer PmHi s3-r is 5'-ATATCGTTTTCATCCCACGGAC-3'.
The target band of the gene amplification of the antimicrobial peptide PmHis3 is detected by agarose gel electrophoresis, as shown in FIG. 1. The PCR product verified by agarose gel electrophoresis was digested, 1. Mu.L of DpnI enzyme was used, and 5. Mu.L of LrCutSmart Buffer was added. The reaction conditions were 37 ℃ for 2h. The linearized fragment of PmHis3-PCBHG is obtained by recovery and purification by using a Cycle Pure Kit PCR purification Kit. The linearized recombinant expression vector PmHis3-PCBHG was ligated into a circular plasmid using Exnase II, with a 5. Mu.L linker system: exnase II 0.5. Mu.L, CE II Buffer 2. Mu.L, ddH 2 O1. Mu.L, plectasin gene 1.5. Mu.L. The reaction was performed at 37 ℃ for 30min to ligate into a loop, and then the product was transformed into E.coli DH 5. Alpha. And cultured in LB solid medium supplemented with bleomycin (zeocin) at 37 ℃ for 16h. After a single colony grows, selecting the single colony for colony PCR and sequencing, and completing the construction of the Trichoderma reesei recombinant expression vector PmHis3-PCBHG of PmHis3 after comparison and verification, as shown in figure 2.
EXAMPLE 3 transformation of Trichoderma recombinant expression vector for antimicrobial peptide PmHis3
Subculturing the Trichoderma reesei host strain Tu6 on a PDA + U solid medium plate at 30 deg.C for 5-6 days, washing off spores using 0.1% Trition solution, inoculating to YEG medium, and culturing in a constant temperature shaker at 30 deg.C and 180rpm for 20h.
Filtering the mature bacteria liquid with sterile filter cloth and funnel after enzymolysis, washing with sterile water, collecting appropriate amount of mycelium, and filtering and sterilizing with enzymolysis liquid with filter with 0.45 μ L pore diameter; incubate at 30 ℃ for 2h on a constant temperature shaker at 80 rpm. Filtering and centrifuging the thallus enzymolysis solution, suspending the protoplast by using a 1M sorbitol solution, centrifuging for 4min at 3500rpm, repeatedly washing for 2 times by using sterile water, and suspending the protoplast by using 1mL of 1M sorbitol solution to finish the preparation of the protoplast.
The recombinant plasmid constructed in example 2 was extracted using an e.z.n.a.plasmid Mini Kit i Kit. mu.L of protoplast, 10. Mu.L of recombinant PmHis3 plasmid, 50. Mu.L of 25% PEG6000 were taken, mixed and ice-cooled for 25min. The resulting system was transferred to a 10mL centrifuge tube containing 2mL of 25% PEG6000, and allowed to stand at room temperature for 20min. Adding the solution into the upper culture medium for trichoderma transformation, mixing, pouring on a flat plate paved with the lower culture medium for trichoderma transformation, and culturing at 30 ℃ for 5-6 days.
Example 4 fermentative expression of antimicrobial peptide PmHis3 in Trichoderma
Inoculating the transformant grown in the trichoderma transformation medium to a new plate for screening, and performing genome extraction and verification on the target gene after hypha and spores grow out. Inoculating the screened positive transformant to a trichoderma fermentation culture medium, firstly growing in a constant-temperature shaking table at 30 ℃ and 180rpm for 2 days, regulating the temperature to 25 ℃ after the strain obviously grows, continuing fermentation and induction expression for 4-5 days, and then centrifuging to obtain a fermentation product. Detecting, screening and separating the fermentation product to obtain the antibacterial peptide PmHis3, and determining that the amino acid sequence is the same as SEQ ID NO.1 and the coding nucleotide sequence is the same as SEQ ID NO.2.
Example 5 antimicrobial Activity assay of the antimicrobial peptide PmHis3
Determination of Minimum Inhibitory Concentration (MIC)
The indicator bacteria used in this example were Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, and Candida albicans.
Determining MIC by adopting a trace double dilution method: preparing bacterial suspension, and respectively diluting the four bacteria cultured to the logarithmic phase of growth to the concentration of 10 5 CFU/mL, mixing the purified antibacterial peptide PmHis3 solution and antibacterial peptide plectasin solution with the bacterial suspension in equal amount in the first column of a 96-well plate, wherein the initial concentration of the antibacterial peptide sample is 150 mu g/mL, and then sequentially diluting in a concentration gradient manner. Adding the bacterial suspension after equal dilution into all samples of a 96-well plate, incubating escherichia coli, pseudomonas aeruginosa and staphylococcus aureus for 18 hours at 37 ℃, and incubating candida albicans for 24 hours at 30 ℃. After incubation, the turbidity of each well was visually observed, wherein the minimum concentration to clarify the wells was the MIC of the antimicrobial peptide PmHis3 for each bacterium.
As shown in Table 1, the antibacterial activity of the recombinant antibacterial peptide PmHis3 is improved relative to plectasin. MIC of both to Escherichia coli is 150 μ g/mL; MICs of the recombinant antibacterial peptide PmHis3 to pseudomonas aeruginosa, staphylococcus aureus and candida albicans are respectively 37.5 mu g/mL, 18.8 mu g/mL and 18.8 mu g/mL, which shows that the antibacterial activity of the recombinant antibacterial peptide PmHis3 to the pseudomonas aeruginosa, staphylococcus aureus and candida albicans is higher than that of the original sequence plectasin.
TABLE 1 MIC (μ g/mL) of plectasin and PmHis3, a plectasin myceliophthorn, to different bacteria
Figure BDA0004047629540000071
Figure BDA0004047629540000081
Determination of zone of inhibition
And (3) coating 150 mu L of the bacterial liquid on a culture medium which is suitable for growth of each bacterium, respectively adding 100 mu L of recombinant antibacterial peptide PmHis3 and plectasin with the concentration of 300 mu g/mL into an Oxford cup, incubating each bacterium for 18h at 37 ℃, and incubating Candida albicans for 24h at 30 ℃. The determination results are shown in table 2, and the antibacterial activity of the recombinant antibacterial peptide PmHis3 to each bacterium is improved.
TABLE 2 inhibition zone size (mm) of plectasin and PmHis3 for different bacteria
Figure BDA0004047629540000082
The data show that the recombinant antibacterial peptide PmHis3 has improved antibacterial activity on bacteria such as gram-negative pseudomonas aeruginosa, gram-positive staphylococcus aureus and candida albicans in fungi which are easy to cause wound infection, and is favorable for being used as a bacteriostatic agent or a bactericide in the treatment of wound infection.
Example 6 thermostability assay of recombinant antimicrobial peptide PmHis3
The indicator bacteria used in this example were staphylococcus aureus, the thermal stability results are shown in fig. 3, where the abscissa is the boiling water bath treatment time, the inhibitory activity of the recombinant antibacterial peptide PmHis3 and plectasin on each bacteria after high temperature treatment was reduced to different degrees, and the PmHis3 could maintain 94% inhibition rate after 30min treatment, which was slightly lower than plectasin. The data show that the recombinant antibacterial peptide PmHis3 has relatively good thermal stability and can resist high temperature for a long time.
Example 7 digestive enzyme stability assay for recombinant antimicrobial peptide PmHis3
The indicator bacteria used in this example were staphylococcus aureus, and the results of the digestive enzyme stability assay of the recombinant antimicrobial peptide PmHis3 are shown in fig. 4 and 5, where fig. 4 is pepsin and fig. 5 is trypsin. The recombinant antibacterial peptide PmHis3 and the original sequence plectasin have better stability in pepsin. Compared with the original sequence, the recombinant antibacterial peptide PmHis3 has better trypsin stability. After 120min of trypsin treatment, the inhibition rate of the PmHis3 on the staphylococcus aureus can still reach more than 50 percent.
Example 8 determination of pH stability of recombinant antimicrobial peptide PmHis3
The indicator used in this example was staphylococcus aureus and the pH stability measurements are shown in fig. 6, where the abscissa is the pH value. The recombinant antibacterial peptide PmHis3 and the original sequence plectasin can keep more than 80% of antibacterial activity after being treated for 120min in alkaline and acidic environments, which shows that the recombinant antibacterial peptide PmHis3 and the original sequence plectasin have good pH stability and tolerance to acid and alkali.

Claims (7)

1. A recombinant antibacterial peptide PmHis3 is characterized in that the amino acid sequence of the recombinant antibacterial peptide PmHis3 is shown in SEQ ID NO. 1.
2. A nucleotide sequence for coding amino acid shown in SEQ ID NO.1, which is characterized in that the nucleotide sequence is shown in SEQ ID NO.2.
3. A recombinant expression vector comprising the nucleotide sequence of SEQ ID No.2 of claim 2.
4. A recombinant bacterium, which contains the nucleotide sequence SEQ ID No.2 of claim 2, wherein the host strain is Trichoderma reesei Tu6.
5. The method for preparing the recombinant antibacterial peptide Pmhis3 as claimed in claim 1, which is characterized in that the method is to firstly construct a recombinant antibacterial peptide Trichoderma reesei expression vector plectasin-pCBHG; constructing a recombinant expression vector PmHis3-pCBHG of the recombinant antibacterial peptide PmHis3 by PCR site-directed mutagenesis by taking plectasin-pCBHG as a template, detecting the PCR fragment by using agarose gel electrophoresis, carrying out sequencing verification on the PCR fragment, and transforming the PCR fragment into escherichia coli DH5 alpha; and transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, and obtaining the recombinant antibacterial peptide PmHis3 through screening a positive transformant and verifying and fermentation expression.
6. The use of the recombinant antibacterial peptide PmHis3 as claimed in claim 1, in the preparation of bacteriostatic or bactericidal agents, wherein said bacteria are staphylococcus aureus, candida albicans and pseudomonas aeruginosa.
7. The use of the recombinant antibacterial peptide PmHis3 of claim 1 in the preparation of feed and feed additives.
CN202310032181.5A 2023-01-10 2023-01-10 Recombinant antibacterial peptide PmHis3, recombinant expression vector, engineering bacterium and application thereof Pending CN115960187A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101189256A (en) * 2005-06-06 2008-05-28 诺维信公司 Polypeptides having antimicrobial activity and polynucleotides encoding same
CN113831395A (en) * 2021-11-26 2021-12-24 中国海洋大学 Recombinant antibacterial peptide Trsub, preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101189256A (en) * 2005-06-06 2008-05-28 诺维信公司 Polypeptides having antimicrobial activity and polynucleotides encoding same
CN113831395A (en) * 2021-11-26 2021-12-24 中国海洋大学 Recombinant antibacterial peptide Trsub, preparation method and application thereof

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