CN115124604A - Recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacterium and application thereof - Google Patents

Recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacterium and application thereof Download PDF

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CN115124604A
CN115124604A CN202210696778.5A CN202210696778A CN115124604A CN 115124604 A CN115124604 A CN 115124604A CN 202210696778 A CN202210696778 A CN 202210696778A CN 115124604 A CN115124604 A CN 115124604A
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牟海津
梁青平
赵迎军
刘哲民
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Weihai Dipusen Biology Technology Co ltd
Ocean University of China
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Abstract

The invention relates to a recombinant antibacterial peptide E-EJ97, 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 E-EJ97 is shown as SEQ ID NO.1, and the recombinant expression vector contains the gene SEQ ID NO.3 of the recombinant antibacterial peptide E-EJ 97. The invention also provides a recombinant trichoderma reesei Tu6 engineering bacterium capable of expressing the recombinant antibacterial peptide E-EJ 97. The recombinant antibacterial peptide E-EJ97 has an inhibiting effect on escherichia coli, salmonella and clostridium perfringens, has good thermal stability, salt ion stability and digestive enzyme stability, and is beneficial to application in preparation of feeds and feed additives.

Description

Recombinant antibacterial peptide E-EJ97, 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 E-EJ97, a recombinant expression vector, an engineering bacterium and application thereof.
Background
In animal husbandry, antibiotics have a broad spectrum of protection against bacterial infections in animals for the purpose of preventing diseases of microbial origin, but overdose and abuse thereof may generate multi-drug resistant bacteria, and the problem of antibiotic residues is becoming serious, so that the use of antibiotics has been prohibited in many countries and regions. With the proposal and popularization of the 'antibiotic-free culture' concept, the antibacterial peptide is used as a natural active substance which can effectively replace antibiotics, has the advantages of safety, specificity, high-efficiency antibacterial activity and the like, and presents a new idea for solving the problem of antibiotic substitutes.
Enterococcus faecalis is a gram-positive bacterium widely present in the intestinal tract and upper respiratory tract of humans and other animals and capable of producing a variety of antibiotics and antimicrobial peptides. Enterococcus faecalis can be added to animal feed as an additive, and therefore can be considered as a non-toxic and harmless beneficial bacterium. Various antibacterial peptides existing in enterococcus faecalis are applied to the aspects of commercial use and medical treatment, for example, the antibacterial peptide ST4SA can inhibit pathogenic bacteria such as listeria monocytogenes, staphylococcus aureus and the like; enterotoxin K1(EntK1) showed significant inhibitory effect against enterococcus faecium. The enterococcus faecalis bacteriocin can show rapid and continuous inhibitory activity to various gram-positive bacteria, and has inhibitory activity to food-borne pathogens such as Listeria monocytogenes; but the antibacterial spectrum is narrow, and the inhibitory activity to gram-negative bacteria is relatively weak. If some unique, efficient and safe antibacterial peptides in enterococcus faecalis can be optimized, improved and produced and prepared in a large scale or can be used as feed additives in the fields of livestock raising, aquatic products and other breeding, a superior solution is provided for 'nonreactive breeding'.
The antibacterial peptide EJ97 is a non-lantibiotic bacteriocin derived from enterococcus faecalis, and belongs to non-leader bacteriocin. The recombinant strain has a simple genetic structure, is easy to express in probiotics, is convenient for large-scale production, and can effectively inhibit common escherichia coli, staphylococcus aureus and listeria monocytogenes in culture. The range of the antibacterial spectrum is narrow, so that the microbes symbiotic with the host are less influenced, and the balance of intestinal flora can be maintained. The antibacterial peptide EJ97 has excellent thermal stability, can still play a role after passing through a higher sterilization environment, and keeps stable structure under a lower-temperature processing, storage and transportation environment; the EJ97 salt ions have good stability, and can still keep activity in an environment with high salt content; is sensitive to proteases. The antibacterial peptide EJ97 has wide prospect and unique advantages when being applied to feed additives. However, the antibacterial peptide EJ97 has the defect of low gram-negative bacteria inhibitory activity, so that after the antibacterial peptide EJ97 is improved, the large-scale industrial production and preparation method of the antibacterial peptide EJ97 is expected to reduce the production cost and promote the application of the antibacterial peptide in the aspects of feed and additives.
Heterologous expression is a common industrial production mode, common host engineering strains such as pichia pastoris and the like need to be added with inducers such as methanol and the like before fermentation production, trichoderma reesei is not only safe and harmless, but also has the advantages of strong promoter, mammalian-like protein modification capability, efficient synthetic secretion mechanism and the like, and has unique advantages in large-scale fermentation.
Disclosure of Invention
The invention relates to a preparation method of a recombinant antibacterial peptide E-EJ97 taking trichoderma reesei Tu6 as an engineering host bacterium and related application of antibacterial property of the recombinant antibacterial peptide.
The invention is realized based on the following technical scheme:
a recombinant antibacterial peptide E-EJ97, the amino acid sequence of the recombinant antibacterial peptide E-EJ97 is shown in SEQ ID NO. 1.
The nucleotide sequence of the amino acid sequence coded by SEQ ID NO.1 is shown as SEQ ID NO.3, the SEQ ID NO.3 is a mutant obtained by translating the amino acid sequence into a coded nucleotide sequence on the basis of the antibacterial peptide EJ97 of the original amino acid sequence SEQ ID NO.2, carrying out artificial synthesis on the nucleotide fragment, and carrying out error-prone PCR reaction on the artificially synthesized fragment.
SEQ ID NO.1MLAKIRVMIKRFPNPYTIAVKLTTYELNWYKQQYGKYPWERPVA
SEQ ID NO.2：MLAKIKAMIKKFPNPYTLAAKLTTYEINWYKQQYGRYPWERPVA
SEQ ID NO.3：atgttggctaagattgtttaaatgattaaggtttttcctaacccttacactattgcttaaaagttgactacttacgaattgaactggtacaagcaacaatacggtagatacaagtgggaaagacctgttgcttaataa
The invention provides a trichoderma reesei recombinant expression vector containing the antibacterial peptide gene.
The invention relates to a recombinant trichoderma reesei Tu6 engineering bacterium capable of expressing the antibacterial peptide E-EJ 97.
The invention comprises a preparation method of recombinant antibacterial peptide E-EJ97, which comprises the following steps: amplifying a gene fragment of the antibacterial peptide EJ97 through an error-prone PCR reaction, detecting the PCR fragment by agarose gel electrophoresis, inserting the PCR fragment into a trichoderma reesei expression vector through in vitro homologous recombination, constructing a recombinant expression vector of the recombinant antibacterial peptide E-EJ97, and transforming the recombinant expression vector into escherichia coli DH5 alpha; transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, and obtaining the recombinant antibacterial peptide E-EJ97 through screening positive transformants and verifying by fermentation expression.
The invention relates to application of a recombinant antibacterial peptide E-EJ97 in preparation of feed or a feed additive.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains a recombinant antibacterial peptide E-EJ97 by an error-prone PCR method, and constructs a recombinant expression vector mutation library of the recombinant antibacterial peptide E-EJ97 in Trichoderma reesei. The invention represents the antibacterial activity of the recombinant antibacterial peptide E-EJ97, and the recombinant antibacterial peptide E-EJ97 has an inhibitory effect on escherichia coli, salmonella and clostridium perfringens. As for the reported original sequence antibacterial peptide, the EJ97 has extremely weak antibacterial activity on gram-negative bacteria such as escherichia coli, salmonella and the like, and the recombinant antibacterial peptide E-EJ97 both has good inhibition effect on escherichia coli and salmonella.
The recombinant antibacterial peptide E-EJ97 has good thermal stability and salt ion stability.
The pepsin and trypsin stability of the recombinant antibacterial peptide E-EJ97 is improved.
The above characteristics are beneficial to the application of the feed additive in feed.
Drawings
FIG. 1 is a schematic diagram of gene amplification nucleic acid electrophoresis of recombinant antimicrobial peptide;
FIG. 2 is a schematic diagram of the construction of a recombinant expression vector;
FIG. 3 is a schematic diagram showing the thermostability of recombinant antimicrobial peptide E-EJ 97;
FIG. 4 is a schematic representation of pepsin stability for recombinant antimicrobial peptide E-EJ 97;
FIG. 5 is a schematic representation of trypsin stability of recombinant antimicrobial peptide E-EJ 97.
Detailed Description
Example 1 error-prone PCR of recombinant antimicrobial peptide E-EJ97 Gene
The amino acid sequence of EJ97 published by CAD35293.1 in NCBI is used as an original amino acid sequence, and is translated into a nucleotide sequence by using SnapGene 3.2.1 software for artificial synthesis. And (3) taking the synthesized nucleotide sequence as a template, and performing error-prone PCR reaction to prepare the E-EJ97 nucleotide fragment. The PCR amplification enzyme used was
Figure BDA0003702926290000041
High fidelity amplification enzyme, PCR reaction system (50 μ L) was:
Figure BDA0003702926290000042
Buffer 25μL；dNTP 1μL；
Figure BDA0003702926290000043
0.5 mu L; pf 2. mu.L; pr 2. mu.L; synthesizing 1 mu L of template; ddH 2 O18.5. mu.L. E-EJ97 gene PCR amplification 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 63 ℃ for 30sec, extension at 72 ℃ for 1min, and 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 61 ℃ for 30sec, elongation at 72 ℃ for 1min, and 5 cycles; denaturation at 94 ℃ for 30sec, and retrogradation at 59 ℃ for 30secStretching with fire at 72 deg.C for 1min, and 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 57 ℃ for 30sec, extension at 72 ℃ for 1min, and 25 cycles; total extension at 72 ℃ for 10 min; 4 ℃ is prepared. Wherein the sequence of a forward primer pEJ97-f of the E-EJ97 gene is 5'-tcttggccacagctcgtgctgaattcatgttggctaagattaaggc-3', and the sequence of a reverse primer pEJ97-r is 5'-tcaggctttcgccacggagcttaatgatgatgatgatgatgagcaacaggtctttcccaagg-3'.
The agarose gel electrophoresis was used to detect the band of interest of the antibacterial peptide E-EJ97 gene amplification, as shown in FIG. 1. The PCR product verified by agarose gel electrophoresis was digested and 5. mu.L of LrCutSmart Buffer was added using 1. mu.L of LDpnI enzyme (filled with 50. mu.L of solution). The reaction conditions were 37 ℃ for 2 h. And (3) recovering and purifying by using a Cycle Pure Kit PCR purification Kit to obtain the PCR gene fragment of the antibacterial peptide E-EJ 97.
Example 2 construction of recombinant expression vector E-EJ97 of Trichoderma reesei, E-EJ97-PCBHG
Linearized primer pairs using trichoderma reesei Tu6 vector backbone PCBHG-f: 5'-gctccgtggcgaaagcct-3' and PCBHG-r: 5'-agcacgagctgtggccaag-3' it was linearized by a PCR reaction using the enzyme
Figure BDA0003702926290000051
Super-Fidelity DNA Polymerase, PCR reaction system (50. mu.L) was: 5 × SF Buffer 25 μ L; dNTP 1 u L;
Figure BDA0003702926290000052
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 vector 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, extension at 72 ℃ for 7min, and 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 7min, and 5 cycles; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 7min, and 25 cycles; total extension at 72 ℃ for 10 min.
After agarose nucleic acid electrophoresis inspection, carrying out template digestion, recovery and purification on the vector framework to obtain a linear vector recovery fragment. Mixing all the above materialsThe PCR gene segment of the antimicrobial peptide E-EJ97 recovered in example 1 was ligated to a linearized vector by in vitro homologous recombination using Exnase II as the homologous recombination ligase, and 5. mu.L of the linker system: exnase II 0.5. mu.L, CE II Buffer 2. mu.L, linear vector backbone 0.5. mu.L, ddH 2 O1 μ L, E-EJ97 gene 1 μ L.
The reaction was performed at 37 ℃ for 30min to ligate into a loop, and then the product was transformed into E.coli DH5 α and cultured in LB solid medium supplemented with bleomycin (zeocin) at 37 ℃ for 16 h. And after a single colony grows out, selecting the single colony for colony PCR and sequencing, and completing construction of the Trichoderma reesei recombinant expression vector E-EJ97-PCBHG of E-EJ97 after comparison and verification, wherein the schematic composition diagram is shown in FIG. 2.
EXAMPLE 3 transformation of recombinant expression vector for Trichoderma for antimicrobial peptide E-EJ97
Subculturing the Trichoderma reesei host strain Tu6 on a PDA + U solid culture medium plate at 30 ℃ for 5-6 days, washing off spores by using a 0.1% Trition solution, inoculating to a YEG culture medium, and culturing in a constant temperature shaking table at 30 ℃ and 180rpm for 20 h.
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 with a constant temperature shaker at 80 rpm. Filtering and centrifuging the thallus enzymolysis liquid, re-suspending the protoplast with 1M sorbitol solution, centrifuging at 3500rpm for 4min, repeatedly washing with sterile water for 2 times, and re-suspending the protoplast with 1mL of 1M sorbitol solution to complete 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. Take 150. mu.L protoplast, 10. mu.L recombinant E-EJ97 plasmid, 50. mu.L 25% PEG6000, mix well and ice-bath for 25 min. The above system was transferred to a 10mL centrifuge tube containing 2mL of 25% PEG6000 and left at room temperature for 20 min. Adding the mixed solution into the trichoderma conversion upper layer culture medium, uniformly mixing, pouring the mixture on a flat plate paved with the trichoderma conversion lower layer culture medium, and culturing for 5-6 days at 30 ℃.
Example 4 fermentative expression of antimicrobial peptide E-EJ97 in Trichoderma
Inoculating the transformant grown in the trichoderma transformation medium to a new plate for screening, extracting the genome of the transformant after hyphae and spores grow out, and verifying the target gene of the transformant. Inoculating the screened positive transformant into a trichoderma fermentation medium, culturing for 2 days under the conditions of 30 ℃ and 180rpm, regulating the temperature to 25 ℃ after the thalli obviously grow, continuing to ferment for 4-5 days, centrifuging and collecting fermentation supernatant, further detecting, screening and separating fermentation products to obtain recombinant antibacterial peptide E-EJ97, and determining the amino acid sequence of the recombinant antibacterial peptide E-EJ97 as shown in SEQ ID No.1 and the coding nucleotide sequence of the recombinant antibacterial peptide E-EJ97 as shown in SEQ ID No. 3.
EXAMPLE 5 antimicrobial Activity assay of antimicrobial peptide E-EJ97
Determination of Minimum Inhibitory Concentration (MIC)
The indicator bacteria used in this example were escherichia coli, salmonella, clostridium perfringens.
The MIC is determined by adopting a trace double dilution method, and the specific implementation method comprises the following steps: preparing bacterial suspension to be tested, and respectively diluting the three bacteria cultured to the logarithmic phase of growth to the concentration of 10 5 CFU/mL, the purified antimicrobial peptide E-EJ97 solution was mixed with the bacterial suspension in equal amounts in the first column of a 96-well plate, at an initial concentration of 200. mu.g/mL, and then sequentially diluted in concentration gradients. The bacterial suspension after equal dilution is added into all samples of the 96-well plate, and the incubation is carried out for 16-18 h at the temperature of 37 ℃. After incubation, the wells were visually inspected for turbidity, with the minimum concentration of antimicrobial peptide E-EJ97 to clarify the wells being the MIC for each bacterium.
The results are shown in Table 1, and the recombinant antibacterial peptide E-EJ97 has better inhibitory activity to three bacteria. MICs for E.coli and Salmonella were 50. mu.g/mL, and MICs for Clostridium perfringens were 12.5. mu.g/mL. Compared with the original sequence antibacterial peptide EJ97, the MIC of EJ97 to escherichia coli and salmonella is higher, and the activity of EJ97 to gram-negative bacteria is weak in reports; the recombinant antibacterial peptide E-EJ97 has a good inhibition effect on both Escherichia coli and salmonella.
TABLE 1 MIC of recombinant antimicrobial peptide E-EJ97 and antimicrobial peptide EJ97 against test bacteria
Figure BDA0003702926290000071
EXAMPLE 6 thermostability assay of recombinant antimicrobial peptide E-EJ97
The heat stability result is shown in fig. 3, the inhibition rate of the recombinant antibacterial peptide E-EJ97 to clostridium perfringens is almost unchanged after being treated in boiling water for 20min, and the inhibition rate can still reach more than 80% after being treated for 25 min; after the heat treatment for 15min, the inhibition rate of the Escherichia coli and the salmonella is still kept above 80%. The data show that the recombinant antibacterial peptide E-EJ97 has relatively good thermal stability and can resist high temperature for a long time.
EXAMPLE 7 determination of salt ion stability of recombinant antimicrobial peptide E-EJ97
The results of the salt ion stability measurement of the recombinant antimicrobial peptide E-EJ97 are shown in Table 2, wherein the salt solutions used were set at physiological concentrations. Adding NaCl, KCl and NH to the control group 4 Cl、ZnCl 2 Then, the MIC of E-EJ97 to Escherichia coli is reduced to 25 mug/mL, which shows that the bacteriostatic effect of the recombinant antibacterial peptide E-EJ97 in the salt solutions is better. The salt ion environment does not influence the bacteriostatic activity of the recombinant antibacterial peptide E-EJ97, which indicates that the antibacterial peptide has salt ion stability.
TABLE 2 salt ion stability of recombinant antimicrobial peptides E-EJ97
Figure BDA0003702926290000081
EXAMPLE 8 digestive enzyme stability assay of recombinant antimicrobial peptide E-EJ97
The results of the digestive enzyme stability assay of the recombinant antimicrobial peptide E-EJ97 are shown in FIGS. 4 and 5, wherein FIG. 4 is pepsin and FIG. 5 is trypsin. Compared with the original sequence antibacterial peptide EJ97 which is completely inactivated in pepsin and trypsin, the recombinant antibacterial peptide E-EJ97 has better digestive enzyme stability. After being treated by pepsin for 15min, the inhibition rate of E-EJ97 on salmonella is almost unchanged; when the treatment time is 180min, the inhibition rate of the recombinant antibacterial peptide E-EJ97 on three tested bacteria can still be kept above 70%. After the recombinant antibacterial peptide E-EJ97 is treated for 180min in a trypsin environment, the inhibition rate of the E-EJ97 on escherichia coli, salmonella and clostridium perfringens is kept at about 80%, and the result shows that the recombinant antibacterial peptide E-EJ97 has digestive enzyme tolerance and has the potential of exerting antibacterial activity in animal gastrointestinal tracts.
Sequence listing
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WEIHAI DIPUSEN BIOLOGY TECHNOLOGY Co.,Ltd.
<120> recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacterium and application thereof
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<210> 1
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Met Leu Ala Lys Ile Arg Val Met Ile Lys Arg Phe Pro Asn Pro Tyr
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Thr Ile Ala Val Lys Leu Thr Thr Tyr Glu Leu Asn Trp Tyr Lys Gln
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atgttggcta agattgttta aatgattaag gtttttccta acccttacac tattgcttaa 60
aagttgacta cttacgaatt gaactggtac aagcaacaat acggtagata caagtgggaa 120
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gctccgtggc gaaagcct 18
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Claims (5)

1. A recombinant antibacterial peptide E-EJ97 is characterized in that the amino acid sequence of the recombinant antibacterial peptide E-EJ97 is shown as SEQ ID NO. 1.
2. A recombinant expression vector characterized in that it contains the gene SEQ ID No.3 of the recombinant antimicrobial peptide E-EJ97 of claim 1.
3. A recombinant Trichoderma reesei Tu6 engineering bacterium, characterized in that the engineering bacterium can express the recombinant antibacterial peptide E-EJ97 of claim 1.
4. A preparation method of recombinant antibacterial peptide E-EJ97 is characterized in that the method is characterized in that a gene shown in SEQ ID NO.3 is inserted into a Trichoderma reesei expression vector through in vitro homologous recombination to construct a recombinant expression vector of the recombinant antibacterial peptide E-EJ97, and the recombinant expression vector is transformed into Escherichia coli DH5 alpha; transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, and obtaining the recombinant antibacterial peptide E-EJ97 through screening positive transformants and verifying by fermentation expression.
5. Use of the recombinant antimicrobial peptide E-EJ97 of claim 1 in the preparation of feed or feed additives.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116023450A (en) * 2023-01-10 2023-04-28 中国海洋大学 Recombinant antibacterial peptide PmHis9, recombinant expression vector, engineering bacteria and application thereof
CN116023450B (en) * 2023-01-10 2024-05-03 中国海洋大学 Recombinant antibacterial peptide PmHis, recombinant expression vector, engineering bacteria and application thereof

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