阅读说明：本技术 一种重组抗菌肽TrSub、制备方法及其应用 (Recombinant antibacterial peptide Trsub, preparation method and application thereof ) 是由 牟海津 梁青平 刘哲民 于 2021-11-26 设计创作，主要内容包括：本发明涉及一种重组抗菌肽TrSub、制备方法及其应用,属于基因工程及生物技术领域,所述重组抗菌肽TrSub的氨基酸序列如SEQ ID NO.1所示。编码SEQ ID NO.1所示氨基酸的核苷酸序列如SEQ ID NO.2所示,本发明还提供一种重组抗菌肽TrSub在里氏木霉中异源表达的制备方法,所述重组抗菌肽TrSub对大肠杆菌、沙门氏菌及产气荚膜梭菌均有抑制作用,且具有良好的热稳定性、耐酸和耐胃蛋白酶特性及较低的溶血活性,有利于其在制备饲料及饲料添加剂中的应用。(The invention relates to a recombinant antibacterial peptide Trsub, a preparation method and application thereof, belonging to the field of genetic engineering and biotechnology, wherein the amino acid sequence of the recombinant antibacterial peptide Trsub is shown as SEQ ID NO. 1. The nucleotide sequence of the amino acid shown in the coded SEQ ID NO.1 is shown in SEQ ID NO.2, the invention also provides a preparation method of heterologous expression of the recombinant antibacterial peptide Trsub in Trichoderma reesei, and the recombinant antibacterial peptide Trsub has inhibitory action on Escherichia coli, Salmonella and Clostridium perfringens, has good heat stability, acid resistance and pepsin resistance and lower hemolytic activity, and is beneficial to the application of the recombinant antibacterial peptide Trsub in preparing feed and feed additives.)

1. A recombinant antibacterial peptide Trsub is characterized in that the amino acid sequence of the recombinant antibacterial peptide Trsub is shown as SEQ ID number 1.

2. The nucleotide sequence of the amino acid shown in SEQ ID number 1 is coded and is characterized in that the nucleotide sequence is shown in SEQ ID number 2.

3. A trichoderma reesei recombinant expression vector comprising the nucleotide sequence SEQ ID number 2 according to claim 2.

4. Recombinant strain comprising the nucleotide sequence SEQ ID number 2 according to claim 2, characterized in that the strain is trichoderma reesei Tu 6.

5. A preparation method of a recombinant antibacterial peptide Trsub is characterized in that the method comprises the steps of amplifying a gene segment of an antibacterial peptide Sublancin168 through an error-prone PCR reaction, detecting the error-prone PCR segment by agarose gel electrophoresis, inserting a Trichoderma reesei expression vector through in vitro homologous recombination, and constructing the recombinant expression vector of the recombinant antibacterial peptide Trsub; and (3) transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, screening a positive transformant and verifying the positive transformant, and then obtaining the recombinant antibacterial peptide Trsub through fermentation expression.

6. The use of the recombinant antibacterial peptide Trsub of claim 5 in the preparation of feed and feed additives.

Technical Field

The invention belongs to the technical field of genetic engineering and biology, and particularly relates to a preparation method and application of a recombinant antibacterial peptide Trsub in Trichoderma reesei.

Background

In animal breeding, bacterial diseases such as bacterial diarrhea and the like seriously harm the health of animals and cause great loss to the breeding. At present, antibiotics are mainly used for treating and preventing the diseases, but overuse and abuse of the antibiotics continuously cause drug-resistant strains and drug resistance, so that the health of human beings is threatened. The policy of banning the use of antibiotics was introduced in china in 2020, which made the emergence and excavation of antibiotic substitutes urgent. Antibacterial peptides (AMPs) are a class of small-molecule peptide substances, which have wide sources, small molecular weights and broad-spectrum antibacterial activity. Compared with the traditional antibiotics, the antibacterial peptide belongs to natural active substances, has no pollution, is not easy to generate drug resistance, and is an antibiotic substitute with great potential. However, the preparation methods by means of biological extraction, chemical synthesis and the like have high cost and low efficiency, are not easy to realize mass preparation, and cannot meet the requirements of industrial production, so that the method for realizing efficient preparation and production of the antibacterial peptide is a hotspot of research.

The antibacterial peptide Sublancin168 is one of metabolites secreted by bacillus subtilis, has a specific structure and certain antibacterial activity, but the antibacterial property of the peptide Sublancin168 needs to be further explored. The antibacterial peptide to be developed and to be applied to the feed industry needs to meet good antibacterial activity, and also needs to have good heat stability capable of tolerating high-temperature processing processes such as granulation in industrial production, digestive enzyme stability capable of tolerating animal pepsin to exert activity, and production and use safety in application to animal feed. Therefore, the antibacterial activity to common pathogenic bacteria in livestock and poultry breeding, the self thermal stability, the pepsin stability and the production and self safety are all the basis for applying the antibacterial peptide as the feed additive.

To meet the requirement of industrial production, the preparation of heterologous expression is widely adopted. Host engineering strains such as escherichia coli and pichia pastoris need an inducer to induce and express foreign proteins, and the produced foreign proteins cannot be directly applied to feed additives. The trichoderma reesei serving as a food-grade safe strain (GRAS) can meet the safety requirement of produced foreign protein, has the advantages of strong promoter, strong protein post-translational processing capability and the like, and can lay a foundation for the efficient production and safety of feed additives. And Escherichia coli, salmonella and clostridium perfringens are used as common pathogenic bacteria in diarrhea of piglets and young chickens, and antibacterial peptides which can inhibit the three bacteria and have good characteristics need to be developed and excavated. Can meet the requirement of the feed for preventing common diseases of animals.

Disclosure of Invention

The invention aims to provide a preparation method of a recombinant antibacterial peptide Trsub in Trichoderma reesei and antibacterial characteristics thereof, wherein the recombinant antibacterial peptide Trsub has antibacterial activity on common pathogenic bacteria in diarrhea of piglets and young chickens, and meets the requirements of heat resistance, pepsin stability and hemolytic activity of feed preparation and animal feed.

The invention is realized by the following technical scheme:

a recombinant antibacterial peptide Trsub, the amino acid sequence of which is shown in SEQ ID number 1; the method specifically comprises the following steps: GAGKAQCAAAWLQCASGGTLGCGGGAVACQNYRQFCR are provided.

The nucleotide sequence of the amino acid sequence coded by the SEQ ID number 1 is shown as SEQ ID number 2, the SEQ ID number 2 is a mutant obtained by translating the amino acid sequence into a coded nucleotide sequence on the basis of the antibacterial peptide subparcin 168 of the original amino acid sequence SEQ ID number 3, carrying out artificial synthesis on the nucleotide fragment, and carrying out error-prone PCR reaction on the artificially synthesized fragment.

The SEQ ID number 2 is ggagctggaaaggctcaatgtgctgctgcttggttgcaatgtgcttcaggaggtactttgggatgtggaggaggagctgttgcttgtcaaaactacagacaattttgtaga.

SEQ ID NO. 3：GLGKAQCAALWLQCASGGTIGCGGGAVAC QNYRQFCR。

The invention also provides a trichoderma recombinant expression vector containing the antibacterial peptide gene.

The invention also provides a recombinant strain containing the antibacterial peptide, wherein the strain is Trichoderma reesei Tu 6.

A preparation method of a recombinant antibacterial peptide Trsub specifically comprises the following steps: amplifying a gene fragment of the antimicrobial peptide Sublancin168 through an error-prone PCR reaction, detecting the error-prone PCR fragment by agarose gel electrophoresis, inserting the error-prone PCR fragment into a trichoderma expression vector through in vitro homologous recombination, and constructing a recombinant expression vector of the recombinant antimicrobial peptide Trsub; and (3) transferring the recombinant expression vector into trichoderma reesei Tu6 by adopting a polyethylene glycol mediated protoplast transformation method, screening a positive transformant and verifying the positive transformant, and then obtaining the recombinant antibacterial peptide Trsub through fermentation expression.

An application of a recombinant antibacterial peptide Trsub in preparing feed and feed additives.

Compared with the prior art, the invention has the beneficial effects that:

a recombinant antibacterial peptide Trsub is obtained by an error-prone PCR method, and a recombinant expression vector mutation library of the Trsub in Trichoderma reesei is constructed.

The invention heterologously expresses the recombinant antibacterial peptide Trsub in Trichoderma reesei Tu6 to obtain an expression product of the recombinant antibacterial peptide Trsub, and provides a preparation method of the recombinant antibacterial peptide Trsub in Trichoderma reesei Tu 6.

The invention represents the antibacterial activity of the expression product of the recombinant antibacterial peptide Trsub, and the expression product has inhibition effect on escherichia coli, salmonella and clostridium perfringens. Compared with the original sequence antibacterial peptide, the recombinant antibacterial peptide Trsub has stronger antibacterial activity to gram-negative bacteria.

The recombinant antibacterial peptide Trsub has good heat stability, acid resistance, pepsin resistance and low hemolytic activity, and the characteristics are favorable for application in feeds and feed additives.

Drawings

FIG. 1 is a schematic diagram of gene amplification nucleic acid electrophoresis of a recombinant antimicrobial peptide Trsub;

FIG. 2 is a schematic diagram of the construction of a recombinant expression vector PCBHG-Trsub;

FIG. 3 is a schematic diagram of protein electrophoresis detection of an expression product and a purified product of the recombinant antibacterial peptide Trsub;

FIG. 4 is a schematic diagram showing the thermostability of the recombinant antimicrobial peptide Trsub;

FIG. 5 is a schematic representation of pepsin stability for the recombinant antimicrobial peptide Trsub;

FIG. 6 is a schematic diagram showing the hemolytic activity of the recombinant antimicrobial peptide Trsub on rabbit blood erythrocytes.

Detailed Description

Example 1 error-prone PCR of recombinant antimicrobial peptide Trsub Gene

Taking the amino acid sequence of the antibacterial peptide Sublancin168 published in GenBank: ACE07988.1 as an original sequence, translating the original sequence into a coded nucleotide sequence by using DNAMAN software, and artificially synthesizing the nucleotide fragment. The artificially synthesized fragment was subjected to error-prone PCR using PrimeSTAR as a PCR amplification enzyme^®High fidelity amplification enzyme, PCR reaction system (50 μ L) was: 5 × PrimeStar^® Buffer 10 μL；dNTP 4 μL；PrimeSTAR^® 0.5 mu L; pf 2. mu.L; pr 2. mu.L; synthesizing 1 mu L of template; ddH₂O30.5. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 sec, annealing at 56 ℃ for 30 sec, elongation at 72 ℃ for 1 min, and 35 cycles; total extension at 72 ℃ for 10 min; 4 ℃ is prepared. After the PCR reaction is completed, agarose gel electrophoresis is performed to detect a target band amplified by the gene of the antimicrobial peptide Trsub, as shown in FIG. 1. The verified PCR product was subjected to template digestion using DpnI enzyme in a digestion system of 1. mu.L of DpnI and 5. mu.L of Fast Digest Buffer, and reacted at 37 ℃ for 2 h. And (3) recovering and purifying the PCR digestion product by using a Cycle Pure Kit PCR purification Kit, wherein the recovered product is an antibacterial peptide Sublancin168 error-prone PCR gene fragment.

Example 2 insertion of error-prone PCR product of antimicrobial peptide Sublancin168 Gene into Trichoderma Tu6 expression vector to construct recombinant expression vector Sub168-PCBHG

Linearized primer pair F using trichoderma Tu6 vector: 5'-gctccgtggcgaaagcct-3' and R: 5'-agcacgagctgtggccaag-3' was linearized by PCR using the enzyme Phanta^®Super-Fidelity DNA Polymerase, PCR reaction system (50. mu.L) was: 5 × SF Buffer 25 μ L; dNTP 1 u L; phanta^® 0.5 mu L; pf 2. mu.L; pr 2. mu.L; the carrier PCBHG1 μ L; ddH₂O mu L; 18.5. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 sec, annealing at 58 ℃ for 30 sec, extension at 72 ℃ for 8 min, and 35 cycles; total extension at 72 ℃ for 10 min; 4 ℃ is prepared. After the nucleic acid electrophoresis test, the template digestion is carried out and the Cycle Pure Kit PCR purification Kit is used for recovery and purification, thus obtaining the linearized vector recovery fragment. The recovered sheet in example 1 was subjected toThe fragment and the linearized vector are subjected to in vitro homologous recombination and connection, the used homologous recombination ligase is Exnase II, and a connection system (50 mu L) is as follows: CE II Buffer 2 μ L; 0.5 mu L of Exnase II; 0.5 mu L of linearized vector; sublancin168 error-prone PCR gene 1. mu.L; ddH₂O1. mu.L. The connection conditions are as follows: 30 min at 37 ℃. And then transforming the homologous recombinant ligation product into escherichia coli DH5 alpha, culturing for 14-16 h at 37 ℃ after coating, picking a single colony for colony PCR and sequencing verification, and completing construction of a recombinant expression vector Sub168-PCBHG after comparison, wherein the composition schematic is shown in figure 2.

Example 3 transformation of Trichoderma recombinant expression vector for antimicrobial peptide Trsub

Subculturing the Trichoderma reesei host strain on a PDA + U solid culture medium plate under the condition of 30 ℃ for 5-6 days. The mature Trichoderma reesei host strain is inoculated to YEG + U culture medium and cultured for 20 h at 30 ℃ and 180 rpm as an expression host. Filtering and culturing mature bacteria liquid, collecting mycelium on filter cloth to sterilized conical flask, adding lyase, cracking at 30 deg.C and 90 rpm for 2 hr, sampling in sterile workbench every 30 min, observing morphology and number of protoplast generated by cracking with blood counting plate under microscope until reaching 10⁸Cleavage was stopped at CFU/mL. The lysate in the Erlenmeyer flask was filtered and centrifuged, washed repeatedly with 1M sorbitol solution and resuspended 3 times, and the final protoplast pellet was collected.

Example 4 fermentative expression of the antimicrobial peptide Trsub in Trichoderma

The recombinant Plasmid constructed in example 2 was subjected to Plasmid extraction using an e.z.n.a. Plasmid Mini Kit i Kit, the extracted recombinant Plasmid was transferred to and mixed with the prepared trichoderma protoplast, and the mixture was added with a polyethylene glycol solution and incubated on ice for 30 min, followed by culture in a PDA solid medium at 30 ℃ for 5-6 days.

Selecting transformants growing in PDA solid culture medium to new plate for screening, performing genome extraction after hypha and spore grow out and verifying target gene, inoculating screened positive transformants to Trichoderma fermentation culture medium at 30 deg.C and 180 rpmThe lower fermentation expression is carried out for 5-6 days, and the temperature is adjusted to be low after the thalli obviously grow. Primarily screening the fermentation product in a 96-well plate, wherein the specific screening process comprises the following steps: primarily taking escherichia coli as an indicator bacterium, culturing the escherichia coli to a logarithmic growth phase, and diluting the escherichia coli to a bacterium concentration of 10 by using an LB liquid culture medium⁵ CFU/mL, adding 1 mL of diluted bacterial suspension into the equivalent antibacterial peptide fermentation product, placing a 96-well plate in a shaking table at 37 ℃ for about 14 h, observing the clarity by naked eyes and determining the OD_600nmThe OD is obtained by preliminary screening_600nmAnd screening a recombinant antibacterial peptide Trsub with a lower value and a clearer sample by visual observation, wherein the amino acid sequence of the recombinant antibacterial peptide Trsub is shown as SEQ ID number 1, and the coded nucleotide is shown as SEQ ID number 2. The products were filtered and collected, detected and analyzed using SDS-PAGE. The samples were selected for subsequent purification and protein concentration determination. SDS-PAGE detection is shown in FIG. 3.

Example 5 antimicrobial Activity assay of the antimicrobial peptide Trsub

Determination of Minimum Inhibitory Concentration (MIC)

The indicator bacteria used in this example were escherichia coli, salmonella, clostridium perfringens.

The specific determination method of MIC is a micro two-fold dilution method: culturing three kinds of bacteria with liquid culture medium to logarithmic phase of growth, collecting each thallus, diluting to thallus concentration of 10⁵CFU/mL is ready for use, and this step is the preparation of bacterial suspension. The purified and collected antibacterial peptide Trsub solution is mixed with the liquid culture medium of the bacterial suspension in the first row of a 96-well plate in equal quantity until the initial concentration of the antibacterial peptide sample is 200 mu g/mL, and the antibacterial peptide Trsub solution is continuously diluted twice in the subsequent rows to form a concentration gradient. Adding equal amount of diluted bacterial suspension into samples with concentration gradient of a 96-well plate, incubating for 16 h in an incubator at 37 ℃, and observing turbidity degree of each hole by naked eyes after culturing, wherein the minimum concentration for clarifying the holes is MIC of the antibacterial peptide Trsub to each bacterium. As shown in Table 1, the recombinant antibacterial peptide Trsub has better inhibitory activity to three bacteria, and compared with the original sequence antibacterial peptide, the recombinant antibacterial peptide Trsub has stronger inhibitory activity to gram-negative bacteria, the MIC to Escherichia coli is 37.5 mug/mL, and the original sequence antibacterial peptide Trsub hasThe antibacterial activity of the antibacterial peptide is more than 100 mu g/mL.

TABLE 1 minimum inhibitory and bactericidal concentrations of the recombinant antibacterial peptide Trsub for different bacteria

Bacterial species	MIC (μg/mL)	MBC (μg/mL)
			Gram-negative bacteria
Escherichia coli	37.5	37.5
			Salmonella	37.5	75
Gram-positive bacteria
			Clostridium perfringens	25	25

Determination of Minimum Bactericidal Concentration (MBC)

According to the results of the MIC determination process, the antibacterial peptide TrSub can make each bacterium present clear concentration, and the concentration of the antibacterial peptide TrSub can be respectively sampled and applied to the corresponding solid medium of each bacterium, and the colonies growing on the plate can be observed after culturing for 16 h, and the concentration of the sample capable of inhibiting 99.9% of the bacterium growth is the MBC of the antibacterial peptide TrSub for each bacterium, and the results are shown in table 1.

Example 6 thermostability assay of recombinant antimicrobial peptide Trsub

And respectively heating the recombinant antibacterial peptide Trsub sample in boiling water for 5 min, 10 min, 15 min, 20 min, 25 min and 30 min, wherein the sample subjected to heat treatment is used as a sample to be detected, and the sample not subjected to heat treatment is used as a reference. Culturing the three indicator bacteria to the logarithmic phase of growth and diluting to the bacteria concentration of 10⁵CFU/mL is ready for use. Mixing the antibacterial peptide samples subjected to heat treatment at different times with the diluted bacterial suspensions, incubating at 37 ℃ for 18 h, measuring the light absorption value at 600 nm, and calculating the inhibition rate of the samples subjected to heat treatment at different times to each bacterium by taking the samples not subjected to heat treatment as a control.

The heat stability results are shown in fig. 4, the inhibition ratios of the recombinant antimicrobial peptide TrSub to each bacterium after the high temperature treatment at different times are almost not changed, the inhibition ratios of the recombinant antimicrobial peptide TrSub to each bacterium after the treatment in boiling water for 20 min are almost not changed, and the inhibition ratios of the recombinant antimicrobial peptide TrSub to each bacterium after the treatment for 25 min can still reach about 80%, which indicates that the recombinant antimicrobial peptide TrSub has good heat stability and can endure the high temperature treatment process of the industrial processing process.

Example 7 pepsin stability assay for the recombinant antimicrobial peptide Trsub

And (3) carrying out digestive enzyme stability determination on the recombinant antibacterial peptide Trsub, and evaluating the resistance of the recombinant antibacterial peptide Trsub to digestive enzymes. The digestive enzyme used in this example was pepsin, which was diluted to 3000U/mL using Gly-HCl buffer at pH 2.0. The antimicrobial peptide Trsub solution was diluted to its MIC using a pepsin solution prepared by dilution, and the examples herein and below, which are not described, refer to the MIC for E.coli. And (3) placing the diluted antibacterial peptide Trsub solution at 37 ℃ and incubating for 30-180 min to serve as a sample to be detected. Mixing the sample to be tested with the three diluted bacterial suspensions, culturing at 37 ℃ for 18 h, measuring the light absorption value at 600 nm, and calculating the inhibition rate of the pepsin treatment on each bacterium after different time by taking the sample which is not treated by the pepsin as a control.

Through determination, the stability of the recombinant antibacterial peptide Trsub to pepsin is good, and the inhibition rate of the recombinant antibacterial peptide Trsub to various bacteria after being treated by pepsin can still keep a high level (more than 80 percent) (figure 5). The good resistance to pepsin means that the recombinant antimicrobial peptide Trsub has the potential to pass through the animal's stomach digestive tract and exert antimicrobial activity, which is the basis for its intended application as a feed additive.

Example 8 measurement of hemolytic Activity of recombinant antimicrobial peptide Trsub

The hemolytic activity assay of the recombinant antimicrobial peptide Trsub was performed using purchased rabbit blood erythrocytes. The rabbit blood cells were washed and resuspended 2-3 times with PBS solution, the final blood cells were collected and mixed with antimicrobial peptide TrSub solution in 96-well plates in duplicate serial dilutions, and incubated at 37 ℃ for 1 h. The incubated mixed sample was centrifuged at 1200 rpm for 10 min, the supernatant was collected and transferred to a new 96-well plate, and the absorbance was measured at 570 nm. In this example, 0.1% Triton X-100 was used as a positive control, and the hemolysis rate was 100%; the PBS solution was used as a negative control, and the hemolysis rate was 0. By formula definition, hemolysis rate (%) = (OD 570 nm of TrSub-OD 570 nm of PBS)/(OD 570 nm of 0.1% Triton X-100-OD 570 nm of PBS) × 100%.

The results are shown in fig. 6, and the hemolytic activity of the recombinant antimicrobial peptide TrSub at MIC concentration and 2MIC concentration is below 10%, which indicates that the screened recombinant antimicrobial peptide TrSub has low hemolytic activity, and the safety of the application is further improved due to the characteristic.

Sequence listing

<110> China oceanic university

<120> recombinant antibacterial peptide Trsub, preparation method and application thereof

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Gly Ala Gly Lys Ala Gln Cys Ala Ala Ala Trp Leu Gln Cys Ala Ser

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Gly Gly Thr Leu Gly Cys Gly Gly Gly Ala Val Ala Cys Gln Asn Tyr

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Arg Gln Phe Cys Arg

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ggatgtggag gaggagctgt tgcttgtcaa aactacagac aattttgtag a 111

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Gly Leu Gly Lys Ala Gln Cys Ala Ala Leu Trp Leu Gln Cys Ala Ser

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agcacgagct gtggccaag 19