Preparation method of two-copy perch antibacterial peptide wb-moronecidin
By constructing two copies of plasmids in Pichia yeast, the problem of insufficient yield of sea bass antimicrobial peptides in the prior art was solved, and the output was increased by more than twice, and the production cost was reduced, making it suitable for industrial production.
Patent Information
- Application Number
- CN202311219113.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-21
- Publication Date
- 2025-03-21
AI Technical Summary
The production of perch antibacterial peptide wb-moronecidin which is not able to meet industrial needs is especially in chemical synthesis and biosynthesis methods.
Through genetic engineering technology, two copies of plasmids were constructed and expressed in Pichia yeast, and the fermentation process was optimized to improve the high-level synthesis of sea bass antimicrobial peptides.
The production of sea bass antimicrobial peptides has been increased by more than 2 times, simplified the production process, reduced costs, and made it more suitable for industrial production.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for preparing a two-copy seabass antimicrobial peptide wb-moronecidin. Background Art
[0002] The sea bass antimicrobial peptide wb-moronecidin was originally isolated from hybrid striped bass ( Hybrid striped bass ) is an antimicrobial peptide isolated from the gills and skin of the oyster shell. It has strong salt tolerance and broad-spectrum activity against various microorganisms. It is composed of 22 amino acid sequences and has good antibacterial effects on a variety of pathogens such as Listeria monocytogenes and Aeromonas veronii.
[0003] At present, whether the seabass antimicrobial peptide wb-moronecidin is synthesized by chemical synthesis or biosynthesis, the yield of seabass antimicrobial peptide wb-moronecidin obtained cannot meet the needs of actual industrial use. The present invention uses seabass antimicrobial peptide genes, forms multi-copy genes by enzyme digestion and enzymatic ligation, constructs a recombinant plasmid for yeast expression, and establishes a yeast expression system for obtaining seabass antimicrobial peptides. Summary of the invention
[0004] The invention adopts genetic engineering technology to construct two copies of a plasmid in series, uses Pichia pastoris as a host, and optimizes the fermentation process to achieve high-level synthesis of seabass antimicrobial peptide wb-moronecidin, which is easy to realize industrialization.
[0005] To achieve the above object, the present invention adopts the following technical solution:
[0006] 1) The full gene of sea bass antimicrobial peptide wb-moronecidin was synthesized and inserted into the pPICZαA vector, and a single-copy wb-moronecidin-pPICZαA recombinant plasmid was successfully constructed. The single-copy recombinant plasmid was double-digested with two groups of restriction endonucleases to obtain two gene fragments containing the antimicrobial peptide wb-moronecidin; at the same time, the pPICZαA plasmid was double-digested and the linearized plasmid was recovered after enzyme digestion.
[0007] 2) Use T4 ligase to connect the target fragments obtained after double enzyme digestion, and then connect the ligation product with the pPICZαA plasmid recovered after double enzyme digestion to obtain a recombinant expression vector with multiple copies, and identify the ligation product.
[0008] 3) Transfer the recombinant expression vector into Pichia pastoris and screen the recombinant yeast expression bacteria.
[0009] 4) Induce the expression of Pichia pastoris with successful homologous recombination to obtain two copies of the sea bass antimicrobial peptide wb-moronecidin protein that is secreted and expressed.
[0010] Preferably, the construction of the pPICZαA expression vector includes the following contents: enzyme digestion of the pPICZαA vector and recovery of the digestion product; connection of the sea bass antimicrobial peptide wb-moronecidin sequence and the vector; and identification of the connection product.
[0011] Preferably, the identification of the ligation product includes preparation of competent cells, transformation reaction, extraction and identification of recombinant plasmids.
[0012] The nucleotide sequence of the sea bass antimicrobial peptide wb-moronecidin gene can be the nucleotide sequence of any gene; the nucleotide sequence is a nucleotide sequence optimized by Pichia pastoris codon preference.
[0013] The present invention selects Pichia pastoris as the host bacteria. Pichia pastoris has the characteristics of high-efficiency secretory expression and post-translational modification of proteins that are unique to eukaryotic organisms. Therefore, in recent years, Pichia pastoris has become a high-efficiency expression system for expressing foreign target proteins, especially foreign proteins of eukaryotic origin.
[0014] The present invention linearizes and digests the plasmid identified by sequencing, and then electrotransforms the plasmid into X33 Pichia pastoris competent cells. The transformed cells are coated on a YPD plate, and a single clone is selected. The clone is fermented and cultured in a small amount. The fermentation broth is separated by SDS-PAGE to obtain a fermentation broth protein expressing the sea bass antimicrobial peptide wb-moronecidin. The fermentation broth protein is subjected to Ni-NTA agarose resin affinity chromatography, and then cleaved by a corresponding protease, and the obtained sea bass antimicrobial peptide wb-moronecidin is purified.
[0015] The beneficial effects of the present invention are: (1) When the present invention uses yeast to express the sea bass antimicrobial peptide wb-moronecidin, the expression yield of the sea bass antimicrobial peptide wb-moronecidin is increased by more than 2 times by connecting two copies in series.
[0016] (2) The present invention adopts genetic engineering technology to construct a plasmid with two copies in series, and uses Pichia pastoris as a host to successfully express two copies of the tandem protein. The method provided by the present invention has the advantages of strong versatility, high expression efficiency, biological activity, low cost, and easy enterprise production.
[0017] (3) Compared with the single copy expression of sea bass antimicrobial peptide wb-moronecidin, the present invention increases the yield of sea bass antimicrobial peptide wb-moronecidin by more than 2 times without increasing the production process, and the production cost is basically the same as the cost of single copy expression. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the complete synthesis map of the single-copy gene of sea bass antimicrobial peptide.
[0019] Figure 2 A schematic diagram for the construction of a two-copy vector.
[0020] Figure 3 The graph shows the antibacterial activity experimental results against Listeria monocytogenes. Numbers 1-6 represent 6 groups of fermentation tank numbers, and the fermentation induction expression culture time is 96 hours. The left figure shows the antibacterial results of single-copy wb-moronecidin, and the right figure shows the antibacterial results of two-copy wb-moronecidin.
[0021] Figure 4 The graph shows the antibacterial activity experimental results against Aeromonas wb-moronecidin. Numbers 1-6 represent 6 groups of fermentation tank numbers, and the fermentation induced expression culture time is 96 hours. The left figure shows the antibacterial results of single-copy wb-moronecidin, and the right figure shows the antibacterial results of two-copy wb-moronecidin. DETAILED DESCRIPTION
[0022] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0023] Peptone, ammonium sulfate, methanol, agar, glucose, potassium phosphate buffer, glycerol, and sorbitol were purchased from Sinopharm Chemical Reagent Co., Ltd. YNB was purchased from Beijing Solable Technology Co., Ltd.; Yeast extract was purchased from OXOID Company; The bacterial strains were purchased from China General Microbiological Culture Collection Center.
[0024] Unless otherwise specified, the reagents used in the following examples of the present invention are all analytically pure reagents and can be purchased from conventional channels.
[0025] The YPD medium of the present invention is prepared by: 1% yeast extract, 2% peptone, 2% glucose, and 2% agar powder is added to the solid medium.
[0026] Preparation of BMMY culture medium in the present invention: Yeast extract 1%, peptone 2%, potassium phosphate buffer (pH=6.0) 100mmol / L, YNB 1.34%, ammonium sulfate 1%, add water to make 100ml culture medium. Pichia induction expression medium, YNB filter sterilization. During shake flask culture, add 0.5% methanol for induction every 24 hours, generally for 72 hours.
[0027] Preparation of BMGY medium: Yeast extract 1%, Peptone 2%, Potassium phosphate buffer (pH 6.0) 100mmol / L, YNB 1.34%, Ammonium sulfate 1%, Glycerol 1%, add water to prepare 100ml culture medium.
[0028] Preparation of sorbitol culture medium 18.217 g of sorbitol was dissolved in 100 ml of sterile water to prepare 1 M sorbitol medium, and the medium was sterilized by filtration through a 0.22 μm filter membrane. Example
[0029] 1. Total synthesis of a single copy gene of sea bass antimicrobial peptide According to the existing amino acid sequence of sea bass antimicrobial peptide and the preference of Pichia pastoris for genetic codons, the sea bass antimicrobial peptide gene was codon optimized, and the whole gene was synthesized and inserted into the pPICZαA vector.
[0030] TTC TTC CAT CAC ATT TTC CGT GGA ATT GTT CAT GTC GGT AAG ACG ATC CATAAG CTT GTG ACT GGA The specific operation is to delete all the sequences from the KEX2 restriction site to TGA of the pPICZαA vector, replace them with the sea bass antimicrobial peptide gene after codon preference optimization, add TAA termination at the end, and then connect the sequence after TGA of the vector. Figure 1 shown. Example
[0031] Construction of two-copy vector 1) Using two sets of restriction endonucleases Bgl II- Age I and Xcm I- BmI double-digested the wb-moronecidin recombinant plasmid, and recovered the target gene fragment after digestion by gel cutting to obtain two gene fragments containing the antimicrobial peptide wb-moronecidin; at the same time, using restriction endonucleases Xcm I and Age I. Double-digest the pPICZαA plasmid and recover the linearized plasmid after digestion; 2) Using T4 ligase, Bgl II- Age I and Xcm I- Bm The target fragment obtained after double digestion with I was ligated at 16°C for 8h. Xcm I and Age The pPICZαA plasmid after double restriction digestion was ligated at 16°C for 8 h and the ligation product was transformed into E. coli In DH5α competent cells, the cells were plated on LB agar plates containing bleomycin (100 μg / mL), cultured at 37°C for 12 h, and single colonies were picked. The upstream primer 5′AOX primer and the downstream primer 3′AOXprimer were used for bacterial liquid PCR, and then the positive clones were sent to Sangon for DNA nucleotide sequencing. 3) Streak the strain containing the correctly sequenced recombinant plasmid, pick out a single clone and add it to LB medium, shake and culture at 37℃ 180rpm for 12h, and then use the kit to extract the recombinant plasmid. Example
[0032] Inducible expression and detection of recombinant yeast Mix 80 μL of yeast competent cells and 5-10μg (10μL) of linearized plasmid and transfer to an electroporation cup, and place on ice for 5 minutes. Immediately after electroporation at 2000V, 200Ω, 25μF, add 1mL of sorbitol to the electroporation cup, mix well, let stand for 1h, add 1mL of YPD liquid culture medium, shake at 230rpm and 29℃ for 2-4h, enrich the cells and spread them on a YPD agar plate containing bleomycin (100 μg / mL), and culture at 28℃ for 3 days. Pick a single clone grown on the YPD plate to extract the yeast genome for PCR identification, and screen the Mut+ recombinant yeast expression strain; 2) Pick a single colony of the Mut+ recombinant yeast expression strain and inoculate it into BMGY medium. Cultivate the culture at 30°C and 280 rpm until the bacterial concentration reaches OD 600=2.0-6.0, the culture medium was replaced with BMMY medium, and methanol was added every 24 hours at a final concentration of 1%. After fermentation for 120 hours, the collected supernatant was analyzed by SDS-PAGE gel. The analysis results showed that the yeast successfully expressed the protein, and there was no significant difference in the amount of protein between different copy numbers. Example
[0033] Comparison of two-copy expression yield and single-copy expression yield Different copy numbers of sea bass antimicrobial peptide expression strains were fermented under the same conditions (BMMY medium was used for fermentation, initial OD 600 The pH value was 2.0-6.0, the rotation speed was 280 rpm, and 1% methanol of the total amount of the culture medium was added every 24 hours for a total fermentation of 120 hours.
[0034] Part of the supernatant was taken for SDS-PAGE detection to measure its protein yield. The fermentation supernatant was purified by HPLC and then identified by mass spectrometry to obtain a pure product, and its concentration was measured using a micro-BCA protein quantitative analysis kit. The comparison of the yield of sea bass antimicrobial peptides with different copy numbers is shown in the following table: Table 1 Comparison of the production of seabass antimicrobial peptide wb-moronecidin with different copy numbers Copy number 1 2 Yield 13mg / L 24mg / L Example
[0035] Listeria monocytogenes and Aeromonas vermifuge were cultured in 25 mL LB liquid medium at 37°C and 180 rpm overnight, then transferred to 25 mL LB liquid medium and cultured under the same conditions for 1.5 h-2 h. OD 600 The value was between 0.4 and 0.6. 30 μL of bacterial solution was added to 100 mL of LB solid culture medium, mixed and poured onto a plate. After solidification, the Oxford cup was placed on the surface, and 80 μL of recombinantly expressed sea bass antimicrobial peptide samples were added. At the same time, the pPICZαA empty vector was used as a blank control group. After overnight culture at 37°C, the size of the transparent circle was measured. A total of 3 groups of experiments were performed. It was found that the recombinantly expressed sea bass antimicrobial peptide wb-moronecidin had a significantly enhanced inhibitory effect on Listeria monocytogenes and Aeromonas veronii.
[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A method for preparing a two-copy seabass antimicrobial peptide wb-moronecidin, mainly comprising the following steps: ① According to the preference of Pichia pastoris for genetic codons, the codons of sea bass antimicrobial peptide wb-moronecidin were optimized, and the whole gene of sea bass antimicrobial peptide wb-moronecidin was synthesized and inserted into the pPICZαA vector, and a single-copy wb-moronecidin-pPICZαA recombinant plasmid was successfully constructed. The single-copy recombinant plasmid was double-digested with two groups of restriction endonucleases to obtain two gene fragments containing the antimicrobial peptide wb-moronecidin; at the same time, the pPICZαA plasmid was double-digested, and the linearized plasmid after enzyme digestion was recovered; ②Use T4 ligase to connect the target fragments obtained after double enzyme digestion, and then connect the ligation product with the pPICZαA plasmid recovered after double enzyme digestion to obtain a recombinant expression vector with multiple copies, and identify the ligation product; ③Transfer the recombinant expression vector into Pichia pastoris and screen the recombinant yeast expression bacteria; ④ Induce the expression of Pichia pastoris with successful homologous recombination, thereby obtaining two copies of the sea bass antimicrobial peptide wb-moronecidin protein for secretory expression; ⑤ The sea bass antimicrobial peptide wb-moronecidin protein was purified by HPLC and then identified by mass spectrometry to obtain the pure product of sea bass antimicrobial peptide wb-moronecidin protein.
2. Use of the seabass antimicrobial peptide wb-moronecidin obtained by the preparation method of a two-copy seabass antimicrobial peptide wb-moronecidin according to claim 1 in the preparation of drugs for inhibiting Listeria monocytogenes and Aeromonas veronii.