Method for synthesizing cobra antibacterial peptide NA30-CATH through multi-copy integration and high-density fermentation and application of cobra antibacterial peptide NA30-CATH
By constructing two copies of plasmids in tandem and expressing them in Pichia yeast, the problem of insufficient yield of cobra antimicrobial peptide NA30-CATH in the prior art was solved, and efficient and low-cost expression and production were achieved.
Patent Information
- Application Number
- CN202311219115.5
- 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 cobra antimicrobial peptide NA30-CATH that is unable to meet industrial needs is the production of cobra antimicrobial peptide, and its synthesis method is complex and costly.
Through genetic engineering technology, two copies of the plasmid were constructed, and Pichia cerevisiae was used as the host to optimize the fermentation process to achieve high-level synthesis of cobra antimicrobial peptide NA30-CATH.
The expression yield of cobra antimicrobial peptide NA30-CATH has been increased to 2-3 times, reducing production costs, and simplifying the process flow, which is suitable for large-scale production.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] The present invention relates to the field of gene engineering technology, and in particular to a method for synthesizing cobra antimicrobial peptide NA30-CATH by multi-copy integration and high-density fermentation and an application thereof. Background Art
[0002] NA30-CATH is an antimicrobial peptide found in the venom of cobra (Naja naja atra). It has strong antimicrobial activity against a variety of bacteria in the presence of 1% NaCl. At the same time, it has no hemolytic activity against human red blood cells at high concentrations. It has gradually become one of the most promising new antimicrobial drugs and is widely used in research and development in the fields of medicine, neurology, etc.
[0003] At present, whether synthesizing NA30-CATH by chemical synthesis or biosynthesis, the yield of NA30-CATH obtained cannot meet the needs of actual industrial use. The present invention uses the NA30-CATH gene, connects it by enzyme digestion to form a multi-copy gene, constructs a recombinant plasmid for yeast expression, and establishes a yeast expression system for obtaining NA30-CATH. 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 the cobra antimicrobial peptide NA30-CATH.
[0005] To achieve the above object, the present invention adopts the following technical solution:
[0006] A. Construction of multi-copy expression vector of recombinant cobra antimicrobial peptide NA30-CATH
[0007] 1) The whole gene of cobra antimicrobial peptide NA30-CATH was synthesized and inserted into the pPICZαA vector, and a single-copy NA30-CATH-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 NA30-CATH; at the same time, the pPICZαA plasmid was double-digested and the linearized plasmid after digestion was recovered.
[0008] 2) Using T4 ligase, the target fragments obtained after double digestion were ligated, and then the ligation product was ligated with the pPICZαA plasmid recovered after double digestion to obtain a recombinant expression vector with multiple copies, and the ligation product was identified.
[0009] B. Preparation of recombinant cobra antimicrobial peptide NA30-CATH strain
[0010] 3) The recombinant expression vector is transferred into Pichia pastoris and the recombinant yeast expression bacteria are screened.
[0011] 4) Inducing expression of Pichia pastoris with successful homologous recombination to obtain two copies of the secretory cobra antimicrobial peptide NA30-CATH protein.
[0012] Preferably, the construction of the pPICZαA expression vector comprises the following contents: enzyme digestion of the pPICZαA vector and recovery of the digestion product; connection of the cobra antimicrobial peptide NA30-CATH sequence and the vector; and identification of the connection product.
[0013] Preferably, the identification of the ligation product includes preparation of competent cells, transformation reaction, extraction and identification of recombinant plasmids.
[0014] The nucleotide sequence of the cobra antimicrobial peptide NA30-CATH gene can be the nucleotide sequence of any gene; the nucleotide sequence is a nucleotide sequence optimized by Pichia pastoris codon preference.
[0015] 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.
[0016] The present invention linearizes and electrotransforms the plasmid identified by sequencing into X33 Pichia pastoris competent cells, spreads the transformed cells on a YPD plate, selects a single clone, performs small-scale fermentation culture on the clone, separates the fermentation broth by SDS-PAGE, and obtains a fermentation broth protein expressing the cobra antimicrobial peptide NA30-CATH. The fermentation broth protein is subjected to Ni-NTA agarose resin affinity chromatography, and then cleaved by a corresponding protease, and the obtained cobra antimicrobial peptide NA30-CATH is purified.
[0017] The present invention also provides an application of the cobra antimicrobial peptide NA30-CATH, specifically the application of the cobra antimicrobial peptide NA30-CATH in the preparation of aquatic antibacterial drugs, especially in the process of largemouth bass breeding.
[0018] The beneficial effects of the present invention are:
[0019] (1) When the present invention uses yeast to express the cobra antimicrobial peptide NA30-CATH, the expression yield of the cobra antimicrobial peptide NA30-CATH is increased by 2-3 times by connecting two copies in series.
[0020] (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 large-scale production, and can be used as an aquaculture feed additive.
[0021] (3) Compared with the single copy expression of cobra antimicrobial peptide NA30-CATH, the present invention increases the yield of cobra antimicrobial peptide NA30-CATH by 2-3 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
[0022] Figure 1 This is the complete synthesis diagram of the single copy gene of cobra antimicrobial peptide.
[0023] Figure 2 A schematic diagram for the construction of a two-copy vector.
[0024] Figure 3 This is the result of the antibacterial activity experiment, which shows the antibacterial results of the cobra antimicrobial peptide NA30-CATH against Escherichia coli (E. coli). The left figure shows the antibacterial results of single-copy NA30-CATH, and the right figure shows the antibacterial results of two-copy NA30-CATH. Numbers 1-6 represent 6 groups of fermentation tank numbers, and the fermentation induction expression culture time is 96 hours.
[0025] Figure 4 This is the result of the antibacterial activity experiment, showing the antibacterial results of the cobra antimicrobial peptide NA30-CATH against Staphylococcus aureus (S. aureus). The left figure shows the antibacterial results of single-copy NA30-CATH, and the right figure shows the antibacterial results of two-copy NA30-CATH. DETAILED DESCRIPTION
[0026] 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.
[0027] Peptone, ammonium sulfate, methanol, agar, glucose, potassium phosphate buffer, glycerol, and sorbitol were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0028] YNB was purchased from Beijing Solable Technology Co., Ltd.;
[0029] Yeast extract was purchased from OXOID Company;
[0030] The bacterial strains were purchased from China General Microbiological Culture Collection Center.
[0031] 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.
[0032] The YPD medium of the present invention is prepared as follows: 1% yeast extract, 2% peptone, 2% glucose, and 2% agar powder is added to the solid medium.
[0033] Preparation of BMMY culture medium in the present invention:
[0034] 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.
[0035] Preparation of BMGY medium:
[0036] 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.
[0037] Preparation of sorbitol culture medium
[0038] 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.
[0039] Example 1
[0040] 1. Total synthesis of single-copy gene of cobra antimicrobial peptide
[0041] According to the existing amino acid sequence of cobra antimicrobial peptide and the preference of Pichia pastoris for genetic codons, the cobra antimicrobial peptide gene was codon optimized, and the whole gene was synthesized and inserted into the pPICZαA vector.
[0042] AAG TTC TTC AAG AAG TTG AAG AAC TCT GTT AGA AAG GCT AAG AAG TTC TTTAAG AAG CCA AAG GTT ATT GGT GTT ACT TTT CCA TTC
[0043] The specific operation is to delete all the sequences from the KEX2 restriction site to TGA of the pPICZαA vector, replace them with the cobra antimicrobial peptide gene after codon preference optimization, add TAA at the end, and then connect the sequence after TGA of the vector. Figure 1 shown.
[0044] Example 2
[0045] Construction of two-copy vector
[0046] 1) The NA30-CATH recombinant plasmid was double-digested with two groups of restriction endonucleases Bgl II-Age I and Xcm I-BamH I, and the target gene fragments after enzyme digestion were cut and recovered to obtain two gene fragments containing the antimicrobial peptide NA30-CATH; at the same time, the pPICZαA plasmid was double-digested with restriction endonucleases Xcm I and Age I, and the linearized plasmid after enzyme digestion was recovered;
[0047] 2) Using T4 ligase, the target fragment obtained after double digestion with Bgl II-Age I and Xcm I-BamH I was ligated at 16°C for 8h, and the target gene fragment obtained after ligation was ligated with the pPICZαA plasmid after double digestion with Xcm I and Age I at 16°C for 8h. The ligation product was transformed into E. coli DH5α competent cells, spread on LB agar plates containing bleomycin (100 μg / mL), and cultured at 37°C for 12h. After that, a single colony was picked, and the upstream primer 5′AOX primer and the downstream primer 3′AOX primer were used for bacterial liquid PCR. Then, the positive clones were sent to Sangon for DNA nucleotide sequencing;
[0048] 3) The strain containing the correctly sequenced recombinant plasmid was streaked and cultured, and a single clone was picked and added to LB medium, and cultured at 37°C and 180 rpm for 12 h, and then the recombinant plasmid was extracted using a kit.
[0049] Example 3
[0050] Inducible expression and detection of recombinant yeast
[0051] 1) Mix 80 μL of yeast competent cells and 5-10 μg (10 μL) of Sac I linearized plasmid and transfer to an electroporation cup and place on ice for 5 minutes. Immediately after electroporation at 1500V, 200Ω, 25μF, add 1 mL of sorbitol to the electroporation cup, mix well and let stand for 1 hour, add 1 mL of YPD liquid culture medium, shake at 230rpm and 29℃ for 1-2 hours, 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;
[0052] 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.
[0053] Example 4
[0054] Comparison of two-copy expression yield and single-copy expression yield
[0055] Different cobra antimicrobial peptide expression strains with different copy numbers were fermented under the same conditions (BMMY medium was used for fermentation, initial OD 600 The fermentation temperature 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.
[0056] 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 cobra antimicrobial peptides with different copy numbers is shown in the following table:
[0057] Table 1 Comparison of the production of NA30-CATH with different copy numbers
[0058] Copy number 1 2 Yield 19mg / L 29mg / L
[0059] Example 5
[0060] Escherichia coli and Staphylococcus aureus 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 600The value is between 0.4-0.6, take 30μL of bacterial solution into 100mL LB solid culture medium, mix well and pour into a plate; after solidification, put the Oxford cup on the surface, add 80μL of recombinant cobra antimicrobial peptide sample, and use pPICZαA empty vector as blank control group, and measure the size of its transparent circle after overnight culture at 37℃, and do 3 groups of experiments in total. It is concluded that the recombinant cobra antimicrobial peptide NA30-CATH has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus.
[0061] Example 6
[0062] Application of cobra antimicrobial peptide NA30-CATH in combating pathogenic bacteria of aquatic animals
[0063] (1) Enrofloxacin was selected as the control antibiotic to conduct a comparative study between the antimicrobial peptide preparation and enrofloxacin.
[0064] (2) Using largemouth bass as the breeding experiment object, comparing the antibacterial effects of antimicrobial peptides and enrofloxacin in resisting pathogenic bacteria of aquatic animals
[0065] In the breeding laboratory, 12 groups of 30 largemouth bass weighing 350 g ± 5 g were collected and placed in 12 1m 3 The experimental pools were divided into experimental group, blank control group, control group 1 and control group 2, each group consisted of 3 experimental pools. The fish were fed 3 times a day at 7:00, 10:00 and 16:00. After 7 days of feeding, the fish were stopped for 1 day and the challenge experiment was conducted. The challenge experiment was performed by intraperitoneal injection with 1.0×10 8 0.1mL of Aeromonas hydrophila solution with cfu / ml concentration.
[0066] After completing the challenge experiment, the experimental group was added with 4.3 mg / kg antimicrobial peptide preparation throughout the treatment course, with 7 days as one course of treatment. The blank control group did not add any antimicrobial peptides or antibiotics. Control group 1 was added with 0.5 mg / kg enrofloxacin throughout the treatment, and control group 2 was added with 5 mg / kg enrofloxacin throughout the treatment.
[0067] After the treatment, the survival rate of fish in each experimental pool was counted, and the results showed:
[0068]
[0069] The survival rate of the experimental group increased by 52.1% compared with the blank control group, the survival rate of the experimental group increased by 13.4% compared with the control group 1, and the survival rate of the experimental group increased by 8.0% compared with the control group 2.
[0070] 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. The method and application of synthesizing cobra antimicrobial peptide NA30-CATH by multi-copy integration and high-density fermentation mainly include the following steps: A. Construction of multi-copy expression vector of recombinant cobra antimicrobial peptide NA30-CATH ① The whole gene of the cobra antimicrobial peptide NA30-CATH was synthesized and inserted into the pPICZαA vector, and a single-copy NA30-CATH-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 NA30-CATH; the pPICZαA plasmid was double-digested at the same time, and the linearized plasmid after 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; B. Preparation of recombinant cobra antimicrobial peptide NA30-CATH strain ①Transfer the recombinant expression vector into Pichia pastoris and screen the recombinant yeast expression bacteria; ② Induce the expression of Pichia pastoris with successful homologous recombination to obtain two copies of the secreted cobra antimicrobial peptide NA30-CATH protein.
2. The use of the multi-copy integration and high-density fermentation method for synthesizing the cobra antimicrobial peptide NA30-CATH according to claim 1, characterized in that: Application of cobra antimicrobial peptide NA30-CATH in the preparation of aquatic antibacterial drugs.