Method for synthesizing hydrophis cyanocinctus antibacterial peptide Hc-CATH through multi-copy integration and high-density fermentation

Abstract

The invention provides a method for synthesizing a hydrophis cyanocinctus antibacterial peptide Hc-CATH through multi-copy integration and high-density fermentation, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a series-connection two-copy plasmid is constructed, pichia pastoris is taken as a host, two-copy series-connection protein is successfully expressed, and the expression yield of the hydrophis cyanocinctus antibacterial peptide Hc-CATH is improved by 2-3 times. According to the present invention, the hydrophis cyanocinctus antibacterial peptide Hc-CATH can be synthesized at a high level through the construction and optimization of the two-copy integration method and the optimization of the fermentation process, and the method has advantages of strong versatility, high expression efficiency, biological activity, low cost, easy large-scale production, and the like, and can be used for the preparation of the hydrophis cyanocinctus antibacterial peptide Hc-CATH.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a method for synthesizing the antimicrobial peptide Hc-CATH of Sea Snake through multi-copy integration and high-density fermentation. Background Art

[0002] The antimicrobial peptide Hc-CATH is isolated from the sea snake ( Hydrophis cyanocinctus ) is a cathelicidin family antimicrobial peptide amplified in the venom gland of a marine reptile (GenBank accession number: KP202856). It is the first cathelicidin family antimicrobial peptide discovered from marine reptiles. In vitro and in vivo experiments have shown that Hc-CATH has excellent antimicrobial and anti-inflammatory activities. Mechanistic studies have found that it can neutralize lipopolysaccharide (LPS) and thus inhibit the occurrence of inflammatory signaling pathways. Hc-CATH is composed of 30 amino acid residues, and its active structure is mainly an amphipathic α-helical structure located in the 1-17 amino acid sequence.

[0003] At present, whether the antimicrobial peptide Hc-CATH of the sea snake is synthesized by chemical synthesis or biosynthesis, the yield of the antimicrobial peptide Hc-CATH obtained by the sea snake cannot meet the needs of actual industrial use. The present invention uses the antimicrobial peptide gene of the sea snake, 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 the antimicrobial peptide of the sea snake. 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 fermentation synthesis of the sea snake antimicrobial peptide Hc-CATH, thereby increasing the yield of the sea snake antimicrobial peptide Hc-CATH.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] 1) The complete gene of the antimicrobial peptide Hc-CATH from the sea snake was synthesized and inserted into the pPICZαA vector, and a single-copy Hc-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 Hc-CATH; 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] 1) Transfer the recombinant expression vector into Pichia pastoris and screen the recombinant yeast expression bacteria.

[0009] 2) Induce expression in Pichia pastoris with successful homologous recombination to obtain two copies of the sea snake antimicrobial peptide Hc-CATH 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 Hc-CATH sequence of the sea snake antimicrobial peptide 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 Hc-CATH gene of the sea snake can be the nucleotide sequence of any gene; the nucleotide sequence is the nucleotide sequence optimized by the codon preference of Pichia pastoris.

[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 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 antimicrobial peptide Hc-CATH of the sea snake. The fermentation broth protein is subjected to Ni-NTA agarose resin affinity chromatography, and then cleaved by a corresponding protease, and the obtained antimicrobial peptide Hc-CATH of the sea snake is purified.

[0015] The beneficial effects of the present invention are: (1) When the present invention uses yeast to express the antimicrobial peptide Hc-CATH of the sea snake, the expression yield of the antimicrobial peptide Hc-CATH of the sea snake is increased by more than 2 times by connecting two copies in series; then the fermentation medium is optimized to further increase the yield of the pure product of the antimicrobial peptide Hc-CATH of the sea snake.

[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 large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the complete synthesis of the single-copy gene of the antimicrobial peptide of the blue sea snake.

[0018] Figure 2 A schematic diagram for the construction of a two-copy vector.

[0019] Figure 3 The inhibition zones are for the antibacterial effect on Escherichia coli. The left picture shows the antibacterial result of a single copy of the antimicrobial peptide Hc-CATH from the sea snake, and the right picture shows the antimicrobial result of two copies of the antimicrobial peptide Hc-CATH from the sea snake. Numbers 1-5 represent 5 groups of fermentation tanks, and the fermentation induction expression culture time is 96 hours.

[0020] Figure 4 The left picture shows the inhibition zone against Staphylococcus aureus. The left picture shows the inhibition result of single-copy antimicrobial peptide Hc-CATH from sea snake, and the right picture shows the inhibition result of two-copy antimicrobial peptide Hc-CATH from sea snake. Numbers 1-5 represent 5 groups of fermentation tank numbers, and the fermentation induction expression culture time is 96h. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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

[0028] Total synthesis of a single copy gene of an antimicrobial peptide from the sea snake Hylocereus cyanobacteria According to the existing amino acid sequence of the antimicrobial peptide of the sea snake, the codon of the antimicrobial peptide of the sea snake was optimized according to the preference of Pichia pastoris for genetic codons, and the whole gene was synthesized and inserted into the pPICZαA vector.

[0029] AAG TTC TTC AGA AAG TTG TTG AAG TCT GTT AGA AGA GCT GTT AAG AAG TTCAGA AAG AAG CCA AGA TTG ATT GGT TTG TCT ACT TTG TTG The specific operation is to delete all the sequences from the KEX2 restriction site to TGA of the pPICZαA vector, replace them with the antimicrobial peptide gene of the sea snake with optimized codon preference, add TAA at the end, and then connect the sequence after TGA of the vector. Figure 1 shown. Example

[0030] Construction of two-copy vector 1) Using two sets of restriction endonucleases Bgl II- Age I and Xcm I- BmI double-digested the Hc-CATH recombinant plasmid, and recovered the target gene fragment after digestion by gel cutting to obtain two gene fragments containing the antimicrobial peptide Hc-CATH; 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 DH5α competent cells were plated on LB agar plates containing bleomycin (100 μg / mL), cultured at 37°C for 12 h, and single colonies were picked. PCR was performed using the upstream primer 5′AOX primer and the downstream primer 3′AOX primer. The positive clones were then sent to Bio-Tech 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

[0031] Inducible expression and detection of recombinant yeast Mix 80 μL of yeast competent cells and 5 μg of Sac I linearized plasmid and transfer to an electroporation cup and place on ice for 5 minutes. Immediately after electroporation, add 200 μL of sorbitol medium to the electroporation cup, mix well, and then spread all the bacterial liquid on a YPD agar plate containing bleomycin (100 μg / mL) and culture at 28°C 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 0.5%. 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

[0032] Comparison of two-copy expression yield and single-copy expression yield Different copy numbers of the antimicrobial peptide expression strains of the sea snake were fermented under the same conditions (using BMMY medium for fermentation, initial OD 600 The pH value was 2.0-6.0, the rotation speed was 280 rpm, and 0.5% methanol of the total amount of the culture medium was added every 24 hours for a total fermentation of 120 hours.

[0033] 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 antimicrobial peptide yield of different copy numbers of the blue ring sea snake is shown in the following table: Table 1 Comparison of the production of antimicrobial peptide Hc-CATH with different copy numbers in the sea snake Copy number 1 2 Yield 12mg / L 27mg / L Example

[0034] 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 600 The value is between 0.4 and 0.6. Take 30 μL of bacterial solution into 100 mL LB solid medium, mix well and pour into a plate; after solidification, put the Oxford cup on the surface, add 50 μL of recombinantly expressed antimicrobial peptide samples of sea snakes, and use pPICZαA empty vector as a blank control group. After overnight culture at 37°C, measure the size of the transparent circle. A total of 3 groups of experiments were performed. It was concluded that the recombinantly expressed antimicrobial peptide Hc-CATH of sea snakes has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0035] 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 synthesizing the antimicrobial peptide Hc-CATH by multi-copy integration and high-density fermentation, mainly comprising the following steps: Step 1: Construction of multi-copy expression vector of Hc-CATH antimicrobial peptide ① The whole gene of the antimicrobial peptide Hc-CATH of the sea snake was synthesized and inserted into the pPICZαA vector, and a single-copy Hc-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 Hc-CATH; at the same time, the pPICZαA plasmid was double-digested 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; Step 2: Preparation of recombinant Hc-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, thereby obtaining two copies of the sea snake antimicrobial peptide Hc-CATH protein for secretory expression; ③ Two copies of the antimicrobial peptide Hc-CATH protein of the sea snake were fermented, and the fermentation supernatant was purified by HPLC and then identified by mass spectrometry to obtain the pure product of the antimicrobial peptide Hc-CATH protein of the sea snake.

2. The method for synthesizing the antimicrobial peptide Hc-CATH from sea snakes by multi-copy integration and high-density fermentation according to claim 1, characterized in that: During the fermentation process in step ③ of step 2, 0.5% methanol of the total amount of the culture medium is added every 24 hours.

3. Use of the antimicrobial peptide Hc-CATH obtained by the method of multi-copy integration and high-density fermentation synthesis of the antimicrobial peptide Hc-CATH according to claim 1 in the preparation of drugs for inhibiting Escherichia coli and Staphylococcus aureus.

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