Obscure pufferfish antimicrobial peptide ToNK-lysin and its application

Abstract

The present invention discloses ToNK-lysin, an antimicrobial peptide from Takifugu obscurus, whose amino acid sequence is shown in SEQ ID NO. 1. The present invention utilizes in vitro recombinant expression technology to obtain ToNK-lysin, an antimicrobial peptide from Takifugu obscurus. This recombinant protein has no cytotoxic effect on pufferfish head kidney cells and can significantly eliminate Vibrio harveyi in pufferfish. Furthermore, the ToNK-lysin recombinant protein can degrade the genomes of Escherichia coli and Vibrio harveyi and significantly inhibit their growth. As an effective immune effector molecule, ToNK-lysin has potential applications in the development of antimicrobial drugs, new immune preparations, and feed additives.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and specifically relates to in vitro recombinant expression technology of the ToNK-lysin gene coding region of Takifugu obscurus, separation and purification of active recombinant protein, cytotoxic effect of recombinant protein, and antibacterial effect of recombinant protein on Escherichia coli and Vibrio harveyi. Background Art

[0002] Pufferfish, known for its white, tender flesh and delicious flavor, is highly valued by consumers and holds a high economic value. However, wild pufferfish resources are becoming increasingly depleted, and farmed pufferfish has become the primary source. The dusky pufferfish (Takifugu obscurus), known as one of the "Three Fresh Delicious Foods of the Yangtze River," is a high-quality fishery resource unique to the middle and lower reaches of the Yangtze River and a major freshwater farmed pufferfish species in China. However, in recent years, frequent diseases and a pressing need for quality improvement have severely hampered the healthy and sustainable development of the aquaculture industry.

[0003] NK-lysin is a structurally conserved antimicrobial peptide primarily secreted by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs). NK-lysin belongs to the saposin-like protein family (SAPLIP). Its N-terminal sequence contains six highly conserved cysteine ​​residues that can form three internal disulfide bonds. All SAPLIPs share a common fold, known as the saposin fold, which is associated with the antimicrobial activity of NK-lysin. Currently, the antimicrobial activity of NK-lysin has not been reported in Takifugu obscurus. Summary of the Invention

[0004] The present invention provides preparation and application of a recombinant protein of a Takifugu obscurus ToNK-lysin gene.

[0005] Primers containing restriction enzyme sites were designed at both ends of the ToNK-lysin gene coding region. The coding region gene was amplified by PCR technology and cloned into the pET-30a expression vector, and then transferred into the host cell Transetta (DE3) to achieve prokaryotic in vitro recombinant expression.

[0006] The amino acid sequence of the recombinant protein of the ToNK-lysin gene of Takifugu obscurus is shown below:

[0007] MPTSSILLLCILVTCSVWPVQARNLTVSTDDDDEHQGGFAIEAGRLPGVCWACKWALKKVKRIIGNSSTSQAIKAKLTSICDHIGLLKSLCRKFVTNHLGVLIEELTTSDDVRTSASTSGPASQRSWRSSPRVVFPHSHI

[0008] Length: 139 amino acids

[0009] Type: Amino Acid

[0010] Chain type: single chain

[0011] Characteristics: Molecular weight is 15.3kDa, isoelectric point is 9.05, and it has one conserved SapB domain.

[0012] The recombinant product was purified by nickel-agarose gel column and dialyzed for renaturation, showing the following activities:

[0013] The ToNK-lysin recombinant protein had no cytotoxic effect on pufferfish head kidney cells and could significantly eliminate Vibrio harveyi in pufferfish.

[0014] The ToNK-lysin recombinant protein can degrade the genomes of Escherichia coli and Vibrio harveyi and significantly inhibit their growth.

[0015] The advantages of the present invention are:

[0016] This study, using in vitro recombinant expression technology, produced the ToNK-lysin protein, an antimicrobial peptide from Takifugu obscurus. This recombinant protein exhibited no cytotoxicity against Takifugu head kidney cells and significantly eliminated Vibrio harveyi from the pufferfish. Furthermore, the ToNK-lysin recombinant protein was able to degrade the genomes of Escherichia coli and Vibrio harveyi and significantly inhibit their growth. As an effective immune effector molecule, ToNK-lysin has potential applications in the development of antimicrobial drugs, novel immune preparations, and feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 :SDS-PAGE detection of NK-lysin recombinant protein from Takifugu obscurus;

[0019] Figure 2: Cytotoxicity of pufferfish NK-lysin recombinant protein to pufferfish head kidney cells;

[0020] Figure 3 :In vivo pathogen clearance experiment of Takifugu obscurus NK-lysin recombinant protein;

[0021] Figure 4 :The effect of NK-lysin recombinant protein from Takifugu obscurus on the growth curves of Escherichia coli and Vibrio harveyi;

[0022] Figure 5 : Degradation of Escherichia coli genome by NK-lysin recombinant protein from Takifugu obscurus; 1. PBS + 10 μl genomic DNA; 2. 50 ng + 10 μl genomic DNA; 3. 100 ng + 10 μl genomic DNA; 4. 200 ng + 10 μl genomic DNA; 5. 250 ng + 10 μl genomic DNA; 6. 300 ng + 10 μl genomic DNA; 7. 10 μl genomic DNA;

[0023] Figure 6 : Degradation of Vibrio harveyi genome by NK-lysin recombinant protein from Takifugu obscurus, including 1. PBS+10μl genomic DNA; 2. 50ng+10μl genomic DNA; 3. 100ng+10μl genomic DNA; 4. 200ng+10μl genomic DNA; 5. 250ng+10μl genomic DNA; 6. 300ng+10μl genomic DNA; 7. 10μl genomic DNA. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0025] Example 1

[0026] The in vitro prokaryotic recombinant expression of the coding region of the Takifugu obscurus NK-lysin (ToNK-lysin) gene comprises the following steps:

[0027] 1. Construction of recombinant vector

[0028] The recombinant vector used in this example is the pET-30a prokaryotic expression vector. PCR technology was used to use gene-specific primers with Kpn I and Xho 1 specific restriction sites added to the 5' end:

[0029] P1(5'-CGGGGTACCATGCCAACCTCCTCGATCCT-3')

[0030] P2(5'-CCGCTCGAGGTGAGAGTGAGGAAAAACCACTC-3')

[0031] The reaction conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 10 minutes. The amplified fragment was purified and recovered, then ligated into the pMD19-T vector. After transformation, positive clones were screened and the plasmids were extracted. The plasmids were double-digested with restriction endonucleases Kpn I and Xho 1, and the resulting target fragments were purified and recovered using a gel recovery kit (Dalian Bao Bioengineering Co., Ltd.). The recovered target fragments were ligated into the expression vector pET-30a, which had been digested with both Kpn I and Xho 1 restriction endonucleases, completing the construction of the expression vector. Specific experimental methods are described in the third edition of Molecular Cloning.

[0032] 2. Expression of Recombinant Protein ToNK-lysin

[0033] Transform the constructed recombinant vector into the expression host bacteria E. coli Transetta (DE3), screen the positive clones, and confirm the correctness of the expression frame by sequencing. Pick a single clone and inoculate it into 400mL of LB liquid culture medium, 220rpm, 37℃ and culture for 4 hours (OD 600 = 0.4-0.8). After adding IPTG (1mmol / L), continue to culture at 22℃ for 12 hours. Centrifuge at 4℃, 12000g for 10 minutes, collect the bacterial precipitate, and freeze it at -20℃ for later use. Centrifuge 3mL of bacterial solution, discard the supernatant, and add 200μL BeyoLytic TM A small amount of soluble protein was extracted using a bacterial active protein extraction reagent (Biyuntian). After centrifugation again, the supernatant was collected and an appropriate amount of protein loading buffer (4:1) was added. The mixture was boiled at 100°C for 10 minutes, centrifuged briefly, and the supernatant was collected for SDS-PAGE analysis of the expressed product.

[0034] 3. Purification and refolding of recombinant protein ToNK-lysin

[0035] The recombinant protein ToNK-lysin in the present invention is a soluble recombinant protein, which is purified using a nickel-agarose gel FF column. The specific steps are as follows:

[0036] Purification and separation of His-tagged recombinant protein using nickel-agarose column

[0037] The steps for purifying soluble recombinant protein are as follows:

[0038] (1) Nickel Sepharose FF column, 1.6 × 20 cm, bed volume 10 mL;

[0039] (2) Equilibrate 2–5 bed volumes with lysis buffer (10 mM NaH2PO4·2H2O, 40 mM Na2HPO4·12H2O, 30 mM NaCl, pH = 7.4–8.0) at a flow rate of 2 mL / min;

[0040] (3) Add 20 ml of bacterial supernatant (using BeyoLytic TM A large amount of soluble protein was extracted using bacterial active protein extraction reagent. After centrifugation, the supernatant was collected and filtered through a 0.45 μm filter membrane and placed in a column at a flow rate of 1 mL / min.

[0041] (4) Add an appropriate amount of lysis buffer (4 times the amount of bacterial supernatant) to equilibrate the nickel column at a flow rate of 6 mL / min;

[0042] (5) Add 8 times the column volume of rinse buffer (1 L lysate + 50 mM imidazole) to rinse the affinity column and elute the impurities at a flow rate of 6 mL / min;

[0043] (6) Add 50 ml (5 times the column volume) of eluent (1 L lysis buffer + 200 mM imidazole) to elute and collect the target protein;

[0044] (7) Wash the column with pure water for 5 column volumes and then with 20% ethanol for 3 column volumes at a flow rate of 2 mL / min. Store the column in a refrigerator at 4°C.

[0045] (8) Detect the expression of fusion protein by SDS-PAGE;

[0046] The purified recombinant protein needs to be dialyzed in pure water, each time at 4°C for 12 hours, repeated 3 times, to obtain the recombinant protein of the obscure puffer NK-lysin (ToNK-lysin) gene.

[0047] Example 2

[0048] The cytotoxicity test of the recombinant protein NK-lysin from Takifugu obscurus is as follows:

[0049] 1. Collection of Takifugu obscurus head kidney cells

[0050] Dissect the obscure pufferfish kidneys and wash them repeatedly with 1x PBS. Mince the kidneys with sterilized scissors and place them in a culture dish containing trypsin for digestion. After 30 minutes, filter the cell suspension through a 300-mesh cell sieve and centrifuge at 300 x g for 10 minutes to collect the cells.

[0051] 2. Cytotoxicity assay

[0052] The collected cells were cultured in a modified cell culture medium M199 (containing Earle's mixed salts, 25mM HEPES, 100mg / L L-glutamine, 1000mg / L D-glucose, 2200mg / L sodium bicarbonate, pH=7.0-7.4) supplemented with double antibodies (0.1% penicillin (100mg / L) and 0.1% streptomycin (100mg / L)). After culturing for 24 hours, fresh culture medium was replaced, and the cells were distributed into 96-well plates and cultured for another 24 hours. After replacing the fresh culture medium again, the OD490 nm absorbance value was measured using a spectrophotometer. Then, ToNK-lysin recombinant protein was added at concentrations of 0, 1, 5, 10, 20, 50 and 100 μg / mL in sequence, with three replicates for each concentration. After continuing to culture for 24 hours, the OD490 nm absorbance value was measured again using a spectrophotometer.

[0053] Example 3

[0054] The specific steps of the bacteria clearance detection of the obscured pufferfish recombinant protein NK-lysin of the present invention are as follows:

[0055] 1. Bacterial treatment

[0056] Six healthy Takifugu obscurus were selected and injected intraperitoneally with 100 μl of Vibrio harveyi (1×10 6 CFU / mL) in pufferfish. Thirty minutes later, six pufferfish were divided into a bacteria + NK-lysin protein treatment group and a bacteria treatment group. In the bacteria + NK-lysin protein treatment group, each fish was injected with 20 μg / g body weight of ToNK-lysin recombinant protein, while the bacteria treatment group was injected with an equal volume of PBS.

[0057] 2. Bacteria removal test

[0058] After 24 hours, the obscure pufferfish were anesthetized and killed. The head kidney and liver tissues were collected separately in a sterile environment, washed twice with 1 ml of sterile 1× PBS buffer, and then homogenized using a tissue homogenizer. The homogenate was diluted 10-fold and spread on agar plates. After overnight incubation, the number of colonies was counted. Each experiment was divided into three replicates and repeated twice. The results showed that the recombinant ToNK-lysin protein significantly eliminated Vibrio harveyi from the head kidney and liver tissues of pufferfish.

[0059] Example 4

[0060] The specific steps for detecting the antibacterial activity of the obscure pufferfish recombinant protein NK-lysin of the present invention are as follows:

[0061] 1. Cultivation and Preparation of Microorganisms

[0062] Escherichia coli (E. coli) was purchased from Invitrogen and inoculated into LB liquid medium, placed in a 37°C constant temperature shaker, and cultured at 220 rpm for 12 hours. Vibrio harveyi (V. harveyi) was isolated from spotted catfish by our laboratory staff and inoculated into 2% salt LB liquid medium, placed in a 30°C constant temperature shaker, and cultured at 220 rpm for 12 hours. E. coli and V. harveyi that had grown to the logarithmic phase were collected and centrifuged. The bacterial pellet was collected and washed with PBS and resuspended to a density of 1×10 4 CFU / ml.

[0063] 2. Determination of antibacterial activity of ToNK-lysin recombinant protein

[0064] 50 μL of ToNK-lysin recombinant protein (100 μg / mL) from Takifugu obscurus was incubated with equal volumes of Escherichia coli and Vibrio harveyi at room temperature for 2 hours. Twenty μL of this mixture was placed in a flat-bottom 96-well plate (Costar, Fisher), and 200 μL of LB and 2216E liquid media, respectively, were added to each well. The cells were incubated with shaking at 37°C for 20 hours in a microplate reader. The OD600 value of each well was measured and recorded every hour. A PBS negative control group and a blank group were also set up. Three replicates were performed for each sample, and the average of the three measurements was plotted. The experiment showed that ToNK-lysin recombinant protein inhibited the growth of E. coli and Vibrio harveyi.

[0065] Example 5

[0066] The specific steps of the degradation detection of the obscured pufferfish recombinant protein NK-lysin of the present invention are as follows:

[0067] 1. Cultivation and Preparation of Microorganisms

[0068] The experimental method is the same as that in Example 4.

[0069] 2. Detection of Degradation of ToNK-lysin Recombinant Protein

[0070] The genomes of Escherichia coli and Vibrio harveyi were extracted using the Vazyme Genome Extraction Kit. Different concentrations of ToNK-lysin recombinant protein (0, 50 ng, 100 ng, 200 ng, 250 ng, and 300 ng) were mixed with 10 μl of genomic DNA (100 ng / μl). After incubation at room temperature for 30 minutes, the degradation of the bacterial genomes was examined by agarose gel electrophoresis. The results showed that ToNK-lysin recombinant protein significantly degraded the genomes of both E. coli and Vibrio harveyi.

[0071] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Antimicrobial peptide from Takifugu obscurus To NK-lysin, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The antimicrobial peptide of Takifugu obscurus according to claim 1 To The preparation method of NK-lysin is characterized in that: The following steps are involved: S1. Construction of the expression vector for the antimicrobial peptide K-lysin from Takifugu obscurus. The 5' end was added Kpn I and Xho 1 Gene-specific primers for specific restriction sites: P1: 5'-CGGGGTACCATGCCAACCTCCTCGATCCT-3'; P2: 5'- CCGCTCGAGGTGAGAGTGAGGAAAAACCACTC-3'; The reaction conditions were as follows: first, pre-denaturation at 94 °C for 5 min, followed by the following cycles: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, for a total of 30 cycles, and finally extension at 72 °C for 10 min; The amplified fragment was purified and recovered, and then connected to the pMD19-T vector; after transformation, positive clones were screened and plasmids were extracted; use Kpn I and Xho 1 The plasmid was double-digested by two restriction endonucleases, and the target fragments produced by the enzyme digestion were purified and recovered; the target fragments were recovered and separated from the target fragments by Kpn I and Xho 1 The expression vector pET-30a digested with two restriction endonucleases was connected to complete the construction of the expression vector; S2. The constructed recombinant vector is transformed into the expression host bacteria DE3, and positive clones are screened, and then purified and renatured to obtain the product.

3. The antimicrobial peptide of Takifugu obscurus according to claim 1 To The application of NK-lysin in antibacterial activities for non-disease diagnosis and treatment purposes, wherein the antibacterial activities are to inhibit Escherichia coli and Vibrio harveyi.

4. The antimicrobial peptide of Takifugu obscurus according to claim 1 To Application of NK-lysin in the preparation of antibacterial drugs or feed additives; the antibacterial drugs are antibacterial drugs against Enterobacter and Vibrio harveyi.

5. The use according to claim 4, characterized in that The application is application on Takifugu obscurus.

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