CN115925863A - Antimicrobial peptide ToNK-lysin from puffer puffer obscura and its application - Google Patents

Abstract

The invention discloses the antibacterial peptide ToNK-lysin from puffer puffer, whose amino acid sequence is shown in SEQ ID NO.1. The present invention utilizes in vitro recombinant expression technology to obtain the antimicrobial peptide ToNK-lysin of puffer puffer, the recombinant protein has no cytotoxic effect on puffer head kidney cells, and can significantly eliminate Vibrio harveyi in puffer; in addition , 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 protein has potential application value in the development of antibacterial drugs, new immune agents and feed additives.

Description

Antimicrobial peptide ToNK-lysin from puffer puffer obscura and its application

technical field

The invention belongs to the technical field of molecular biology, in particular to the in vitro recombinant expression technology of the ToNK-lysin gene coding region of puffer pufferfish obscura, the separation and purification of active recombinant protein, the cytotoxic effect of recombinant protein and the effect of recombinant protein on Escherichia coli and Harvey Bacteriostatic activity of Vibrio.

Background technique

Pufferfish is deeply loved by consumers because of its white and tender meat and delicious taste, and has high economic value. At present, wild puffer resources are depleting day by day, and cultured puffer has become the main source. Takifugu obscurus, known as one of the "three delicacies of the Yangtze River", is a unique high-quality fishery germplasm resource in the middle and lower reaches of the Yangtze River. These problems have seriously hindered the healthy and green development of the aquaculture industry.

NK-lysin protein is a structurally conserved antibacterial peptide, which is mainly secreted by cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTLs) and natural killer cells (natural killer cells, NKs). NK-lysin belongs to the saponin-like protein family (SAPLIP), and its N-terminal sequence contains six highly conserved cysteine residues, which can form three internal disulfide bonds. All SAPLIPs share a common fold, called the saposin fold, that is associated with the antibacterial activity of NK-lysin proteins. At present, the antibacterial effect of NK-lysin protein has not been reported in Puffer puffer obscura.

Contents of the invention

The invention provides the preparation and application of a recombinant protein of the ToNK-lysin gene of puffer puffer obscura.

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

The amino acid sequence of the recombinant protein of Fugu obscurus ToNK-lysin gene is as follows:

MPTSSILLLCILVTCSVWPVQARNLTVSTDDDDEHQGGFAIEAGRLPGVCWACKWALKKVKRIIGNSSTSQAIKAKLTSICDHIGLLKSLCRKFVTNHLGVLIEELTTSDDVRTSASTSGPASQRSWRSSPRVVFPHSHI

Length: 139 amino acids

Type: amino acid

Chain type: single chain

Features: The molecular weight is 15.3kDa, the isoelectric point is 9.05, and it has a conserved SapB domain.

The recombinant product was purified by nickel agarose gel column, and after refolding by dialysis, it exhibited the following activities:

ToNK-lysin recombinant protein has no cytotoxic effect on puffer head kidney cells, and can significantly eliminate Vibrio harveyi in puffer;

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

The advantages that the present invention has:

The present invention uses in vitro recombinant expression technology to obtain the antibacterial peptide ToNK-lysin protein of puffer puffer, the recombinant protein has no cytotoxic effect on puffer head kidney cells, and can significantly eliminate Vibrio harveyi in puffer; In addition, 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 protein has potential application value in the development of antibacterial drugs, new immune agents and feed additives.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Figure 1: SDS-PAGE detection of NK-lysin recombinant protein of puffer puffer obscura;

Figure 2: Cytotoxicity experiment of fugu obscurus NK-lysin recombinant protein on puffer head kidney cells;

Figure 3: In vivo pathogen removal experiment of the NK-lysin recombinant protein of Pufferfish obscura;

Figure 4: Effects of Fugu obscurus NK-lysin recombinant protein on the growth curves of Escherichia coli and Vibrio harveyi;

Figure 5: The degradation of the Escherichia coli genome by the NK-lysin recombinant protein of Pufferfish obscurus; wherein 1. PBS+10 μl genomic DNA; 2.50ng+10 μl genomic DNA; 3.100ng+10 μl genomic DNA; 4.200ng+10 μl genomic DNA; 5.250ng+10μl genomic DNA; 6.300ng+10μl genomic DNA; 7.10μl genomic DNA;

Figure 6: The degradation of Vibrio harvelii genome by NK-lysin recombinant protein of Pufferfish obscurus, in which 1. PBS+10μl genomic DNA; 2.50ng+10μl genomic DNA; 3.100ng+10μl genomic DNA; 4.200ng+10μl Genomic DNA; 5.250 ng+10 μl genomic DNA; 6.300 ng+10 μl genomic DNA; 7.10 μl genomic DNA.

Detailed ways

The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

The in vitro prokaryotic recombinant expression of puffer puffer NK-lysin (ToNK-lysin) gene coding region comprises the following steps:

1. Construction of recombinant vector

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

P1 (5'-CGGGGTACCATGCCAACCTCCTCGATCCT-3')

P2 (5'-CCGCTCGAGGTGAGAGTGAGGAAAAACCACTC-3')

The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycle: 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1 minute, a total of 30 cycles, and finally 72°C extension for 10 minutes. The amplified fragment was purified and recovered, and connected to the pMD19-T vector. After transformation, screen positive clones and extract plasmids; use Kpn I and Xho 1 two kinds of restriction endonucleases to carry out double enzyme digestion on plasmids, and use gel recovery purification kit (Dalian Bao Biological Engineering Co., Ltd.) to digest the target fragments Purification and recovery; the recovered target fragment was ligated with the expression vector pET-30a digested by two restriction enzymes, Kpn I and Xho 1, to complete the construction of the expression vector. For specific experimental methods, refer to "Molecular Cloning Third Edition".

2. Expression of recombinant protein ToNK-lysin

The constructed recombinant vector was transformed into expression host strain E. Coli Transetta (DE3), and positive clones were screened, and the correctness of the expression frame was confirmed by sequencing. A single clone was picked and inoculated in 400 mL of LB liquid medium at 220 rpm and cultured at 37° C. for 4 hours (OD 600 =0.4-0.8). After adding IPTG (1 mmol/L), culture was continued at 22° C. for 12 hours. Centrifuge at 12,000 g for 10 minutes at 4°C to collect bacterial pellets and freeze them at -20°C for later use. Take 3mL bacterial liquid and centrifuge, discard the supernatant, add 200μL BeyoLytic ^TM Bacterial Active Protein Extraction Reagent (Biyuntian) to extract a small amount of soluble protein. The supernatant was collected after centrifugation again, and an appropriate amount of protein loading buffer (4:1) was added, boiled at 100°C for 10 minutes, centrifuged slightly, and the supernatant was taken for SDS-PAGE to detect the expression product.

3. Purification and renaturation of recombinant protein ToNK-lysin

In the present invention, the recombinant protein ToNK-lysin is a soluble recombinant protein, which is purified by nickel agarose gel FF column, and the specific operation steps are as follows:

Purification and Separation of Recombinant Protein with His Tag by Nickel Sepharose Column

The purification steps of soluble recombinant protein are as follows:

(1) Nickel Sepharose FF packed column, 1.6×20cm, column bed volume is 10mL;

(2) Equilibrate 2-5 bed volumes with lysate (10mM NaH ₂ PO ₄ 2H ₂ O, 40mM Na ₂ HPO ₄ 12H ₂ O, 30mM NaCl, pH=7.4-8.0) at a flow rate of 2mL/min;

(3) Add 20ml of bacterial supernatant (use ^BeyoLyticTM Bacterial Active Protein Extraction Reagent to extract a large amount of soluble protein, take the supernatant after centrifugation, and filter through a 0.45μm filter membrane) into the column, and the flow rate of the column is 1mL/ min;

(4) Add an appropriate amount of lysate (4 times the bacterial liquid supernatant) to balance the nickel column left and right, and the flow rate is 6mL/min;

(5) Add 8 times column bed volume rinse solution (1L lysate + imidazole 50mM) to rinse the affinity column to elute impurity proteins, the flow rate is 6mL/min;

(6) Add 50ml (5 times column bed volume) eluent (1L lysate + imidazole 200mM) to elute and collect the target protein;

(7) Wash 5 column bed volumes with pure water, and then wash 3 column bed volumes with 20% ethanol at a flow rate of 2 mL/min. The columns were stored in a refrigerator at 4°C.

(8) detect the expression of fusion protein with SDS-PAGE;

The purified recombinant protein needs to be dialyzed in pure water for 12 hours each time at 4° C. and repeated three times to obtain the recombinant protein of the NK-lysin (ToNK-lysin) gene of Puffer obscurus.

Example 2

The cytotoxicity detection of the recombinant protein NK-lysin of puffer obscura, the specific steps are as follows:

1. Collection of Kidney Cells from Fugu obscurus

Fugu obscurus was dissected to take the kidneys, and the kidneys were washed repeatedly with 1×PBS. Mince the kidneys with sterilized scissors and place in a trypsin-supplemented Petri dish for digestion. After 30 minutes, filter the cell solution with a 300-mesh cell sieve, centrifuge at 300×g for 10 minutes, and collect the cells.

2. Cytotoxicity test

The collected cells were placed in (0.1% penicillin (100mg/L) and 0.1% streptomycin (100mg/L)) modified cell 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) are cultivated. After culturing for 24 hours, fresh culture medium was replaced, and the cells were distributed into 96-well plates, and cultured for 24 hours. After replacing the fresh culture medium again, measure the OD490 nm absorbance value with a spectrophotometer. Then, ToNK-lysin recombinant protein was added at concentrations of 0, 1, 5, 10, 20, 50 and 100 μg/mL in sequence, and three replicates were set for each concentration. After continuing to cultivate for 24 hours, the OD490 nm absorbance value was measured again with a spectrophotometer.

Example 3

The specific steps of the bacteria removal detection of the Fugu obscura recombinant protein NK-lysin of the present invention are as follows:

1. Bacteria treatment

Select 6 puffer puffers in good health and inject 100 μl Vibrio harvelii (1×10 ⁶ CFU/mL) into puffer puffer intraperitoneally. After 30 minutes, the 6 puffer fish were divided into bacteria + NK-lysin protein treatment group and bacteria treatment group. In the bacteria + NK-lysin protein treatment group, each fish was injected with ToNK-lysin recombinant protein at a dose of 20 μg/g body weight. The bacteria treatment group was injected with an equal volume of PBS.

2. Bacteria removal test

After 24 hours, puffer obscura was anesthetized and sacrificed. The head kidney and liver tissues were collected separately in a sterile environment, washed twice with 1 ml sterile 1×PBS buffer solution, and the head kidney and liver tissues were homogenized using a tissue homogenizer. The homogenate was diluted 10 times and spread on agar plate. After culturing overnight, the number of colonies was counted. Each experiment was divided into triplicates and experiments were repeated twice independently. Experiments showed that ToNK-lysin recombinant protein significantly eliminated Vibrio harveyi in puffer head kidney and liver tissues.

Example 4

The specific steps for detecting the antibacterial activity of the recombinant protein NK-lysin of puffer puffer obscura of the present invention are as follows:

1. Culture and preparation of microorganisms

Escherichia coli (E.coli) was purchased from Invitrogen, inoculated in LB liquid medium, placed on a constant temperature shaker at 37°C, and vibrated at 220rpm for 12 hours; Vibrio harveyi (V.harveyi) was isolated from Ph. Migrating, inoculated in LB liquid medium with 2% salt, placed in a constant temperature shaker at 30°C, and cultured with vibration at 220rpm for 12 hours; collected E. Wash and resuspend in PBS to a density of 1×10 ⁴ CFU/ml.

2. Determination of antibacterial activity of ToNK-lysin recombinant protein

Take 50 μL of Fugu obscura ToNK-lysin recombinant protein (100 μg/mL) and incubate with an equal volume of Escherichia coli and Vibrio harveyi at room temperature for 2 h. Take 20 μL of the above mixture in a flat-bottomed 96-well plate (Costar, Fisher), add 200 μL LB and 2216E liquid medium to each well, shake and culture in a microplate reader at 37°C for 20 hours, detect and record each well every 1 hour OD600 value. In addition, PBS negative control group and blank group were set up. Three repetitions were set up for each sample, and the average value of the three measurement results was used for graphing. Experiments show that ToNK-lysin recombinant protein can inhibit the growth of Escherichia coli and Vibrio harveyi.

Example 5

The specific steps of the degradation detection of the recombinant protein NK-lysin of puffer puffer in the present invention are as follows:

1. Culture and preparation of microorganisms

Experimental method is the same as embodiment 4.

2. ToNK-lysin recombinant protein degradation detection

The genomes of Escherichia coli and Vibrio harveyi were extracted using Vazyme Genome Extraction Kit. Mix different concentrations of ToNK-lysin recombinant protein (0, 50ng, 100ng, 200ng, 250ng and 300ng) with a total volume of 10μl of genomic DNA (100ng/μl), incubate at room temperature for 30min, and use agarose gel electrophoresis Check the degradation of the bacterial genome. The results showed that the ToNK-lysin recombinant protein significantly degraded the genomes of Escherichia coli and Vibrio harveyi.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. The amino acid sequence of the fugu obscurus antibacterial peptide ToNK-lysin is shown in SEQ ID No. 1.

2. The method for preparing the fugu obscurus antimicrobial peptide ToNK-lysin of claim 1, which comprises the following steps:

s1, constructing a fugu obscurus antimicrobial peptide K-lysin expression vector;

gene-specific primers with Kpn I and Xho 1 specific cleavage sites added to the 5' end were used:

P1(5’-CGGGGTACCATGCCAACCTCCTCGATCCT-3’)

P2(5’-CCGCTCGAGGTGAGAGTGAGGAAAAACCACTC-3’)；

the reaction conditions are as follows: first, pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 30 cycles in total, and final extension at 72 ℃ for 10 minutes;

purifying and recovering the amplified fragment, and connecting the amplified fragment with a pMD19-T vector; screening positive clones after transformation, and extracting plasmids;

carrying out double enzyme digestion on the plasmid by using two restriction enzymes Kpn I and Xho 1, and purifying and recovering a target fragment generated by enzyme digestion; the recovered target fragment is connected with an expression vector pET-30a cut by Kpn I and Xho 1 restriction enzymes to complete the construction of the expression vector;

s2, transforming the constructed recombinant vector into an expression host bacterium DE3, screening positive clones, purifying and renaturing to obtain the recombinant vector.

3. The use of the fugu obscurus antimicrobial peptide ToNK-lysin of claim 1 for bacteriostasis.

4. The use of claim 3, wherein the bacteriostatic action is that of inhibiting the activity of E.coli and Vibrio harveyi.

5. Use of the fugu obscurus antimicrobial peptide ToNK-lysin of claim 1 for degrading the genome of Escherichia coli (Escherichia coli) and Vibrio harveyi (Vibrio harveyi).

6. Use of the fugu obscurus antimicrobial peptide ToNK-lysin of claim 1 in the preparation of an antimicrobial medicament, vaccine or feed additive.

7. The use as claimed in claim 6, wherein the use is in takifugu obscurus.

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