Antibacterial peptide for normal temperature preservation of pig sperm, and preparation method and application thereof

Abstract

The application discloses an antibacterial peptide for normal-temperature preservation of pig semen and a preparation method and application thereof. The amino acid sequence of the antibacterial peptide C8-TAT9 is shown in SEQ ID No. 1. The antibacterial activity, the ability of inhibiting bacterial growth in normal-temperature preservation of the obtained antibacterial peptide, and the influence on sperm activity rate and activity are detected, and it is found that the antibacterial peptide C8-TAT9 not only has obvious inhibiting effect on gram-negative bacteria such as escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis and enterococcus faecalis and gram-positive bacteria, but also has high efficient inhibiting effect on methicillin-resistant staphylococcus aureus; therefore, the antibacterial peptide C8-TAT9 of the application can efficiently inhibit the growth of bacteria in normal-temperature preservation of semen, and has no significant influence on the pig sperm activity rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to an antibacterial peptide for normal-temperature preservation of pig semen, a preparation method and application. BACKGROUND

[0002] Due to the fact that the ratio of cholesterol to phospholipid in the cytoplasmic membrane of pig sperm is significantly lower than that of other mammals, pig sperm is more vulnerable to low temperature, and 99% of artificial insemination worldwide uses liquid semen preserved at normal temperature (17℃). Pig semen is extremely susceptible to bacterial contamination during collection and transportation, and the semen contains rich nutrients, which provide favorable growth conditions for bacteria. Studies have shown that 75% of pig semen is contaminated by at least one bacterium, and bacterial spermatozoa is one of the main problems of pig semen preservation. In order to control bacterial contamination, antibiotics are usually added to the semen diluent, and according to statistics, about 12.8 million liters of semen containing antibiotics are used worldwide each year. However, in recent years, bacterial drug resistance has been increasing year by year, and more than 90% of bacteria isolated from semen have drug resistance to antibiotics added to the semen. Therefore, in order to maintain the long-term development of pig semen preservation technology and protect human health, green and safe, non-residual non-antibiotic bacteriostatic strategies are the trend of future development. Antimicrobial peptides (AMPs) are a kind of amphiphilic small molecular oligopeptides with broad-spectrum antibacterial, antiviral and antifungal activity, which are usually composed of no more than 50 positively charged amino acids and hydrophobic amino acids. They are ubiquitous in the epithelial barrier and systemic induced defense of multicellular eukaryotes, and are the first line of defense of multicellular organisms. Antimicrobial peptides have diverse biological functions, and can directly kill bacteria, fungi, viruses and even cancer cells. Antimicrobial peptides may have multiple mechanisms of action, in addition to damaging bacterial membranes, antimicrobial peptides may also kill bacteria by inducing bacterial programmed death pathways or binding to dsDNA. This mechanism makes it difficult for bacteria to develop resistance to antimicrobial peptides, and antimicrobial peptides have become one of the most promising alternatives to antibiotics. However, the use of antimicrobial peptides in liquid preservation technology of pig semen also faces problems such as low biological activity, high toxicity to sperm and poor stability. Therefore, it is imperative to design a new type of broad-spectrum and efficient antimicrobial peptide for pig semen normal-temperature preservation with low toxicity. SUMMARY

[0003] Based on the above deficiencies, the application provides an antibacterial peptide C8-TAT9 for normal-temperature preservation of pig semen, which has high antibacterial activity and can effectively inhibit the growth of bacteria in semen without significantly affecting sperm motility and viability.

[0004] The technical scheme adopted by the application is as follows: an antibacterial peptide C8-TAT9 for normal-temperature preservation of pig semen, the amino acid sequence of which is shown in SEQ ID No. 1.

[0005] Further, the molecular formula of the antibacterial peptide C8-TAT9 as described above is shown as formula (I):

[0006]

[0007] Another object of the present application is to provide a preparation method of the antibacterial peptide C8-TAT9 as described above, which comprises the following steps: connecting a middle-chain saturated fatty acid n-octanoic acid C8 and a self-assembled core sequence of amyloid β protein: KLVFFK to provide hydrophobicity, connecting a polyethylene glycol PEG8 to a side chain by using an α-amino group of lysine and -NH2 on the R group as a linking site of the main chain and the side chain respectively to reduce cytotoxicity, connecting a sequence of a cell-penetrating peptide TAT9: RKKRRQRRR to the main chain to provide positive charge, obtaining an amino acid sequence of the polypeptide as shown in SEQ ID No. 1, synthesizing the polypeptide by using a solid-phase chemical synthesis method, purifying the polypeptide by using a reversed-phase high-performance liquid chromatography and identifying the polypeptide by using a mass spectrometry, detecting in-vitro antibacterial activity, the ability to inhibit the growth of bacteria in pig semen and the sperm motility, and finally naming the polypeptide as the antibacterial peptide C8-TAT9.

[0008] Another object of the present application is to provide an application of the antibacterial peptide C8-TAT9 as described above in the preparation of a medicine for inhibiting the growth of gram-negative bacteria and / or gram-positive bacteria in the normal-temperature preservation of pig semen.

[0009] Further, the gram-negative bacteria as described above is Escherichia coli, Salmonella or Pseudomonas aeruginosa.

[0010] Further, the gram-positive bacteria as described above is methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.

[0011] Advantages and beneficial effects of the present aspect: the present application simultaneously provides the hydrophobicity required for antibacterial by using two motifs of medium-chain saturated fatty acid n-octanoic acid and amyloid beta protein (Aβ) self-assembly core sequence KLVFFK, avoids the high cytotoxicity caused by the use of long-chain fatty acids or a large number of hydrophobic amino acids alone, further utilizes the cell penetrating peptide TAT9 to provide the positive charge required for the antibacterial peptide molecule while increasing the cell penetrating ability of the antibacterial peptide molecule, effectively avoids the cytotoxicity caused by membrane damage, and further adds a PEG8 branch sequence to the lysine core to reduce cytotoxicity. The obtained antibacterial peptide is subjected to antibacterial activity, inhibition of bacterial growth in semen storage at room temperature, and influence on sperm motility, and it is found that the antibacterial peptide C8-TAT9 not only has obvious inhibitory effect on gram-negative bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, and gram-positive bacteria, but also has high inhibitory effect on methicillin-resistant Staphylococcus aureus; the antibacterial peptide C8-TAT9 can efficiently inhibit the growth of bacteria in semen storage at room temperature, and has no significant effect on the motility of pig sperm. Therefore, in summary, the antibacterial peptide C8-TAT9 is an antibacterial peptide with high potential in the application of pig semen storage at room temperature. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Mass spectrum of the antibacterial peptide C8-TAT9 for pig semen storage at room temperature;

[0013] Figure 2 High performance liquid chromatogram of the antibacterial peptide C8-TAT9 for pig semen storage at room temperature;

[0014] Figure 3 Figure of the effect of different concentrations of antibacterial peptide C8-TAT9 treatment on the total bacterial load in semen stored at room temperature (17℃);

[0015] Figure 4 Figure of the effect of different concentrations of antibacterial peptide C8-TAT9 treatment on sperm motility. DETAILED DESCRIPTION

[0016] The present application will be further described in detail below in conjunction with the embodiments and drawings, but the embodiments of the present application are not limited thereto.

[0017] Example 1

[0018] Design of antibacterial peptide:

[0019] The amino acid sequence of the antibacterial peptide C8-TAT9 for pig semen storage at room temperature is:

[0020] C8-KLVFFKK(PEG8)RKKRRQRRR

[0021] The medium-chain saturated fatty acid octanoic acid and the amyloid β protein (Aβ) self-assembly core sequence KLVFFK were connected to provide hydrophobicity; the lysine α-amino group and the -NH2 on the R group were used as the linking sites of the main chain and side chain of the dendrimer peptide, respectively, and polyethylene glycol PEG8 was connected to the side chain of the dendrimer peptide to reduce cytotoxicity. The sequence of the cell-penetrating peptide TAT9: RKKRRQRRR was connected to the main chain of the dendrimer peptide to provide a positive charge. The dendrimer peptide was named antimicrobial peptide C8-TAT9, and the sequence was: C8-KLVFFKK(PEG8)RKKRRQRRR, as shown in Table 1.

[0022] Table 1 Main parameters of antimicrobial peptide C8-TAT9

[0023]

[0024] The antimicrobial peptide C8-TAT9 is an octaned branched antimicrobial peptide with a positive charge of 10 and a molecular weight of 2780.50.

[0025] Its structural formula is:

[0026]

[0027] Example 2

[0028] The antimicrobial peptide was synthesized using a peptide synthesizer by solid phase chemical synthesis, with the following specific steps:

[0029] 1. Preparation of the polypeptide backbone is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to the Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); follow this procedure from the C-terminus to the N-terminus until the synthesis is complete, obtaining a resin with the side chain protection removed from the Fmoc group;

[0030] 2. Use hydrazine hydrate to remove the Dde protecting group of the Fmoc-Lys(Dde)-OH side chain and repeat step 1 to complete the side chain PEG8 link.

[0031] 3. Add a cleavage reagent to the polypeptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with TFA (trifluoroacetic acid) to precipitate, mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether. Precipitate at -20°C for 3 hours to precipitate a white powder. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the polypeptide. The cleavage reagent is a mixture of TFA, water, and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0032] 4. Use 0.2 mol / L sodium sulfate (adjusted to pH = 7.5 with phosphoric acid) to equilibrate the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and apply gradient elution (eluent: methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30) on a C18 reverse-phase atmospheric pressure column at a flow rate of 1 ml / min and a detection wave of 220 nm to collect the main peak and freeze-dry; further purification is performed using a reverse-phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12 minutes, the flow rate is 1 ml / min, and the main peak is collected as above and freeze-dried;

[0033] 5. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. The molecular weights shown in the mass spectra (see Appendix Figure 1 ) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide is greater than 95% (see Appendix Figure 2 ).

[0034] Example 3

[0035] The designed and synthesized antimicrobial peptide C8-TAT9 was tested for its in vitro antibacterial activity, ability to inhibit bacterial growth in pig semen, and sperm motility.

[0036] 1. Determination of antimicrobial activity: The minimum inhibitory concentration (MIC) of several antimicrobial peptides was determined using the broth microdilution method. Single bacterial colonies were cultured overnight in MHB medium and transferred to fresh MHB medium until they reached mid-logarithmic phase. The bacterial solution was then centrifuged and resuspended in MHB to a final concentration of 1×10 5 CFU mL -1 50 μL of the solution was transferred to each well of a 96-well plate. 50 μL of BSA containing varying peptide concentrations was added to each well of the plate. The final peptide concentration in the 96-well plate ranged from 0.125 to 64 μM. After incubation at 37°C for 22-24 hours, the minimum inhibitory concentration (MIC) was determined by measuring absorbance at 492 nm (OD = 492 nm) using a microplate reader. The results are shown in Table 2.

[0037] Table 2 Antimicrobial activity of C8-TAT9

[0038]

[0039] Note: a GM is the geometric mean.

[0040] As can be seen from Table 2, the antimicrobial peptide C8-TAT9 exhibits high antibacterial activity against gram-negative, gram-positive bacteria and drug-resistant bacteria, and the geometric mean (GM) of the MIC can reach 2.33 μM.

[0041] 2. Inhibition of the ability of pig semen bacteria to grow: sperm samples were collected on the same day and sent to the laboratory within 30 minutes, and the quality of fresh semen was determined to meet the requirements of subsequent experiments. The quality of semen was determined by the following criteria: sperm motility ≥ 80%, sperm concentration ≥ 3.0 x 10 8 / mL, normal morphology ≥ 80%. The preheated BTS base diluent was isothermally diluted with the original semen that passed the quality test to achieve a final concentration of 1-1.5 billion sperm / mL. The diluted semen was divided into four groups, and 0, 2, 8, and 32 μM of antimicrobial peptide C8-TAT9 was added, respectively. The samples were stored at 17°C in the dark, and were gently shaken every 8-12 hours. The antimicrobial peptide was added to the semen at t = 0 h, and at t = 0, 24, and 72 h, 100 μL of semen was randomly taken from each treatment group in a 1.5 mL centrifuge tube, diluted 1000 times with BTS base diluent, and 50 μL was spread on Mueller-Hinton agar (MHA) medium. After incubation at 37°C for 18 h, the number of bacteria was counted and the bacterial load was calculated. The results of the determination of the ability of antimicrobial peptide C8-TAT9 to inhibit the growth of pig semen bacteria are shown in Table 3. Figure 3 The lower the bacterial load, the stronger the ability of antimicrobial peptide C8-TAT9 to inhibit bacterial growth. As can be seen from Table 3, Figure 3 antimicrobial peptide C8-TAT9 at a concentration of 8 μM and above can effectively inhibit the growth of bacteria in pig semen, especially within 24 h, with an inhibition rate as high as 95%, indicating that antimicrobial peptide C8-TAT9 has a high potential for semen storage at room temperature.

[0042] 3. Sperm motility assay: To test whether the antimicrobial peptide has cytotoxicity to sperm, we determined the sperm motility after the addition of the antimicrobial peptide to the pig sperm. The experiment was divided into four groups, and 0, 2, 8, 32 μΜ of the antimicrobial peptide C8-TAT9 was added, respectively. The samples were stored at 17 °C in the dark, and were gently shaken every 8-12 h. The antimicrobial peptide was added to the sperm at t = 0 h. At t = 0, 24 and 72 h, 100 μΐ^of sperm was randomly selected from each treatment group in a 1.5 mL centrifuge tube, and was incubated in a 37 °C water bath for 10-20 min. Then 5-10 μΐ^of sperm was dropped on a preheated special glass slide, and the computer-aided sperm analysis system (CASA) was used to determine the sperm motility. Five fields of view were randomly selected for each test, and the test was repeated three times. Finally, the mean value of the sperm motility was obtained, and the test results are shown in Figure 4 . The results show that the addition of the antimicrobial peptide C8-TAT9 has no significant effect on the sperm motility compared with the control group.

[0043] The above results show that the hybridization of n-octanoic acid, KLVFFK sequence, polyethylene glycol PEG8 and cell penetrating peptide TAT9 on the same molecule by using the lysine amino group as the core of the dendrimer peptide multiple binding sites can design an antimicrobial peptide with high activity and low toxicity. Comprehensive analysis of the above results, the minimum inhibitory concentration of the antimicrobial peptide C8-TAT9 to various gram-negative bacteria and gram-positive bacteria including drug-resistant bacteria can reach the micromolar level, which exhibits high inhibitory capacity. At the same time, the use of the antimicrobial peptide C8-TAT9 in the preservation of sperm at room temperature can efficiently inhibit the growth of bacteria in the sperm, and has no significant effect on the sperm motility compared with the control group. It is shown that the newly designed antimicrobial peptide C8-TAT9 has high "replacement" potential in the preservation of sperm at room temperature.

Claims

1. An antimicrobial peptide C8-TAT9 for storage of pig semen at room temperature, characterized in that: Its molecular formula is shown in formula (I): ， Formula (I).

2. The method for preparing an antimicrobial peptide C8-TAT9 for storage of pig semen at room temperature according to claim 1, characterized in that: The method is as follows: a medium-chain saturated fatty acid, n-octanoic acid C8, is connected to the amyloid β protein self-assembly core sequence: KLVFFK to provide hydrophobicity, the α-amino group of lysine and the -NH2 on the R group are used as the linking sites of the main chain and the side chain, respectively, polyethylene glycol PEG8 is connected to the side chain to reduce cytotoxicity, the sequence of the cell-penetrating peptide TAT9: RKKRRQRRR is connected to the main chain to provide a positive charge, and the molecular formula of the obtained polypeptide is shown in formula (I). The polypeptide is synthesized by solid-phase chemical synthesis, purified by reverse-phase high-performance liquid chromatography and identified by mass spectrometry, and then tested by in vitro antibacterial activity, ability to inhibit bacterial growth in pig semen, and sperm motility. Finally, it is named antimicrobial peptide C8-TAT9.

3. Use of the antimicrobial peptide C8-TAT9 for storage of porcine semen at room temperature as claimed in claim 1 in the preparation of a drug for inhibiting the growth of Gram-negative bacteria and / or Gram-positive bacteria during storage of porcine semen at room temperature.

4. The use according to claim 3, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella or Pseudomonas aeruginosa.

5. The use according to claim 3, characterized in that: The Gram-positive bacteria are methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.

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