Antibacterial peptide for normal-temperature preservation of boar semen as well as preparation method and application of antibacterial peptide

Abstract

The invention discloses an antibacterial peptide for normal-temperature preservation of boar semen as well as a preparation method and application of the antibacterial peptide. The amino acid sequence of the sequence of the antibacterial peptide C8-TAT9 is as shown in SEQ ID No. 1. The antibacterial activity of the obtained antibacterial peptide, the bacterial growth inhibition capability of the antibacterial peptide in normal-temperature preservation of semen and the influence on the motility and activity of sperms are found, and the antibacterial peptide C8-TAT9 not only has the antibacterial activity on escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus aureus and the like, but also has the antibacterial activity on sperms. Gram-negative bacteria such as staphylococcus epidermidis, enterococcus faecalis and the like and gram-positive bacteria can be obviously inhibited, and an efficient inhibition effect on methicillin-resistant staphylococcus aureus can be realized; therefore, the antibacterial peptide C8-TAT9 disclosed by the invention can efficiently inhibit the growth of bacteria when the semen is stored at normal temperature, and has no obvious influence on the motility rate of the pig sperms.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide for storing pig semen at room temperature, a preparation method and an application thereof. Background Art

[0002] Because the ratio of cholesterol to phospholipids in the plasma membrane of pig sperm is significantly lower than that of other mammals, pig sperm is more vulnerable to low temperature damage. 99% of artificial insemination worldwide uses liquid semen stored at room temperature (17°C). Pig semen is easily contaminated by bacteria during collection and transportation. At the same time, semen is rich in nutrients. These nutrients and high storage temperature provide favorable growth conditions for bacteria. Studies have shown that 75% of pig semen is contaminated by at least one bacteria, and bacterial sperm disease has become one of the main problems in pig semen preservation. In order to control bacterial contamination, antibiotics are usually added to semen diluents. According to statistics, about 12.8 million liters of semen containing antibiotics are used worldwide each year. However, in recent years, bacterial resistance has increased year by year. Among the bacteria isolated from semen, more than 90% are resistant to the antibiotics added to semen. Therefore, in order to maintain the long-term development of pig semen preservation technology and protect human health, green, safe, and non-residue non-antibiotic antibacterial strategies have become the general trend of future development. Antimicrobial peptides (AMPs) are amphiphilic small molecule oligopeptides with broad-spectrum antibacterial, antiviral and antifungal activities, 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 eukaryotic organisms and are the first line of defense for 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 destroying bacterial membranes, antimicrobial peptides may also kill bacteria by inducing bacterial programmed death pathways or binding to dsDNA. This mechanism of action makes it difficult for bacteria to develop resistance to antimicrobial peptides, and antimicrobial peptides have become one of the most promising antibiotic substitutes. However, the use of antimicrobial peptides as an alternative to antibiotics in the liquid storage technology of pig semen also faces problems such as low biological activity, high toxicity to sperm and poor stability. Therefore, the design of new antimicrobial peptides for room temperature storage of pig semen with broad spectrum, high efficiency and low toxicity is imperative. Summary of the invention

[0003] Based on the above shortcomings, the present invention provides an antimicrobial peptide C8-TAT9 for storage of pig semen at room temperature. The antimicrobial peptide C8-TAT9 has high antimicrobial activity, can effectively inhibit the growth of bacteria in semen, and has no obvious effect on sperm motility and vitality.

[0004] The technical solution adopted by the present invention is as follows: an antimicrobial peptide C8-TAT9 for storing pig semen at room temperature, whose amino acid sequence is shown in SEQ ID No.1.

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

[0006]

[0007] Another object of the present invention is to provide a method for preparing the antimicrobial peptide C8-TAT9 as described above, as follows: using a medium-chain saturated fatty acid n-octanoic acid C8 to connect with the amyloid β protein self-assembly core sequence: KLVFFK to provide hydrophobicity, and then using the α-amino group of lysine and the -NH2 on the R group 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, and the sequence of the cell penetrating peptide TAT9: RKKRRQRRR is connected to the main chain to provide a positive charge. The amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1, and the polypeptide is synthesized by solid phase chemical synthesis, and then purified by reversed-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, and finally named as antimicrobial peptide C8-TAT9.

[0008] Another object of the present invention is to provide the use of the antimicrobial peptide C8-TAT9 as described above in the preparation of a drug for inhibiting the growth of Gram-negative bacteria and / or Gram-positive bacteria in pig semen stored at room temperature.

[0009] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Salmonella or Pseudomonas aeruginosa.

[0010] Furthermore, the Gram-positive bacteria mentioned above are methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.

[0011] Advantages and beneficial effects of this aspect: The present invention uses two motifs, namely, octanoic acid of a medium-chain saturated fatty acid and KLVFFK, a core sequence of amyloid β protein (Aβ) self-assembly, to simultaneously provide the hydrophobicity required for antibacterial properties, thereby avoiding the high cytotoxicity caused by the use of long-chain fatty acids or a large number of hydrophobic amino acids alone. At the same time, the cell-penetrating peptide TAT9 is further utilized to increase the cell-penetrating ability of the antimicrobial peptide molecules while providing the positive charge required by the antimicrobial peptide molecules, thereby effectively avoiding the cytotoxicity caused by membrane damage. The PEG8 branch sequence is further added through the lysine core to achieve the purpose of reducing cytotoxicity. The obtained antimicrobial peptides were tested for their antimicrobial activity, ability to inhibit bacterial growth during semen storage at room temperature, and effect on sperm motility. It was found that the antimicrobial peptide C8-TAT9 not only had a significant inhibitory effect on Gram-negative and Gram-positive bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis, but also had a highly effective inhibitory effect on methicillin-resistant Staphylococcus aureus; the antimicrobial peptide C8-TAT9 could effectively inhibit bacterial growth during semen storage at room temperature, and had no significant effect on pig sperm motility. Therefore, on the whole, the antimicrobial peptide C8-TAT9 is an antimicrobial peptide with high potential in the application of pig semen storage at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the mass spectrum of the antimicrobial peptide C8-TAT9 used for the storage of pig semen at room temperature;

[0013] Figure 2 This is the HPLC chromatogram of the antimicrobial peptide C8-TAT9 used for the storage of pig semen at room temperature;

[0014] Figure 3 This is a graph showing the effect of different concentrations of antimicrobial peptide C8-TAT9 treatment on the total bacterial load in semen stored at room temperature (17°C);

[0015] Figure 4 This graph shows the effect of different concentrations of antimicrobial peptide C8-TAT9 on sperm motility. DETAILED DESCRIPTION

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

[0017] Example 1

[0018] Design of antimicrobial peptides:

[0019] The amino acid sequence of the antimicrobial peptide C8-TAT9 used for the storage of pig semen 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, and 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. The preparation of the main chain of the polypeptide is carried out one by one from the C-terminus to the N-terminus, and is completed by 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 the 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); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and the side chain protection resin with the Fmoc group removed is obtained;

[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 branched PEG8 linking.

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

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

[0033] 5. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization 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 antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the microbroth dilution method. Single bacterial colonies were cultured in MHB medium overnight and transferred to new MHB to grow to the mid-logarithmic phase. The bacterial solution was then centrifuged and resuspended in MHB to a final concentration of 1×10 5 CFU mL -1 , and transferred it to a 96-well plate, 50 μL per well. 50 μL of BSA containing different concentrations of peptides was added to the above 96-well plate, and 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 light absorption value was measured at 492 nm (OD = 492 nm) using an ELISA reader to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0037] Table 2 Antibacterial activity of antimicrobial peptide C8-TAT9

[0038]

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

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

[0041] 2. Determination of the ability to inhibit bacterial growth in pig semen: The sperm sample is collected on the same day and sent to the laboratory within 30 minutes to determine whether the quality of fresh semen meets the requirements of subsequent experiments. The criteria for judging semen quality are: sperm motility ≥ 80%, sperm concentration ≥ 3.0×10 8 / mL, normal morphology ≥80%. The preheated BTS basic diluent was isothermally diluted with the original semen that passed the quality inspection to reach a final concentration of 100 million to 150 million sperm / mL. The diluted semen was divided into four groups, and 0, 2, 8, and 32 μM of the antimicrobial peptide C8-TAT9 were added respectively. The samples were stored in the dark at 17°C and inverted and gently shaken every 8 to 12 hours. The time when the antimicrobial peptide was added to the semen was recorded as t = 0h. At t = 0, 24 and 72h, 100 μL of semen was randomly drawn from each treatment group in a 1.5mL centrifuge tube, diluted 1000 times with BTS basic diluent, and 50 μL was aspirated and spread on Mueller-Hinton agar (MHA) medium. After incubation at 37°C for 18 hours, the number of colonies was counted and the bacterial load was calculated. The results of the test on the ability of antimicrobial peptide C8-TAT9 to inhibit bacterial growth in pig semen are shown in Figure 3 The lower the bacterial load, the stronger the ability of antimicrobial peptide C8-TAT9 to inhibit bacterial growth. Figure 3 It can be seen that the antimicrobial peptide C8-TAT9 can effectively inhibit the growth of bacteria in pig semen at a concentration of 8 μM and above, especially within 24 hours, with an inhibition rate of up to 95%, indicating that the antimicrobial peptide C8-TAT9 has extremely high application potential in semen storage at room temperature.

[0042] 3. Sperm motility test: In order to test whether antimicrobial peptides will produce cytotoxicity to sperm, we measured the sperm motility after antimicrobial peptides were added to pig semen. The experiment was divided into four groups, and 0, 2, 8, and 32 μM of antimicrobial peptide C8-TAT9 were added respectively. The samples were stored in the dark at 17°C and inverted and gently shaken every 8 to 12 hours. The time when the antimicrobial peptide was added to the semen was recorded as t = 0h. At t = 0, 24 and 72h, 100 μL of semen was randomly drawn from each treatment group into a 1.5mL centrifuge tube and incubated in a 37°C water bath for 10 to 20 minutes. Then 5 to 10 μL of semen was dropped onto a preheated special slide, and the computer-assisted sperm analysis system (CASA) was used to detect sperm motility. Five fields of view were randomly selected for detection each time, and the test was repeated three times. Finally, the average value was calculated to obtain the sperm motility index. The test results are shown in Figure 4 The results showed that the addition of antimicrobial peptide C8-TAT9 had little effect on sperm motility compared with the control group.

[0043] The above results show that by using multiple binding sites of dendritic macromolecular peptides with lysine amino groups as the core to hybridize octanoic acid, KLVFFK sequence, polyethylene glycol PEG8 and cell penetrating peptide TAT9 on the same molecule, an antimicrobial peptide with high activity and low toxicity can be designed. Comprehensive analysis of the above results shows that the minimum inhibitory concentration of the antimicrobial peptide C8-TAT9 against a variety of Gram-negative bacteria and Gram-positive bacteria, including drug-resistant bacteria, can reach the micromolar level, showing efficient inhibition; at the same time, the antimicrobial peptide C8-TAT9 can effectively inhibit the proliferation of bacteria in semen when used in semen preservation at room temperature, and has no significant effect on sperm motility compared with the control group. This shows that the newly designed antimicrobial peptide C8-TAT9 has a high "antibiotic replacement" potential in semen preservation at room temperature.

Claims

1. An antimicrobial peptide C8-TAT9 for storage of pig semen at room temperature, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

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

3. 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 amino acid sequence of the obtained polypeptide is shown in SEQ ID No.

1. The polypeptide is synthesized by solid phase chemical synthesis, purified by reversed-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, and finally named as antimicrobial peptide C8-TAT9.

4. Use of the antimicrobial peptide C8-TAT9 for storage of pig 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 pig semen at room temperature.

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

6. The use according to claim 4, characterized in that: The Gram-positive bacteria are methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.

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