CN104163861B - Reptile antibacterial peptide Alligatorin5 and applications thereof - Google Patents

Abstract

The invention discloses a reptile-derived antibacterial peptide Alligatorin5, an amino acid sequence thereof and applications of the antibacterial peptide in the antibacterial field. The antibacterial peptide Alligatorin5 is derived from Chinese alligator. By searching a Chinese alligator gene database, screening and analyzing, a mature peptide sequence of the antibacterial peptide is obtained. The amino acid sequence of the antibacterial peptide Alligatorin5 is shown as SEQ ID NO.1 in a sequence table. By comparative analysis, the antibacterial peptide Alligatorin5 is obviously different from amino acid sequences of antibacterial peptides known at present, and belongs to a novel antibacterial pepetide. Antibacterial experiments show that: the antibacterial peptide Alligatorin5 has high antibacterial activity for gram positive bacteria, gram negative bacteria and funguses, and is broad in antibacterial spectrum and efficient; the antibacterial peptide Alligatorin5 has characteristics of low hemolytic activity, salt tolerance, heat tolerance, good thermal stability, and the like, and can be used for the fields of medicines, cosmetics, food fresh-keeping and the breeding industry.

Description

A reptile antimicrobial peptide Alligatorin5 and its application

technical field

The invention relates to a reptile-derived antimicrobial peptide and its application, more specifically, relates to a reptile-derived alligator antibacterial peptide and its application, and belongs to the technical field of biomedicine.

Background technique

Traditional antibiotics have achieved remarkable results in the treatment of bacterial infectious diseases, but in recent years, with the abuse of traditional antibiotics in the fields of medicine and aquaculture, microorganisms have become increasingly resistant to traditional antibiotics, and microbial resistance It has become a difficult problem that seriously threatens human health. The response to microbial resistance has always been to use new or alternative antimicrobial agents that have not been used against drug-resistant microorganisms. Drug resistance, thus requiring the continuous development of new antimicrobial agents. Developing a drug with a new antibacterial mechanism is a new direction to solve the problem of microbial tolerance.

Antimicrobial peptides are small molecule polypeptides that can kill bacteria, fungi, viruses, and protozoa. Antimicrobial peptides have the characteristics of small molecular weight, simple structure, and strong bactericidal activity. The bactericidal mechanism of most antimicrobial peptides is by acting on the phospholipid bilayer on the bacterial cell membrane, destroying the integrity of the cell membrane and forming a transmembrane channel on the cell membrane, resulting in the dissolution of the cell content and cell death. This unique bactericidal mechanism is generally less likely to cause microbial resistance. Moreover, antimicrobial peptides are generally non-toxic to mammalian normal cells and tissues, and there is no residual problem. Therefore, antimicrobial peptides are expected to become a new type of highly effective antibacterial drug and have broad development and application prospects.

Contents of the invention

The purpose of the present invention is to solve the shortcomings of traditional antibiotics, and provides an antibacterial peptide Alligatorin5 derived from reptile alligator alligator, and also provides the amino acid sequence of the antibacterial peptide and its application in antibacterial aspects. Reptile antimicrobial peptide Alligatorin5 has a broad-spectrum and high-efficiency antibacterial effect, has strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has small molecular weight, simple synthesis, low hemolytic activity, and salt tolerance , thermal tolerance and thermal stability, it can be used in the fields of medicine, cosmetics, food preservation and aquaculture.

For realizing the purpose of the present invention, the present invention provides following technical scheme:

The antimicrobial peptide Alligatorin5 in the present invention is an antimicrobial peptide derived from reptile Chinese alligator. The reptile antimicrobial peptide Alligatorin5 is composed of 22 amino acids, with a molecular weight of 2557.1 Da and an isoelectric point of 12.03. The amino acid sequence is shown in the sequence table SEQ ID NO :1 shows: Thr Arg Arg Lys Phe Trp Lys Lys Val Leu Asn GlyAla Leu Lys Ile Ala Pro Phe Leu Leu Gly, all amino acids are L-type;

The present invention also provides a chemical preparation method of the antimicrobial peptide Alligatorin5:

According to the obtained amino acid sequence of the reptile antimicrobial peptide Alligatorin5 mature peptide, its complete sequence was combined with an automatic peptide synthesizer (433A, Applied Biosystems), and desalted and purified by HPLC reverse-phase column chromatography; its purity was identified by high performance liquid chromatography (HPLC) The molecular weight is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point is determined by isoelectric focusing electrophoresis, and the amino acid sequence structure is determined by an automatic amino acid sequencer;

In addition, the present invention provides the application of the antibacterial peptide Alligatorin5 in the preparation of antibacterial drugs or compositions, antibacterial growth inhibitory drugs or compositions, preservatives, animal feed additives or cosmetic additives.

The invention has the advantages of providing a reptile antimicrobial peptide Alligatorin5 derived from alligator alligator and its antibacterial application. The antimicrobial peptide Alligatorin5 has a broad-spectrum and high-efficiency antibacterial effect, and has the characteristics of small molecular weight, simple synthesis, low hemolytic activity, good salt tolerance, heat tolerance and thermal stability, and can be used in medicine, cosmetics, food preservation and field of farming.

detailed description

Example 1:

Discovery of reptile antimicrobial peptide Alligatorin5

Through retrieval, a gene sequence (XP_006026473.1) was found from the Chinese alligator genome data on the website of the National Center for Biotechnology Information (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov), with a length of 477bp. The encoded polypeptide precursor consists of 158 amino acid residues. The gene and its encoded polypeptide precursor are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 3:

The blastX software (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) of the gene sequence on the NCBI website was used for sequence comparison and analysis, and the results showed that the The encoded product of the gene may be the precursor of antimicrobial peptides of the alligator cathelicidins family. The polypeptide precursor sequence encoded by the gene was further sequenced using the protein blast software (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) of the NCBI website Compare and compare with the antimicrobial peptide sequences of other animal-derived cathelicidins family found before, and determine that the enzyme cleavage site in the maturation process of the antimicrobial peptide is -L ¹³⁵ -V ¹³⁶ -, so as to obtain the mature peptide sequence of the antimicrobial peptide as TRRKFWKKVLNGALKIAPFLLG (single-letter abbreviation sequence of amino acids), named Alligatorin5. The amino acid sequence of Alligatorin5 is significantly different from that of all known antimicrobial peptides.

Example 2

Chemical Synthesis of Antimicrobial Peptide Alligatorin5

(1) The full sequence of Alligatorin5 was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and purified by HPLC C ₁₈ reverse-phase column chromatography desalting. (2) The molecular weight was determined by conventional matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). (3) The purity of the purified Alligatorin5 was identified by high performance liquid chromatography (HPLC)>95%, the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, 2557.1 Da), and the isoelectric point was 12.03 by isoelectric focusing electrophoresis , using an automatic amino acid sequencer to determine its amino acid sequence structure is consistent with the natural Alligatorin5.

Example 3:

Detection of antibacterial activity of antimicrobial peptide Alligatorin5

(1) Pick the test strains preserved on the slant (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC27853 were purchased from China General Microorganism Culture Collection Management Center, and other strains were collected from local hospitals) and spread evenly Spread on the MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) plate, place a sterilized filter paper piece with a diameter of 0.5 cm on the surface of the medium, and add 2 mg/ml dissolved in sterilized deionized water dropwise. Alligatorin5 sample solution 10μl, incubate upside down at 37°C for 18-20 hours, observe whether the inhibition zone is formed or not. If the sample has antibacterial activity, a clear and transparent bacteriostatic zone will be formed around the filter paper, and the larger the bacteriostatic zone, the stronger the antibacterial activity of the sample. The results showed that the antimicrobial peptide Alligatorin5 had antibacterial activity against the strains listed in Table 2. (2) Determination of the Minimum Inhibitory Concentration (Minimum Inhibitory Concentration) by the 2-fold dilution method: the strains with the bacteriostatic zone in the above experimental step (1) were selected for the MIC determination experiment. The test strains were inoculated into MH liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), and then cultured in an incubator with shaking at 37°C to logarithmic growth phase, and then cultured to logarithmic growth with fresh MH liquid medium. The culture medium of strains at the stage was diluted to 2×10 ⁵ cfu/ml for use.

Add 100 μl of MH liquid medium to each well of a sterile 96-well plate, and then add 100 μl of Alligatorin5 sample solution diluted to a certain concentration with MH liquid medium to the first well and filtered through a 0.22 μm filter membrane, and mix well Afterwards, 100 μl was taken and added to the second well, and serially diluted (see Table 1), 100 μl was aspirated from the ninth well and discarded, and the tenth well was used as a control tube.

Table 1 Dilution method

Add 100 μl of the diluted strain culture solution to each well, then place the 96-well plate in an incubator at 37° C. for 18 hours with slow shaking, and measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is seen. The results are shown in Table 2.

It can be seen from Table 2 that Alligatorin5 exhibited strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, including a large number of clinically isolated pathogenic bacteria. Its MIC value is in the range of 4.69-75 μg/ml.

Table 2 Antibacterial activity of Chinese alligator antimicrobial peptide Alligatorin5

Example 4:

Determination of Antimicrobial Peptide Alligatorin5 Bactericidal Speed

Escherichia coli ATCC25922 was cultured at 37°C for 12 hours with MH liquid medium (Qingdao Haibo Biotechnology Co., Ltd.), and then diluted with fresh MH liquid medium to form a bacterial suspension of 10 ⁶ cfu/ml. The Alligatorin5 sample dissolved in sterilized deionized water was added to the bacterial suspension so that the final concentration was 5×MIC (46.9 μg/ml). Place the bacterial solution added with the Alligatorin5 sample in an incubator at 37°C for shaking culture, take 50 μl of the bacterial solution at 0, 10, 20, 30, 45, 60, 90, 120 and 180 minutes to dilute 1000 times, and then take 50 μl of the diluted bacterial solution was spread on the MH solid medium, and the colonies were counted after culturing overnight at 37°C in an incubator. In this example, meropenem was used as a positive control, and sterilized deionized water was used as a negative control. The experimental results are shown in Table 3.

It can be seen from Table 3 that the bactericidal effect of Alligatorin5 is extremely rapid, and all E. coli can be killed within 20 minutes. The positive control, meropenem, required 180 minutes to kill all bacterial cells.

Table 3 Antibacterial peptide Alligatorin5 sterilization speed

Example 5:

Determination of Hemolytic Activity of Antimicrobial Peptide Alligatorin5

The collected fresh human blood was mixed with Alfred's solution for anticoagulation, washed twice with normal saline and resuspended to a suspension of 10 ⁷ -10 ⁸ cell/ml. The above-mentioned diluted erythrocyte suspension was mixed with a certain concentration of Alligatorin 5 sample solution dissolved in normal saline, incubated at 37°C for 30 minutes, then centrifuged at 1000rpm for 5 minutes, and the light absorption value of the supernatant was measured at a wavelength of 540nm. Negative control uses physiological saline, positive control uses Triton X-100, and the hemolysis percentage is calculated according to the following formula: hemolysis percentage H%=A _sample -A _{negative control} /A _{positive control} ×100%. Calculate the half hemolytic concentration HC ₅₀ of Alligatorin5 according to the experimental results. The results showed that Alligatorin5 had low hemolytic activity, and its half hemolytic concentration HC ₅₀ value was 210.05 μg/ml, which was far greater than the MICs value of Alligatorin5 against microorganisms, indicating that Alligatorin5 had great application potential.

Embodiment 6:

Antimicrobial Peptide Alligatorin5 Salt Tolerance, Heat Tolerance and Thermostability Determination

(1) Determination of salt tolerance of the antimicrobial peptide Alligatorin5: Escherichia coli ATCC25922 was cultured in MH liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) 150, 200 and 400 mM NaCl were diluted to 10 ⁶ cfu/ml in fresh MH broth. Alligatorin5 sample solutions with different concentration gradients were prepared with MH liquid medium containing different sodium chloride concentrations. As shown in Table 4. The MIC value of Alligatorin5 was determined by the 2-fold dilution method described in Example 2 above. The results are shown in Table 4.

(2) Determination of the thermal tolerance of the antimicrobial peptide Alligatorin5: Dissolve Alligatorin5 in sterilized deionized water, prepare a sample solution with a concentration of 2 mg/ml, and place it in an incubator at 4°C, 20°C, 37°C, and 50°C , 70° C., and 90° C. were incubated for 1 hour each, and then the incubated Alligatorin5 sample solution was taken, and the MIC value of Alligatorin5 against Escherichia coli ATCC25922 was determined by the 2-fold dilution method described in Example 2. The results are shown in Table 5.

(3) Determination of the thermal stability of the antimicrobial peptide Alligatorin5: Dissolve Alligatorin5 in sterilized deionized water, prepare a sample solution with a concentration of 2 mg/ml, and incubate in an incubator at 37° C. for 0-96 hours. Sample solutions of Alligatorin5 were taken at 0, 6, 12, 24, 48, 72 and 96 hours respectively, and the MIC value of Alligatorin5 against Escherichia coli ATCC25922 was determined by the 2-fold dilution method described in Example 2. The results are shown in Table 6.

It can be seen from Table 4 that Alligatorin5 has strong salt tolerance, and the concentration of salt ions has no effect on its activity. When it is lower or higher than the human physiological salt concentration (150mM NaCl), the MICs value of Alligatorin5 is between 9.38-18.75μg/ml, with little fluctuation, and its antibacterial activity remains basically unchanged.

It can be seen from Table 5 that Alligatorin5 has strong heat tolerance. After the Alligatorin5 solution was placed at 90°C for 1 hour, its MICs value remained unchanged, indicating that its antibacterial activity would not change.

As can be seen from Table 6, the Alligatorin5 solution has good thermal stability. After the Alligatorin5 solution was placed at 37°C for 96 hours, its MICs ranged from 9.38 to 18.75, with a small fluctuation range, and its antibacterial activity hardly changed. Many traditional antibiotics, such as cephalosporins, are extremely unstable in solution and lose their activity within a few hours. Compared with some traditional antibiotics, Alligatorin5 can be used in a wider range.

Table 4 contains the Alligatorin5 solution of different sodium chloride concentrations to the MIC of Escherichia coli ATCC25922

NaCl concentration (mM) MIC (μg/ml)

0 9.38 50 15.6 100 15.6 150 15.6 200 18.75 400 9.38

Table 5 The MIC of Alligatorin5 solution to Escherichia coli ATCC25922 after incubation at different temperatures

temperature(℃) Alligatorin5MIC (μg/ml) 4 9.38 20 9.38 37 9.38 50 9.38 70 9.38 90 9.38

Table 6 The MIC of Alligatorin5 solution to Escherichia coli ATCC25922 after incubation at 37°C for different times

time (h) Alligatorin5MIC (μg/ml) 0 9.38 6 9.38 12 18.75 twenty four 12.5 48 9.38 72 9.38 96 12.5

As can be seen from the above examples, the reptile antimicrobial peptide Alligatorin5 from Chinese alligator in the present invention can be obtained by chemical synthesis, and the antimicrobial peptide is effective against Gram-positive bacteria, Gram-negative bacteria and fungi, including some The pathogenic bacteria isolated clinically all had strong antibacterial activity. Secondly, the antimicrobial peptide has the characteristics of small molecular weight, simple synthesis, low hemolytic activity, good salt tolerance, heat tolerance and thermal stability, and can be used in the fields of medicine, cosmetics, food preservation and aquaculture.

Claims (2)

1. A reptile antimicrobial peptide Alligatorin5, characterized in that, said antimicrobial peptide is derived from Chinese alligator, consists of 22 amino acids, has a molecular weight of 2557.1Da, and an isoelectric point of 12.03, and the amino acid sequence is as shown in the sequence table SEQ ID NO:1 Thr Arg Arg Lys Phe Trp Lys Lys Val Leu Asn Gly Ala Leu Lys Ile Ala Pro PheLeu Leu Gly, all amino acids are L-type. 2. The application of a reptile antimicrobial peptide Alligatorin5 as claimed in claim 1 in the preparation of antibacterial drugs or compositions, antibacterial growth inhibitory drugs or compositions, preservatives, animal feed additives or cosmetic additives.