阅读说明：本技术 一种长翼蝠来源抗菌肽ms-cath及其应用 (Hepialus longissimus source antibacterial peptide MS-CATH and application thereof ) 是由 周江 罗暄巾 王义鹏 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种长翼蝠来源抗菌肽MS-CATH及其在抗菌方面的应用。本发明的抗菌肽MS-CATH来源于长翼蝠。通过检索长翼蝠基因数据库,筛选分析获得抗菌肽成熟肽序列,其氨基酸序列如序列表中SEQ ID NO.1所示。通过对比分析,该天然抗菌肽MS-CATH与目前已知所有抗菌肽的氨基酸序列存在明显差异,属于一种新型抗菌肽。抗菌实验结果表明,本发明的抗菌肽MS-CATH对革兰氏阳性细菌、革兰氏阴性细菌和真菌具有极强的抗菌活性,杀菌作用迅速,此外具有溶血活性低的特点,可应用于制备抗菌、抑制细菌生长的药物、防腐剂、兽药、动物饲料以及化妆品中。(The invention discloses a Hepialus longissimus derived antimicrobial peptide MS-CATH and application thereof in antibiosis. The antibacterial peptide MS-CATH of the invention is derived from Hepialus longissimus. The mature peptide sequence of the antibacterial peptide is obtained by searching a gene database of the flying wing, screening and analyzing, and the amino acid sequence of the mature peptide sequence is shown as SEQ ID NO.1 in a sequence table. Through comparative analysis, the natural antibacterial peptide MS-CATH has obvious difference with the amino acid sequences of all currently known antibacterial peptides, and belongs to a novel antibacterial peptide. The antibacterial peptide MS-CATH has strong antibacterial activity on gram-positive bacteria, gram-negative bacteria and fungi, has the characteristic of quick sterilization effect and low hemolytic activity, and can be applied to preparation of antibacterial and bacteria growth inhibiting medicines, preservatives, veterinary medicines, animal feeds and cosmetics.)

1. A natural antibacterial peptide MS-CATH is characterized in that the antibacterial peptide is derived from Hepialus longissimus, consists of 41 amino acids, has the molecular weight of 4460.21Da and the isoelectric point of 11.73, the amino acid sequence table of the antibacterial peptide is shown in SEQ ID NO.1, and all the amino acids are L-type.

2. The use of the natural antimicrobial peptide MS-CATH of claim 1 for the preparation of an antimicrobial medicament, wherein: the natural antibacterial peptide MS-CATH is used as the only effective component or one of the effective components.

3. The use of the natural antimicrobial peptide MS-CATH of claim 1 in the preparation of a medicament for inhibiting bacterial growth, wherein: the natural antibacterial peptide MS-CATH is used as the only effective component or one of the effective components.

4. Use of the natural antimicrobial peptide MS-CATH of claim 1 for the preparation of a preservative.

5. Use of the natural antimicrobial peptide MS-CATH of claim 1 in the preparation of an animal feed additive.

6. Use of the natural antimicrobial peptide MS-CATH of claim 1 for the preparation of a cosmetic additive.

Technical Field

The invention relates to a natural antibacterial peptide and application thereof, in particular to a natural antibacterial peptide MS-CATH from Hepialus longissimus and application thereof, belonging to the technical field of biomedicine.

Background

The traditional antibiotics have remarkable effects on treating bacterial infection diseases, but in recent years, along with the abuse of the traditional antibiotics in the fields of medicine, breeding industry and the like, microorganisms have stronger and stronger tolerance to the traditional antibiotics, and meanwhile, some super bacteria with extremely strong drug resistance appear, and the problem of microbial drug resistance becomes a problem seriously threatening the health of human beings. There is a continuing need for new antimicrobial agents that can be developed because the resistance to microbial agents has been addressed by the use of new or alternative antimicrobial agents that have not been used by resistant microorganisms and that are further resistant to such new antibiotics after a period of use. The development of a novel antibacterial mechanism medicament is a new direction for solving the problem of microbial tolerance.

The antibacterial peptide is a small molecular polypeptide and has the effect of killing bacteria, fungi, viruses, protozoa and the like. The antibacterial peptide has the characteristics of small molecular weight, simple structure, strong bactericidal activity and the like. 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 transmembrane channels in the cell membrane, causing the cell contents to be dissolved out and leading to cell death. This unique bactericidal mechanism is generally not susceptible to microbial resistance. Moreover, the antibacterial peptide has no toxicity to normal cells and tissues of mammals generally and has no residue problem, so the antibacterial peptide is expected to become a novel high-efficiency antibacterial drug and has wide development and application prospects.

Disclosure of Invention

The invention aims to overcome the defects of the traditional antibiotics, provides a natural antibacterial peptide MS-CATH from Hepialus longissimus, and also provides an amino acid sequence of the antibacterial peptide and application thereof in the aspect of antibiosis. The batus longwing antibacterial peptide MS-CATH has broad-spectrum and efficient antibacterial action, has extremely strong antibacterial activity on gram-positive bacteria, gram-negative bacteria and fungi, has the characteristics of small molecular weight, simple synthesis and low hemolytic activity, and can be applied to the fields of medicines, cosmetics, food preservation and breeding industry.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

the natural antibacterial peptide MS-CATH is from pteropteris pedunculata, consists of 41 amino acids, has molecular weight of 4460.21Da and isoelectric point of 11.73. The amino acid sequence is Lys Leu Arg Gly Leu Leu Arg Gly Leu Val Arg Lys Gly Gly Arg Lys Ile Gly Glu Gly Leu Glu Gly Phe Gly Arg Arg Ile Lys Asn Phe Phe Ser Lys Ser Ser Pro Gly Glu Ala Ser shown in SEQ ID NO.1 of the sequence table, and all amino acids are L-type.

The invention also provides a chemical preparation method of the antibacterial peptide MS-CATH, which comprises the following steps:

synthesizing the full sequence of the obtained natural antibacterial peptide MS-CATH mature peptide by using an automatic polypeptide synthesizer (433A, Applied Biosystems), and desalting and purifying by HPLC reverse phase column chromatography; the purity is identified by high performance liquid chromatography HPLC method, the molecular weight is determined by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF), 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 invention provides the application of the natural antibacterial peptide MS-CATH in preparing antibacterial drugs, wherein the natural antibacterial peptide MS-CATH is used as the only effective component or one of the effective components.

The natural antibacterial peptide MS-CATH is applied to preparing the medicine for inhibiting the growth of bacteria, and the natural antibacterial peptide MS-CATH is used as the only effective component or one of the effective components.

The natural antibacterial peptide MS-CATH is applied to the preparation of preservatives.

The natural antibacterial peptide MS-CATH is applied to the preparation of animal feed additives.

The natural antibacterial peptide MS-CATH is applied to the preparation of cosmetic additives.

The invention has the advantages of providing the natural antibacterial peptide MS-CATH from the Hepialus longissimus and the application thereof in the aspect of antibiosis. The antibacterial peptide MS-CATH has the characteristics of broad-spectrum and efficient antibacterial action, rapid antibacterial action, small molecular weight, simple synthesis and low hemolytic activity, and can be applied to the fields of medicines, cosmetics, food preservation and breeding industry.

Drawings

FIG. 1 hemolytic activity of MS-CATH.

Detailed Description

Example 1:

discovery of natural antibacterial peptide MS-CATH

1) Extracting total RNA of lung tissues of Hepialus longus:

firstly, 100mg of lung tissue of Hepialus longissimus is taken and put into a mortar, liquid nitrogen is added into the mortar to be ground into powder, the powder is transferred into an EP tube, 1ml of total RNA extraction buffer (Trizol, a product of Life company, USA) is added into the powder, the mixture is fully mixed, and then the mixture is centrifuged at 12000rpm for 10min at 4 ℃.

② centrifuging and taking supernatant, adding 0.2ml chloroform solution, mixing vigorously, standing at room temperature for 10 minutes, then centrifuging at 4 ℃, 12000rpm for 10 minutes, and discarding the precipitate.

③ adding isopropanol with the same volume into the supernatant, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 4 ℃ and 12000rpm, collecting the precipitate, washing the precipitate with 75% (V/V) ethanol once, and drying in the air, wherein the precipitate at the bottom of the tube is the total RNA of the skin of the Wuyi torreya.

2) Two-chain synthesis of lung tissue cDNA of Hepialus longus: synthesized using the In-Fusion SMARTERTM directive cDNA Library Construction Kit from CLONTECH.

(1) First strand cDNA Synthesis (reverse transcription of mRNA):

adding 1 mul of gross RNA of lung tissue of the Hepialus longissimus, 1 mul of 3 'end one-strand synthesis Primer (3' In-Fusion SMARTer CDS Primer) and 2.5 mul of RNase-free water into a PCR tube of the RNase-free to ensure that the total volume reaches 4.5 mul, mixing uniformly, centrifuging for a short time (2000rpm for 30s), and preserving the temperature for 3 minutes at 72 ℃ after centrifuging; after incubation, the tubes were incubated at 42 ℃ for 2 minutes.

② adding the following reagents (all prepared In-Fusion SMARTERTM directed cDNA Library Construction Kit of CLONTECH Co., Ltd.) into the centrifuge tube, 2.0. mu.l of 5 Xfirst strand buffer, 0.25. mu.l of 100mM DTT, 1.0. mu.l of 10mM dNTP Mix, 1.0. mu.l of SMARTer V Oligonucleoside, 0.25. mu.l of RNase Inhibitor and 1.0. mu.l of SMARTScribere Reverse Transcriptase, mixing the reagents In the centrifuge tube and centrifuging briefly (2000rpm, 30s), keeping the temperature at 42 ℃ for 90min, and then keeping the temperature at 68 ℃ for 10 min. After the incubation treatment, the centrifuge tube was placed on ice to stop the synthesis of the first strand. Mu.l of the first strand of the synthesized cDNA was taken from the centrifuge tube and used.

(2) Amplifying the second strand by long-terminal polymerase chain reaction (LD-PCR) (all reagents are provided In-Fusion SMARTERTM directive cDNA Library Construction Kit of CLONTECH)

Mu.l of cDNA first strand (reverse transcription of mRNA), 80. mu.l of deionized water, 10. mu.l of 10 × Advantage 2PCR buffer, 2. mu.l of 50 × dNTP Mix, 2. mu.l of 5 'PCR primer, 2. mu.l of CDS III/3' PCR primer and 2. mu.l of 50 × Advantage 2Polymerase Mix were mixed in a PCR tube preheated at 95 ℃.

Amplifying in a PCR instrument according to the following procedures:

at 95 ℃ for 1 min; 18 cycles: 95 deg.C, 15sec, 65 deg.C, 30sec, 68 deg.C, 6 min. After the circulation was completed, the cDNA double strand synthesized in the centrifuge tube was stored at-80 ℃.

(3) Cloning of the long-wing bats antibacterial peptide coding gene:

the forward primer 5-FCATH is designed and synthesized according to the signal peptide conserved region of the cDNA sequence of the batcathelicidins family antibacterial peptide submitted in NCBI database: 5'-ATGGAGACCCAGGGGGCCAGCCCG-3', the reverse primer is 3 ' -PCR primer In the In-Fusion SMARTERTM directive cDNA Library Construction Kit of CLONTECH, and the sequence is 5'-CGGGGTACGATGAGACACCAT-3'. The PCR reaction was performed under the following conditions: 95 ℃ for 4min, 95 ℃ for 30sec, 57 ℃ for 30sec and 72 ℃ for 1min, 30 cycles. After the amplification, the target fragment was recovered with a gel recovery kit (Tiangen). The recovered target fragment was ligated to pMD19-T vector (Takara, Dalian) to transform DH 5. alpha. competent cells. Plates were plated and screened for ampicillin, and single colonies were picked and the insert size was determined by PCR using M13 primer. Positive colonies were picked, plasmids were extracted by shake culture, and nucleotide sequencing was performed using an Applied Biosystems DNA sequencer, model ABI PRISM 377.

The gene sequence was expressed using the blastX software of the NCBI website (http:// blast.st-va. NCBI. nlm. nih. gov/blast. cgipram ═ blas tx:&PAGE_TYPE＝BlastSearch&LINK _ LOC ═ blustonome) shows that the coded product may be the precursor of the peptide of hepcidins family. The polypeptide precursor sequence encoded by the gene was further modified by protein blast software of NCBI website (http:// blast.st-va. NCBI. nlm. nih. gov/blast. cgiprogram. blast. blas tp)&PAGE_TYPE＝BlastSearch&LINK _ LOC ═ blustonome), and comparing and analyzing with the sequences of the previously discovered antimicrobial peptides of other animal-derived cathelicidins families, and determining that the enzyme cutting site is-N in the maturation process of the antimicrobial peptide¹³²-V¹³³The mature peptide sequence KLRGLLRGLVRKGGRKIGEGLEGFGRRIKNFFSKSSPGEAS (amino acid one letter sequence) of the antibacterial peptide is obtained and named as MS-CATH. The amino acid sequence of MS-CATH is obviously different from that of all the currently known antibacterial peptides.

The precursor encoding MS-CATH consists of 167 amino acid residues. The sequence length is 591 bases.

Example 2

Chemical synthesis method of antibacterial peptide MS-CATH

(1) The full sequence of MS-CATH was synthesized by HPLC C using an automated polypeptide synthesizer (433A, Applied Biosystems)₁₈Desalting and purifying by reverse phase column chromatography. (2) The molecular weight is determined by conventional matrix-assisted laser desorptionTime of flight mass spectrometry (MALDI-TOF) method. (3) The purity of the purified MS-CATH is identified by a High Performance Liquid Chromatography (HPLC) method>95 percent, the isoelectric point determined by isoelectric focusing electrophoresis is 10.16, and the amino acid sequence structure is determined to be consistent with the natural MS-CATH by an automatic amino acid sequencer.

Example 3:

detection of antibacterial activity of antibacterial peptide MS-CATH

(1) The test strains preserved on the inclined plane were respectively picked and evenly spread on a MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), a sterilized filter paper sheet with a diameter of 0.5cm was placed on the surface of the medium, 10. mu.l of a 2mg/ml MS-CATH sample solution dissolved in sterilized deionized water was added dropwise, inverted culture was carried out at 37 ℃ for 18-20 hours, and whether the zone of inhibition was formed or not was observed. If the sample has antibacterial activity, clear and transparent inhibition zones can be formed around the filter paper sheet, and the larger the inhibition zone is, the stronger the antibacterial activity of the sample is. The results showed that the antimicrobial peptide MS-CATH has antimicrobial activity against all the strains listed in table 1.

(2) The Minimum Inhibitory Concentration (Minimum Inhibitory Concentration) was determined by 2-fold dilution: and (3) selecting the bacterial strain with the inhibition zone in the experiment step (1) to carry out an MIC determination experiment. The test strains were inoculated into MH liquid medium (purchased from Qingdao Haibo Biotech Co., Ltd.), then cultured with shaking at 37 ℃ in an incubator to logarithmic growth phase, and then the culture broth of the strains cultured to logarithmic growth phase was diluted to 2X 10 with fresh MH liquid medium⁵cfu/ml is ready for use.

100 mul MH liquid culture medium is added into each hole of a sterile 96-hole plate in advance, then 100 mul MS-CATH sample solution which is diluted to a certain concentration by using the MH liquid culture medium and filtered by a filter membrane with the hole of 0.22 mu m is added into the first hole, 100 mul MS-CATH sample solution is added into the second hole after being mixed evenly, the mixture is diluted by times, 100 mul MS-CATH sample solution is sucked out from the 9 th hole and discarded, and the 10 th hole is a control tube.

After adding 100. mu.l of the diluted strain culture solution to each well, the 96-well plate was placed in an incubator at 37 ℃ and cultured with slow shaking for 18 hours, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible. The results are shown in Table 1.

As can be seen from Table 1, the antimicrobial peptide MS-CATH shows very strong antimicrobial activity against gram-positive bacteria, gram-negative bacteria and fungi, including a large number of clinically isolated pathogenic bacteria. The MIC value was in the range of 4.69-18.75. mu.g/ml.

TABLE 1 antibacterial Activity of Hepialus longissimus antibacterial peptide MS-CATH

Example 4:

determination of sterilization rate of antibacterial peptide MS-CATH

Escherichia coli ATCC25922 was cultured in MH broth (Qingdao Haibo Biotech Co., Ltd.) at 37 ℃ for 12 hours, and then diluted to 10 with fresh MH broth⁶cfu/ml bacterial suspension. A sample of MS-CATH dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 5 × MIC (46.9 μ g/ml). And placing the bacterial liquid added with the MS-CATH sample in an incubator at 37 ℃ for shake culture, taking 50 mu l of bacterial liquid to dilute 1000 times in 0, 15, 30, 60, 120 and 180 minutes respectively, then taking 50 mu l of diluted bacterial liquid to coat on an MH solid culture medium, and counting colonies after culturing overnight at 37 ℃ in the incubator. Ampicillin was used as a positive control and sterilized deionized water was used as a negative control in this example. The results of the experiment are shown in table 2.

As can be seen from the comparison in Table 2, the sterilization speed of MS-CATH is faster and even better than that of the positive control ampicillin, and all bacterial cells can be killed within 30 minutes;

TABLE 2 antibacterial peptide MS-CATH bactericidal rate

Example 5:

determination of hemolytic Activity of MS-CATH

The collected fresh C57 mouse blood was mixed with Ashi solution for anticoagulation, washed 2 times with physiological saline and resuspended to 10⁷-10⁸cell/ml suspension. Mixing the diluted erythrocyte suspension with MS-CATH sample solution dissolved in normal saline with a certain concentration, keeping the temperature at 37 ℃ for 30min, centrifuging at 1000rpm for 5min, and measuring the light absorption value of the supernatant at the wavelength of 540 nm. The negative control uses physiological saline, the positive control uses TritonX-100, and the percentage of hemolysis is calculated according to the following formula: percent hemolysis H% ═ a sample-a negative control/a positive control × 100%. The results in FIG. 1 show that the percentage of hemolysis is 1% and 1.3% at MS-CATH concentrations up to 100. mu.g/ml and 200. mu.g/ml, respectively, indicating that MS-CATH has very low hemolytic activity to mammalian erythrocytes.

It can be seen from the above examples that the batus longatus antimicrobial peptide MS-CATH of the present invention can be obtained by chemical synthesis, and the antimicrobial peptide has strong antimicrobial activity on gram-positive bacteria, gram-negative bacteria and fungi, including some clinical isolated pathogenic bacteria, and has rapid bactericidal action. The antibacterial peptide has the characteristics of small molecular weight, simple synthesis and low hemolytic activity, and can be applied to the fields of medicines, cosmetics, food preservation and breeding industry.

Sequence listing

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