CN113480631A

CN113480631A - Horseshoe head bats source antibacterial peptide RF-CATH2 and application thereof

Info

Publication number: CN113480631A
Application number: CN202110944826.3A
Authority: CN
Inventors: 周江; 罗暄巾; 王义鹏
Original assignee: Guizhou Education University
Current assignee: Guizhou Education University
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-10-08
Anticipated expiration: 2041-08-17
Also published as: CN113480631B

Abstract

The invention discloses horseshoe head batus source antibacterial peptide RF-CATH2 and application thereof in antibiosis. The antibacterial peptide RF-CATH2 is derived from horsetail bats. The mature peptide sequence of the antibacterial peptide is obtained by searching a horsetail batus gene database, screening and analyzing, and the amino acid sequence of the mature peptide sequence is shown as SEQID NO.1 in a sequence table. Through comparative analysis, the natural antibacterial peptide RF-CATH2 has obvious difference with the amino acid sequences of all currently known antibacterial peptides, and belongs to a novel antibacterial peptide. The antibacterial peptide RF-CATH2 has strong antibacterial activity on gram-positive bacteria, gram-negative bacteria and fungi, has the characteristics of quick bactericidal action and low hemolytic activity, and can be applied to preparation of antibacterial and bacteria growth inhibiting medicines, preservatives, veterinary medicines, animal feeds and cosmetics.

Description

Horseshoe head bats source antibacterial peptide RF-CATH2 and application thereof

Technical Field

The invention relates to horseshoe brier source antibacterial peptide and application thereof, in particular to natural antibacterial peptide RF-CATH2 derived from horseshoe brier 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 RF-CATH2 from horsetail bats, and further provides an amino acid sequence of the antibacterial peptide and application thereof in the aspect of antibiosis. The horseshoe batus antibacterial peptide RF-CATH2 has broad-spectrum and efficient antibacterial action, has strong antibacterial activity on gram-positive bacteria, gram-negative bacteria and fungi, has the characteristics of quick antibacterial action, small molecular weight, simple synthesis and low hemolytic activity, and can be applied to the fields of medicines, cosmetics, food fresh keeping and breeding industry.

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

the natural antibacterial peptide RF-CATH2 is derived from pterocarpus marsupium of pterocardia, and the antibacterial peptide RF-CATH2 is composed of 34 amino acids, has a molecular weight of 3872.67Da and an isoelectric point of 11.76. The amino acid sequence is Lys Leu Gly Arg Trp Leu Gly Lys Leu Ile Gln Lys Gly Gly Gln Lys Ile Gly Gln Gly Leu Glu Asn Ile Gly Arg Arg Ile Lys Gly Phe Phe Phe Lys 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 RF-CATH2, which comprises the following steps:

according to the amino acid sequence of the mature peptide of the obtained natural antibacterial peptide RF-CATH2, combining the whole sequence by an automatic polypeptide synthesizer (433A, Applied Biosystems), 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 horseshoe brier source antibacterial peptide RF-CATH2 in preparing antibacterial drugs, and horseshoe brier source antibacterial peptide RF-CATH2 is used as the only effective component or one of the effective components.

The application of horseshoe brier source antibacterial peptide RF-CATH2 in preparing medicines for inhibiting bacterial growth comprises horseshoe brier source antibacterial peptide RF-CATH2 as the only effective component or one of the effective components.

The batus ferrugineus source antibacterial peptide RF-CATH2 is applied to the preparation of preservatives.

The horsemanspipe herb derived antibacterial peptide RF-CATH2 is applied to the preparation of animal feed additives.

The horseshoe batus source antibacterial peptide RF-CATH2 can be used for preparing cosmetic additives.

The invention has the advantages of providing the natural antibacterial peptide RF-CATH2 derived from horseshoe brier and the application thereof in the aspect of antibiosis. The antibacterial peptide RF-CATH2 has the characteristics of broad-spectrum efficient antibacterial action, rapid bactericidal 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 RF-CATH 2.

Detailed Description

Example 1:

discovery of horseshoe bateria cepharantha derived antibacterial peptide RF-CATH2

1) Extracting total RNA of horsetail batus lung tissues:

firstly, 100mg of horsetail bats lung tissue 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 in America) 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) The horsetail batus lung tissue cDNA double-chain synthesis: 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 horsetail bats lung tissue total RNA, 1 mul of 3 'end one-strand synthesis Primer (3' In-Fusion SMARTer CDS Primer) and 2.5 mul of RNase-free water into an RNase-free PCR tube to make the total volume reach 4.5 mul, mixing uniformly, centrifuging for a short time (2000rpm, 30s), and preserving heat at 72 ℃ for 3 minutes 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 a 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 × Advantage2Polymerase 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 a horsehead hepcidin encoding 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 ═ blastX:&PAGE_TYPE＝BlastSearch&LINK _ LOC ═ blustonome) shows that the coded product may be the precursor of horsehead horseshoe caterpillar family antibacterial peptide. 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)&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 KLGRWLGKLI QKGGQKIGQG LENIGRRIKGFFFK (amino acid one letter sequence) of the antibacterial peptide is obtained and named as RF-CATH 2. The amino acid sequence of the RF-CATH2 is obviously different from that of all the currently known antibacterial peptides.

The precursor encoding RF-CATH2 consists of 167 amino acid residues. The sequence length is 615 bases.

Example 2

Chemical synthesis method of antibacterial peptide RF-CATH2

(1) The full sequence of RF-CATH2 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 desorption ionization time of flight mass spectrometry (MALDI-TOF). (3) The purity of the purified RF-CATH2 was verified by HPLC>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 RF-CATH2 by an automatic amino acid sequencer.

Example 3:

detection of antibacterial activity of antibacterial peptide RF-CATH2

(1) The test strains preserved on the inclined planes are respectively picked and evenly spread on a MH solid culture medium (purchased from Qingdao Haibo biotechnology, Inc.) plate, a sterilized filter paper sheet with the diameter of 0.5cm is placed on the surface of the culture medium, 10 mul of RF-CATH2 sample solution dissolved in 2mg/ml of sterilized deionized water is dripped, inverted culture is carried out at 37 ℃ for 18-20 hours, and whether the inhibition zone is formed or not is 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 RF-CATH2 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 RF-CATH2 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, after uniform mixing, 100 mul is added into the 2 nd hole, the dilution is carried out in multiple times, 100 mul 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 2.

As can be seen from Table 2, the antimicrobial peptide RF-CATH2 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 horseshoe bats antibacterial peptide RF-CATH2

Example 4:

determination of Sterilization Rate of antimicrobial peptide RF-CATH2

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 RF-CATH2 dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 5 × MIC (46.9 μ g/ml). The bacterial liquid added with the RF-CATH2 sample is placed in an incubator at 37 ℃ for shake culture, 50 mu l of bacterial liquid is taken to dilute 1000 times in 0, 10, 20, 30, 45, 60, 90, 120 and 180 minutes respectively, then 50 mu l of diluted bacterial liquid is taken to coat on MH solid culture medium, and bacterial colonies are counted after the bacterial liquid is cultured in the incubator at 37 ℃ overnight. 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 rate of RF-CATH2 is faster and even better than that of the positive control ampicillin, and all bacterial cells can be killed within 15 minutes;

TABLE 2 Sterilization rate of the antimicrobial peptides RF-CATH2

Example 5:

determination of hemolytic Activity of RF-CATH2

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 RF-CATH2 sample solution with certain concentration dissolved in normal saline, 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 percent hemolysis was 23.3% and 24.6% at concentrations of RF-CATH2 up to 100. mu.g/ml and 200. mu.g/ml, respectively, indicating that RF-CATH2 has a lower hemolytic activity on mammalian erythrocytes.

As can be seen from the above examples, the horseshoe brier antibacterial peptide RF-CATH2 of the present invention can be obtained by chemical synthesis, and has strong antibacterial activity against gram-positive bacteria, gram-negative bacteria and fungi, including some clinically isolated pathogenic bacteria. 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.

The antibacterial peptide RF-CATH2 with conventional dosage and conventional adjuvants are processed by the existing process to obtain corresponding medicine, additive, etc.

Sequence listing

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<120> horseshoe head bats source antibacterial peptide RF-CATH2 and application thereof

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Leu Leu Gly Ala Thr Leu Gly Leu Leu Ile Gly Leu Gly Gly Gly Leu

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aaggagactg tgtgtcccag gaagctgcgg aggacacagc agccaatgga gcagtgtgac 300

ttcaaggaga atgggctggt gaaacagtgt gtggggacag tcactctgga ccaggtcgat 360

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atcaaaggtt tttttttcaa atgatgagcc cagggaagag tcctaagggc ctgctttgcc 540

ctggcttggg ctactggatt ctgaaaaata aattcttgta aaagctaaaa aaaaaaaaaa 600

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atggagaccc agggggccag cccg 24

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Claims

1. An horseshoe brier source antibacterial peptide RF-CATH2 is characterized in that the antibacterial peptide is derived from horseshoe brier, consists of 34 amino acids, has a molecular weight of 3872.67Da and an isoelectric point of 11.76, and an amino acid sequence table of the horseshoe brier source antibacterial peptide is shown in SEQ ID NO: 1: lys Leu Gly Arg Trp Leu Gly Lys LeuIle Gln Lys Gly Gly Gln LysIle Gly Gln Gly Leu Glu AsnIle Gly Arg ArgIle Lys Gly Phe Phe Phe Lys, all amino acids are L-form.

2. The use of batryticatus head source antibacterial peptide RF-CATH2 in the preparation of antibacterial medicine according to claim 1, wherein: horseshoe bats derived antibacterial peptide RF-CATH2 as the only effective component or one of the effective components.

3. The use of batryticatus head-derived antimicrobial peptide RF-CATH2 as claimed in claim 1 in the preparation of a medicament for inhibiting bacterial growth, wherein: horseshoe bats derived antibacterial peptide RF-CATH2 as the only effective component or one of the effective components.

4. Use of batula equine derived antibacterial peptide RF-CATH2 as defined in claim 1 for the preparation of a preservative.

5. Use of batryticatus head source antibacterial peptide RF-CATH2 as claimed in claim 1 in preparing animal feed additive.

6. Use of batula equine derived antibacterial peptide RF-CATH2 as claimed in claim 1 in the preparation of cosmetic additives.