Antibacterial peptide CnCATH as well as coding gene and application thereof

Abstract

The invention relates to an antibacterial peptide CnCATH as well as a coding gene and application thereof, and belongs to the technical field of biomedicine. The novel antibacterial peptide is extracted from sika deer bone marrow, and the amino acid sequence is as shown in SEQ ID NO. 2. The antibacterial peptide shows strong antibacterial activity on various microorganisms including gram-negative bacteria, gram-positive bacteria and fungi, the sterilization speed of the antibacterial peptide is obviously improved compared with that of traditional antibiotics, the bacterial strain is not prone to drug resistance, endotoxin can be further neutralized, and the concentration of the endotoxin can be further reduced. The novel sika deer-derived antibacterial peptide provided by the invention has the advantages of small molecular weight, simple artificial synthesis, low drug resistance tendency and high broad-spectrum bactericidal activity, and provides a new strategy for microbial anti-infection treatment and prevention and treatment of diseases caused by endotoxin.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial peptide CnCATH, its encoding gene, and its applications. Background Technology

[0002] In recent years, with the large-scale and inappropriate use of traditional antibiotics, microorganisms have developed increasingly strong resistance to them. Currently, the main approach to combating microbial resistance relies on the development of novel or alternative antibiotics, which requires continuous research and development of new antimicrobial agents.

[0003] Antimicrobial peptides are naturally occurring small polypeptides synthesized in vivo. They are important functional molecules in the innate immune system of multicellular organisms, exhibiting direct killing effects against bacteria, fungi, viruses, and protozoa. Their core mechanism lies in disrupting the cell membranes of microorganisms. Due to their advantages such as small molecular weight, simple structure, strong bactericidal activity, unique bactericidal mechanism, and low likelihood of inducing drug resistance, they are considered highly promising alternatives to traditional antibiotics and have great potential for development and application.

[0004] Even-toed ungulates are diverse, widely distributed, and live in complex environments, making them vulnerable to pathogens. Antimicrobial peptides, as important molecules in the innate immune system of vertebrates, are abundant and diverse in even-toed ungulates. However, currently discovered antimicrobial peptides mainly originate from Bovidae and Suidae, with fewer findings from other even-toed ungulates. Furthermore, antimicrobial peptides from Bovidae and Suidae suffer from high similarity, low activity, large molecular weight, and poor stability. Current research indicates differences in the types, structures, and activities of antimicrobial peptides among different families of even-toed ungulates; therefore, the discovery of new antimicrobial peptides from other families of even-toed ungulates is of great significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention extracts a novel antimicrobial peptide from sika deer bone marrow tissue, the amino acid sequence of which is shown in SEQ ID NO.2. This antimicrobial peptide exhibits strong antimicrobial activity against a variety of microorganisms, including Gram-negative bacteria, Gram-positive bacteria, and fungi. Its bactericidal speed is significantly improved compared to traditional antibiotics, and it is less likely to induce drug resistance in strains. Furthermore, it can neutralize endotoxins and reduce endotoxin concentrations.

[0006] The first objective of this invention is to provide an antimicrobial peptide derived from sika deer, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A second objective of this invention is to provide the application of the above-mentioned antimicrobial peptide in the preparation of antimicrobial agents.

[0008] A third object of the present invention is to provide an antimicrobial composition comprising the above-mentioned antimicrobial peptide.

[0009] A fourth objective of this invention is to provide the encoding gene for the aforementioned antimicrobial peptide.

[0010] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0011] A fifth objective of this invention is to provide an expression vector containing the above-described coding gene.

[0012] A sixth object of the present invention is to provide the use of the above-mentioned antimicrobial peptides or the above-mentioned antimicrobial compositions in killing bacteria or fungi.

[0013] Furthermore, the bacteria include one or more of Escherichia coli, Acinetobacter baumannii, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Staphylococcus epidermidis, or Enterococcus faecalis, and the fungi include Candida albicans and / or Candida glabrata.

[0014] A seventh object of the present invention is to provide the use of the above-mentioned antimicrobial peptide in neutralizing endotoxins.

[0015] Furthermore, the concentration of the antimicrobial peptide is 20-80 μg / mL.

[0016] An eighth object of the present invention is to provide a medicament for neutralizing endotoxins, the medicament comprising the above-mentioned antimicrobial peptide.

[0017] The beneficial effects of this invention are:

[0018] The novel antimicrobial peptides screened from sika deer bone marrow have clear sequences, small molecular weights, and are simple to synthesize artificially. They exhibit strong antimicrobial activity and high bactericidal speed against a variety of microorganisms, including Gram-negative bacteria, Gram-positive bacteria, and fungi. They are not prone to inducing drug resistance in strains and can further neutralize endotoxins. This provides a new strategy for antimicrobial anti-infection treatment and prevention and treatment of diseases caused by endotoxins, and has broad application prospects. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0020] Figure 1 This is the verification result of the neutralizing LPS activity of the antimicrobial peptide CnCATH in Example 5 of the present invention;

[0021] Figure 2This is the test result of the ability of the antimicrobial peptide CnCATH in Example 6 of the present invention to induce drug resistance in Escherichia coli ATCC25922;

[0022] Figure 3 This is the test result of the ability of the antimicrobial peptide CnCATH in Example 6 of the present invention to induce drug resistance in Staphylococcus aureus CMCC26003. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0024] Experimental strains: All clinically isolated strains were collected from the Affiliated Hospital of Soochow University.

[0025] Example 1: Cloning of the CnCATH antimicrobial peptide encoding gene from sika deer

[0026] I. Total RNA extraction from sika deer bone marrow:

[0027] (1) Take 200mg of bone marrow tissue from sika deer (Cervus nippon), put it in a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1mL of total RNA extraction buffer (Trizol, a product of Life Sciences, USA), mix thoroughly, and then centrifuge at 4℃, 12000rpm for 10min.

[0028] (2) Centrifuge and take the supernatant, add 0.2 mL of chloroform solution, mix vigorously, let stand at room temperature for 10 min, and then centrifuge at 4℃ and 12000 rpm for 10 min to remove the precipitate.

[0029] (3) Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 min, centrifuge at 4℃ and 12000 rpm for 10 min, collect the precipitate, wash once with 75% (V / V) ethanol, air dry, and the precipitate at the bottom of the tube is the total RNA of sika deer bone marrow.

[0030] II. Second-strand synthesis of cDNA from sika deer bone marrow

[0031] Using Takara's In-Fusion SMARTer TM Synthesized by Directional cDNA Library Construction Kit.

[0032] (1) First-strand cDNA synthesis:

[0033] Add 1 μL of total RNA from sika deer bone marrow, 1 μL of 3' end single-strand synthetic primer (3'-In-Fusion SMARTer CDS Primer), and 2.5 μL of RNase-free water to an RNase-free PCR tube to make a total volume of 4.5 μL. After mixing, briefly centrifuge (2000 rpm, 30 s) and incubate at 72 °C for 3 min. After incubation, incubate the centrifuge tube at 42 °C for 2 min.

[0034] Add the following reagents (all In-Fusion SMARTer) to the centrifuge tubes mentioned above. TM The following reagents are provided in the Directional cDNA Library Construction Kit: 2.0 μL 5× first-strand buffer, 0.25 μL 100 mM DTT, 1.0 μL 10 mM dNTP Mix, 1.0 μL SMARTer VOligonucleotide, 0.25 μL RNase Inhibitor, and 1.0 μL SMARTScribe Reverse Transcriptase. Mix the reagents in the centrifuge tube and centrifuge briefly (2000 rpm, 30 s). Incubate at 42 °C for 90 min, then at 68 °C for 10 min. After incubation, place the centrifuge tube on ice to stop first-strand synthesis. Take 2 μL of the synthesized cDNA first strand from the centrifuge tube for later use.

[0035] (2) The second strand was amplified using long-terminated polymerase chain reaction (LD-PCR) (all reagents used were In-Fusion SMARTer). TM (Included in the Directional cDNA Library Construction Kit)

[0036] Mix 2 μL of cDNA first strand, 80 μL of deionized water, 10 μL of 10×Advantage 2 PCR buffer, 2 μL of 50×dNTP mixture, 2 μL of 5' PCR primers, 2 μL of CDS III / 3' PCR primers, and 2 μL of 50×Advantage2Polymerase Mix in a PCR tube preheated to 95°C.

[0037] Amplify using the following procedure in a PCR instrument:

[0038] 95℃, 1 min; 18 cycles: 95℃, 15 sec, 65℃, 30 sec, 68℃, 6 min. After cycling, store the synthesized double-stranded cDNA in the centrifuge tube at -80℃.

[0039] (3) Cloning of the gene encoding the antimicrobial peptide CnCATH from sika deer:

[0040] Based on the conserved sequence of the signal peptide region of antimicrobial peptides in the cathelicidins family of even-toed ungulates, a forward degenerate primer, 5'-atggaraccmagwggsccagcc-3', was artificially designed and synthesized. The reverse primer was the 3'-PCR primer from the In-Fusion SMARTer™ Directional cDNA Library Construction Kit, with the sequence 5'-CGGGGTACGATGAGACACCAT-3'. PCR was performed under the following conditions: 95℃ for 4 min, 95℃ for 30 s, 57℃ for 30 s, and 72℃ for 1 min, for 30 cycles. After amplification, the target fragment was recovered using a gel extraction kit. The recovered target fragment was ligated into the pMD19-T vector (Takara, Dalian) and transformed into TOP10 competent cells. The cells were plated and screened with ampicillin. Single colonies were picked and the insert size was detected by PCR using the M13 primer. Positive colonies were picked, plasmids were extracted by shaking, and samples were sent for nucleotide sequencing.

[0041] Measurement results:

[0042] The gene encoding the sika deer antimicrobial peptide CnCATH precursor, from its 5' to 3' end, is shown in SEQ ID NO.1. The gene sequence encoding the sika deer antimicrobial peptide CnCATH precursor is 593 bases long, of the following types: nucleic acid, single-stranded, linear, cDNA, and derived from sika deer bone marrow.

[0043] Example 2: Preparation of CnCATH, an antimicrobial peptide from sika deer

[0044] (1) Chemical synthesis method of sika deer antimicrobial peptide CnCATH: Based on the amino acid sequence deduced from the encoding gene, its full sequence was synthesized using an automated peptide synthesizer (433A, Applied Biosystems) and purified by HPLC reverse-phase column chromatography.

[0045] (2) Molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0046] (3) The purity of the purified antimicrobial peptides was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automated amino acid sequencer.

[0047] Sika deer antimicrobial peptide CnCATH is a small molecule polypeptide encoded by a gene, consisting of 34 amino acid residues, with a molecular weight of 4183.97 Da and an isoelectric point of 11.80. Its amino acid sequence is shown in SEQ ID NO.2.

[0048] Example 3: Detection of the antibacterial activity of CnCATH, an antimicrobial peptide from sika deer

[0049] The minimum inhibitory concentration (MIC) of the samples against different bacteria was determined using the standard two-fold dilution method. Microbial strains were inoculated into MH liquid medium (Oxoid, UK) and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial culture was then diluted to 2 × 10⁻⁶ with fresh MH liquid medium. 5 CFU / mL was prepared for use. 100 μL of MH liquid medium was added to each well of a sterile 96-well plate (Thermo, USA). Then, 100 μL of the sika deer antimicrobial peptide CnCATH sample solution diluted with MH liquid medium was added to the first well. After mixing, 100 μL was added to the second well, and so on, serially diluted. 100 μL was aspirated from the ninth well and discarded. The tenth well was used as a control tube.

[0050] The 96-well plates were incubated at 37°C with slow shaking for 18 hours. The absorbance was measured at 600 nm, and the minimum inhibitory concentration (MIC) of the samples against different microorganisms was calculated. In this experiment, Omiganan (amino acid sequence: ILRWPWWPWRRK-NH2, Phase 3 clinical trial, trade name: Omiganan), a modified bovine cathelicidin family antimicrobial peptide indolicidin, Iseganan (amino acid sequence: RGGLCYCRGRFCVCVGR-NH2, Phase 3 clinical trial, trade name: Iseganan), a modified porcine cathelicidin family antimicrobial peptide protegrin-1, and the antibiotic ampicillin were used as controls. The results are shown in Table 1.

[0051] Table 1. Antibacterial activity of CnCATH, an antimicrobial peptide from sika deer.

[0052]

[0053]

[0054] As shown in Table 1, the sika deer antimicrobial peptide CnCATH exhibits extremely strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi, including a variety of clinically isolated pathogens. The MIC values ​​range from 1.17 to 4.69 μg / mL, superior to the positive control ampicillin, and comparable to the antimicrobial peptides Omiganan and Iseganan, both derived from ungulates and currently in phase 3 clinical trials. For certain strains, such as *Escherichia coli* ATCC25922, *Acinetobacter baumannii* ATCC19606, *Enterococcus faecalis* ATCC29212, *Klebsiella pneumoniae* 9409, and *Enterococcus faecium*, CnCATH shows superior antimicrobial activity compared to Omiganan and Iseganan, indicating its significant development potential.

[0055] Example 4: Determination of the bactericidal rate of CnCATH, an antimicrobial peptide from sika deer

[0056] Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 were cultured in MH liquid medium (Oxoid, UK) at 37°C for 12 hours, and then diluted with fresh MH liquid medium to a concentration of 10. 6 CFU / mL bacterial suspension. CnCATH sample dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 5×MIC. The bacterial suspension with added CnCATH sample was incubated at 37°C with shaking. At 0, 10, 20, 30, 45, 60, 90, 120, and 180 minutes, 50 μL of the bacterial suspension was taken and diluted 1000-fold. Then, 50 μL of the diluted bacterial suspension was spread onto MH solid medium and incubated overnight at 37°C. Colony counting was then performed. Ampicillin was used as a positive control, and sterile deionized water as a negative control. The experimental results are shown in Tables 2 and 3.

[0057] Table 2. Bactericidal rate of sika deer antimicrobial peptide CnCATH against Escherichia coli ATCC25922

[0058]

[0059] Table 3. Bactericidal rate of sika deer antimicrobial peptide CnCATH against Staphylococcus aureus CMCC26003

[0060]

[0061] As can be seen from the comparison in Tables 2 and 3, CnCATH has an extremely fast bactericidal speed, killing Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 within 10 minutes, which is much faster than the positive control ampicillin. This is a huge advantage of CnCATH compared with traditional antibiotics.

[0062] Example 5: Determination of the activity of sika deer antimicrobial peptide CnCATH in neutralizing endotoxin (LPS)

[0063] Using ToxinSensor TM The Limulus amebocyte lysate (LAL) reagent endotoxin test kit (Genscript, Nanjing) was used to perform a neutralization experiment of the antimicrobial peptide CnCATH from sika deer with LPS. The following steps were performed according to the manufacturer's instructions: (1) A standard curve was prepared to obtain the linear relationship between absorbance at 545 nm and LPS concentration under standard conditions. The standard curve was plotted with absorbance as the ordinate (Y) and endotoxin content as the abscissa (X). When the correlation coefficient R of the standard curve was... 2 Experimental data are only valid when ≥0.980. (2) Dissolve LPS in pyrogen-free water to 1 EU / mL, prepare 2 mg / mL antimicrobial peptide stock solution in pyrogen-free water, and incubate 1 mL of LPS solution with 30 μL of antimicrobial peptide stock solution for 5 min and 60 min respectively, and use pyrogen-free water as negative control. First, take 100 μL of each sample to be tested after co-incubation, and make three parallel controls for each sample. Shake and mix for 30 seconds to prevent LPS from forming polymers. Add 100 μL of Limulus Amebocyte Lysate (LAL) reagent, shake gently to mix, incubate at 37°C for 10 min, and immediately add 100 μL of chromogenic matrix. Shake gently to mix, incubate at 37°C for 6 min, and immediately add 500 μL of reaction stop solution. Mix well, and then add 500 μL of azo dye #2 and azo dye #3 in sequence. After standing for 5 min, take 200 μL of each reagent and add it to a 96-well microtiter plate. Finally, use a full-wavelength microplate reader to measure the absorbance at 545 nm. (3) Substitute the measured absorbance of the sample into the standard curve formula to calculate the X value, which is the concentration of endotoxin in the sample. Statistical analysis of the data was performed using GraphPad Prism software.

[0064] The results are as follows Figure 1 As shown, CnCATH exhibits extremely strong concentration-dependent LPS neutralization activity. The LPS neutralization activity gradually increases with increasing peptide concentration; 80 μg / mL of CnCATH can neutralize 99.8% of LPS toxicity. This indicates that CnCATH has great potential for treating LPS-induced diseases, such as endotoxemia.

[0065] Example 6: Determination of the ability of sika deer antimicrobial peptide CnCATH to induce drug resistance

[0066] Single colonies of *Escherichia coli* ATCC25922 and *Staphylococcus aureus* CMCC26003 were picked from MH plates and transferred to MH liquid medium. The culture was incubated at 37°C with shaking until the logarithmic growth phase. The bacterial concentration was measured and diluted with fresh MH liquid medium to the desired concentration for later use. Appropriate amounts of CnCATH and the positive control antibiotic ampicillin were weighed and prepared as stock solutions using MH liquid medium. Fresh culture was added to sterile 1.5 mL EP tubes, along with 10 μL of the diluted bacterial solution and 10 μL of the prepared antimicrobial peptide or antibiotic sample, to achieve a final drug concentration of 0.5 × MIC and a final volume of 1 mL. The culture was incubated at 37°C and 150 rpm for 24 h. For each subsequent generation, fresh culture was added to sterile 1.5 mL EP tubes, along with 10 μL of the previous generation bacterial solution and 10 μL of the prepared drug sample, to achieve a final drug concentration of 0.5 × MIC and a final volume of 1 mL. The culture was incubated at 37°C and 150 rpm for 24 h. The MIC value of each generation of strain was determined by the two-fold dilution method and compared with the MIC value of the original strain to assess the development of drug resistance.

[0067] The results are as follows Figure 2 and Figure 3 As shown, after 60 passages at a drug concentration of 0.5 × MIC, the sensitivity of *E. coli* ATCC25922 and *Staphylococcus aureus* CMCC26003 to CnCATH remained almost unchanged, with the MIC value increasing only 2-fold. In contrast, the MIC values ​​of ampicillin against *E. coli* ATCC25922 and *Staphylococcus aureus* CMCC26003 increased by 32-fold and 64-fold, respectively, indicating that *E. coli* ATCC25922 and *Staphylococcus aureus* CMCC26003 developed significant resistance to ampicillin. These experimental results demonstrate that, compared to the antibiotic ampicillin, long-term use of CnCATH is less likely to lead to bacterial resistance, which is a significant advantage of CnCATH over traditional antibiotics.

[0068] Example 7: Determination of the hemolytic activity of the antimicrobial peptide CnCATH from sika deer

[0069] Freshly collected human blood was mixed with Abercrombie's solution for anticoagulation, washed twice with physiological saline, and resuspended to a final concentration of 10 μL. 7 -10 8 A suspension of erythrocytes at a concentration of 1 cell / mL was prepared. This diluted erythrocyte suspension was mixed with a sample of sika deer antimicrobial peptide CnCATH dissolved in physiological saline, incubated at 37°C for 30 min, and then centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was measured at 540 nm. Physiological saline was used as the negative control, and Triton X-100 was used as the positive control. The hemolysis percentage was calculated using the following formula: Hemolysis percentage H% = (A sample - A negative control / A positive control) × 100%.

[0070] The results showed that the hemolysis percentages of the sika deer antimicrobial peptide CnCATH at concentrations of 100 μg / mL and 200 μg / mL were 5.36% and 9.88%, respectively. This indicates that the sika deer antimicrobial peptide CnCATH has extremely low hemolytic activity against human erythrocytes, suggesting its great development potential and laying the foundation for its future development and utilization.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antimicrobial peptide derived from sika deer, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

2.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial agents.

3. An antibacterial composition, characterized in that: The antimicrobial composition comprises the antimicrobial peptide of claim 1.

4. The gene encoding the antimicrobial peptide of claim 1.

5. The encoding gene according to claim 4, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

6. An expression vector comprising the encoding gene of claim 4 or 5.

7. The use of the antimicrobial peptide of claim 1 or the antimicrobial composition of claim 3 in killing bacteria or fungi.

8. The application according to claim 7, characterized in that: The bacteria include one or more of Escherichia coli, Acinetobacter baumannii, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Staphylococcus epidermidis, or Enterococcus faecalis, and the fungi include Candida albicans and / or Candida glabrata.

9. The use of the antimicrobial peptide according to claim 1 in neutralizing endotoxins.

10. A drug for neutralizing endotoxins, characterized in that: The drug comprises the antimicrobial peptide of claim 1.

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