Boma-CATH, the host defense peptide of the giant toad, its gene and applications

Abstract

This invention relates to a host defense peptide, Boma-CATH, from the giant toad (Toadus spp.), its gene, and its applications, belonging to the biomedical field. Boma-CATH is a polypeptide encoded by the defense peptide gene of the giant toad, an amphibian endemic to China. It has a molecular weight of 3124.38 Daltons and an isoelectric point of 5.2, and its amino acid sequence is shown in SEQ ID NO:1. The gene encoding the precursor of Boma-CATH (GenBank accession No. OR797636) consists of 690 nucleotide sequences, as shown in SEQ ID NO:2, where nucleotides 430–510 encode the mature giant toad host defense peptide, Boma-CATH. The giant toad host defense peptide Boma-CATH can be used to prepare drugs for treating skin tumors, including malignant melanoma and squamous cell carcinoma.

Description

Technical Field

[0001] This invention relates to a host defense peptide Boma-CATH from the giant toad (Bombina maxima), its gene, and its applications, belonging to the biomedical field. Background Technology

[0002] Cancer is one of the leading diseases threatening human life, imposing a huge physical and economic burden on humanity worldwide. Surgery, radiotherapy, and chemotherapy are currently the main treatments for cancer. However, traditional methods have drawbacks such as a lack of specific drug delivery systems and the inability to distinguish between cancerous and normal cells, potentially leading to systemic toxic side effects. Therefore, there is an urgent need for more targeted and effective new drugs to address this problem. In recent years, research has discovered that many host defense peptides also have anti-tumor effects. Compared to other radiotherapy and chemotherapy drugs, host defense peptides have high biocompatibility, low side effects, and are readily available, which provides more possibilities for cancer treatment.

[0003] Amphibians represent one of the most important resource groups in the exploration of host defense peptides. Their exposed skin allows them to secrete a variety of novel and functionally complex host defense peptides to resist invasion by various microorganisms in the environment. These bioactive peptides participate extensively in various physiological activities of the body and possess diverse pharmacological activities, such as antimicrobial, antioxidant, immunomodulatory, wound repair, and antitumor effects. Cathelicidins are important members of the natural host defense peptide family, playing a crucial role in regulating host defense and immunity. Currently, only two major classes of cathelicidins with antitumor activity have been reported: LL-37 from humans and Cath-1, Cath-2, and Cath-3 from chickens. No reports have been made of amphibian-derived cathelicidins with antitumor activity. The giant toad (Bombina maxima), an amphibian belonging to the family Bombinaidae and endemic to China, has not yet reported potent antitumor peptides of the cathelicidin family in its skin. Summary of the Invention

[0004] The purpose of this invention is to provide a host defense peptide Boma-CATH from the giant toad (Bombina maxima) and its gene, as well as its application in the preparation of drugs for treating melanoma and squamous cell carcinoma of the skin. This host defense peptide Boma-CATH from the giant toad (Bombina maxima) has potent anti-skin-associated tumor activity.

[0005] The host defense peptide Boma-CATH of this invention is a polypeptide encoded by the defense peptide gene of the Chinese amphibian endemic species, the giant toad (Boma spp.), with a molecular weight of 3124.38 Daltons and an isoelectric point of 5.2. The primary structure of the polypeptide (amino acid sequence SEQ ID NO: 1) is as follows:

[0006]

[0007] The gene encoding the host defense peptide Boma-CATH precursor consists of 690 nucleotides (SEQ ID NO:2), and its sequence from the 5' end to the 3' end is as follows:

[0008]

[0009] Nucleotides 430–510 encode the host defense peptide Boma-CATH from the mature giant toad.

[0010] The application of the host defense peptide Boma-CATH from the giant toad of the present invention in the preparation of drugs for treating skin tumors, including malignant melanoma and squamous cell carcinoma of the skin.

[0011] The beneficial effects of this invention are as follows:

[0012] This invention provides a novel host defense peptide, Boma-CATH, with potent anti-skin cancer activity. This host defense peptide has significant effects against skin tumors, including malignant melanoma and squamous cell carcinoma, and can be used in the preparation of therapeutic drugs for skin tumors such as malignant melanoma and squamous cell carcinoma. Attached Figure Description

[0013] Figure 1 Figure showing experimental results demonstrating the strong anti-skin tumor activity of the host defense peptide Boma-CATH from the giant toad.

[0014] In the figure: A represents the antitumor activity of Boma-CATH against tumor cells such as B16, SK-MEL-110, A375, and A431; B represents the cytotoxicity of Boma-CATH against normal cells such as HSF and HaCaT; where: the absorbance value at 450 nm was measured for cytotoxicity, and each group of values ​​represents data from three independent experiments.

[0015] Figure 2 Figure showing the experimental results of the host defense peptide Boma-CATH in the giant toad significantly inhibiting tumor growth;

[0016] In the figure: A shows the procedure for operating tumor-bearing mice; B shows the tumor size image; C shows the tumor weight statistics; D shows the tumor growth curve; E shows the mouse weight change curve; where: the values ​​represent the mean ± standard deviation of at least three independent experiments, *P<0.05, **P<0.01, ***P<0.001, ns, no statistical difference. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings.

[0018] I. Cloning and amino acid sequence determination of the Boma-CATH gene, a host defense peptide from the giant toad.

[0019] 1. Total RNA extraction from the skin of the giant toad (Bulbophrum fasciatus)

[0020] Live *Bulbophyllum fasciatus* were cleaned with water and flash-frozen in liquid nitrogen for 10 hours. 300 mg of skin tissue was taken and 3 ml of Trizol solution was added, then homogenized in a 20 ml glass homogenizer for 30 minutes. An equal volume of phenol / chloroform solution was added, and the mixture was vigorously mixed. The mixture was incubated at room temperature for 10 minutes, then centrifuged at 12000 rpm for 10 minutes at 4°C, and the precipitate was discarded. An equal volume of isopropanol was added to the supernatant, and the mixture was incubated at room temperature for 10 minutes, then centrifuged at 12000 rpm for 10 minutes at 4°C. The precipitate was washed once with 75% ethanol, air-dried, and the precipitate at the bottom of the tube was the total RNA from *Bulbophyllum fasciatus* skin.

[0021] 2. Purification of skin mRNA from the giant webbed toad

[0022] mRNA isolation and purification were performed using PROMEGA (USA) mRNA Isolation Systems Kit

[0023] Total RNA extracted from the skin of *Bulbophyllum fasciatus* was dissolved in 500 μl of DEPC water and incubated at 65°C for 10 minutes. Then, 3 μl of Oligo(dT) probe and 13 μl of 20×SSC solution were added, mixed thoroughly, and allowed to cool to room temperature; this was called solution A. Magnetic beads (SA-PMP) were gently tapped to mix, and the mixture was allowed to adhere to a magnetic rack for 30 seconds. The supernatant was discarded, and 0.3 ml of 0.5×SSC was added, allowing the mixture to adhere to a magnetic rack for 30 seconds. Finally, 0.1 ml of 0.5×SSC was added to suspend the mixture; this was called solution B. Add solution A to solution B, incubate at room temperature for 10 minutes, then allow to adhere to a magnetic rack for 30 seconds. Discard the supernatant. Wash four times with 0.1×SSC solution, discarding the supernatant again. Resuspend the sample in 0.1 ml of DEPC-treated water, allowing it to adhere to a magnetic rack for 30 seconds. Transfer the supernatant to a new test tube, add 0.15 ml of DEPC-treated water to resuspend the sample, and allow it to adhere to a magnetic rack for 30 seconds. Transfer the supernatant back to the previous test tube. The supernatant will contain purified toad skin mRNA. Add 1 / 10 volume of 3M sodium acetate (pH 5.2) and an equal volume of isopropanol. Incubate at -70°C for 30 minutes, then centrifuge at 12000 rpm for 10 minutes at 4°C. Discard the supernatant and dissolve the precipitate in 10 μl of DEPC-treated water.

[0024] 3. Construction of cDNA library from the skin of the giant toad

[0025] Using CLONTECH's Creator TM SMART TM cDNA Library Construction Kit (cDNA Library Construction Kit)

[0026] (a) cDNA first-strand synthesis (mRNA reverse transcription)

[0027] Add 1 μl of *Bombyx mori* skin mRNA, 1 μl of SMART IV oligonucleotides, and 1 μl of CDS III / 3' PCR primers to a sterile 0.5 ml centrifuge tube, and add 2 μl of deionized water to bring the total volume to 5 μl. Mix the reagents in the centrifuge tube and centrifuge at 12000 rpm for 15 seconds, then incubate at 72°C for 2 minutes. Incubate the centrifuge tube on ice for 2 minutes. Add the following reagents to the centrifuge tube: 2.0 μl of 5× first-strand buffer, 1.0 μl of 20 mM dithiothreitol, 1.0 μl of 10 mM dNTP mixture, and 1.0 μl of PowerScript reverse transcriptase. Mix the reagents in the centrifuge tube and centrifuge at 12000 rpm for 15 seconds, then incubate at 42°C for 1 hour. Place the centrifuge tube on ice to stop the first-strand synthesis. Take 2 μl of the synthesized cDNA first strand from the centrifuge tube for later use.

[0028] (b) Amplification of the second strand using long-terminated polymerase chain reaction (LD-PCR)

[0029] Preheat the PCR instrument to 95℃. In a centrifuge tube, combine 2 μl of cDNA first strand (mRNA reverse transcription), 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 E. coli polymerase. Amplify the PCR using the following program: 95℃ for 20 seconds; 22 cycles (95℃ for 5 seconds; 68℃ for 6 minutes). After cycling, recover the synthesized double-stranded cDNA from the centrifuge tube.

[0030] (c) PCR product recovery

[0031] Using PROMEGA The SV Gel and PCR Clean-Up System kit was used for extraction and recovery, and the steps are as follows:

[0032] Add an equal volume of membrane binding buffer to the cDNA double strands obtained by PCR, invert and mix well. Transfer the mixture to a centrifuge purification column and incubate at room temperature for 5 minutes to allow the DNA to fully bind to the silica membrane. Centrifuge at 12000 rpm for 30 seconds and discard the waste liquid in the collection tube. Add 700 μl of elution buffer (containing ethanol) to the centrifuge purification column and centrifuge at 12000 rpm for 30 seconds, discarding the waste liquid in the collection tube. Repeat step 2. Centrifuge at 12000 rpm for 5 minutes, then transfer the centrifuge purification column to a new centrifuge tube. Add 30 μl of ultrapure water and incubate at room temperature for 5 minutes. Centrifuge at 12000 rpm for 30 seconds; the solution at the bottom of the tube is the purified cDNA double strands.

[0033] (d) Preparation of Escherichia coli DH5α competent cells

[0034] Pick a single DH5α colony and inoculate it into 3 ml of Luria-Bertani (LB) medium without ampicillin. Incubate overnight at 37°C. The next day, take the above bacterial culture and inoculate it into 50 ml of LB medium at a 1:100 ratio. Shake at 37°C for 2 hours. When OD 600Harvest the bacterial culture when the pH reaches 0.35. Transfer the bacteria to a sterile, single-use, ice-cold 50ml polypropylene tube and place it on ice for 10 minutes to cool the culture to 0°C. Centrifuge at 4100 rpm for 10 minutes at 4°C to recover the cells. Discard the culture medium and invert the tube for 1 minute to allow the last trace amount of culture medium to drain completely. Resuspend each cell pellet in 50ml of initial culture medium and 30ml of ice-cold 0.1mol / L CaCl2-MgCl2 solution (80mmol / L MgCl2, 20mmol / L CaCl2). Centrifuge at 4100 rpm for 10 minutes at 4°C to recover the cells. Discard the culture medium and invert the tube for 1 minute to allow the last trace amount of culture medium to drain completely. Resuspend each cell pellet in 2ml of ice-cold 0.1mol / L CaCl2 for 50ml of initial culture and aliquot for later use.

[0035] (e) Enzyme digestion, ligation, and transformation of ligation products

[0036] Add 1 μl of Takara pMD18-T vector and 4 μl of *Bombyx mori* cDNA double-stranded solution to a microcentrifuge tube, bringing the total volume to 5 μl. Add 5 μl (equal volume) of ligase buffer mixture. Incubate at 16°C for 2 hours. Add 10 μl of the total volume to 100 μl of DH5α competent cells and incubate on ice for 30 minutes. Heat at 42°C for 90 seconds, then incubate on ice for 1 minute. Add 890 μl of LB medium that has been preheated to 37°C and incubate at 37°C with gentle shaking for 60 minutes. Spread 200 μl of the mixture onto LB medium containing X-Gal, IPTG, and Amp and incubate at 37°C for 16 hours to form single colonies. Wash each LB plate with 5 ml of LB liquid medium and freeze with 30% glycerol. The constructed cDNA contains approximately 1 × 10⁻⁶ cells / mL. 6 A single clone.

[0037] 4. Cloning and sequencing of host defense peptide genes in the giant webbed toad

[0038] Based on the reported highly conserved cathelin domain sequence of amphibian cathelicidins, a 3' reverse primer, Boma-CATH-R1 (5'-WSCRCAGRYCTTCACCTCC-3' (W=A / T; S=G / C; R=A / G; Y=C / T)), was designed and combined with the 5' PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') provided in the kit to amplify the 5' fragment of the cDNA encoded by cathelicidin. PCR conditions were: 95℃ pre-denaturation for 2 min, 92℃ denaturation for 10 s, 50℃ annealing for 30 s, 72℃ extension for 40 s, repeated 30 times, followed by a final extension at 72℃ for 10 min. The PCR product was purified by gel electrophoresis and cloned into the pMD19-T vector (Takara, Japan) for DNA sequencing.

[0039] Based on the 5' end sequence obtained from sequencing, a 5' forward primer, Boma-CATH-F1 (5'-ATGGTCTTGTGTGTAGGTCTT-3'), was designed and combined with the 3' PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCG-3') provided in the kit to amplify the full-length cDNA sequence encoding cathelicidin. The PCR reaction conditions were as described above. Finally, the PCR product was cloned into the pMD19-T vector and sequenced. The specific sequencing steps are as follows: DNA sequencing was performed using an ABIPRISM 377 Applied Biosystems DNA sequencer. The final amplified PCR product was recovered using a DNA gel recovery kit (DP209-02, Tiangen, China). The recovered fragment was ligated into the pMD19-T vector, and the ligation product was transformed into *E. coli* DH5α competent cells. After transformation, the transformation product was plated onto LB (Luria-Bertani) solid medium containing AMP, and the plates were inverted and incubated at 37°C for 16 hours. Single colonies were selected as templates for colony PCR. The PCR products were determined by agarose gel electrophoresis, and positive clones containing the target fragment were selected, labeled, and cultured in LB liquid medium for amplification. The bacterial culture was then sent to a biotechnology company for DNA sequencing.

[0040] Gene sequencing results indicate that the gene encoding the host defense peptide precursor of the giant toad (Bulbophrum fasciatus) consists of 690 nucleotides (SEQ ID NO: 2) (GenBank Accession Number: OR797636), and the sequence from the 5' end to the 3' end is as follows:

[0041]

[0042] Nucleotides 430–510 encode the host defense peptide Boma-CATH from the mature giant toad. 5. Amino acid sequence determination and synthesis of the giant toad host defense peptide Boma-CATH.

[0043] Sequencing results show that the full-sequence primary structure of the host defense peptide Boma-CATH from the giant toad is: PVIRHYYGEYSTAAKHPPEDIAEETH. The giant toad host defense peptide Boma-CATH was synthesized by Shanghai Jietai Biotechnology Co., Ltd., and the peptide purity is greater than 95%.

[0044] II. Application of Boma-CATH, a host defense peptide from the giant toad, in the preparation of drugs for the treatment of melanoma and squamous cell carcinoma of the skin.

[0045] 1. The host defense peptide Boma-CATH from the giant webbed toad exhibits strong anti-skin tumor activity (including malignant melanoma and squamous cell carcinoma).

[0046] To investigate the tumor-killing ability of Boma-CATH, we used the CCK-8 assay to examine its cytotoxic effects on four skin-related tumors: melanoma cells B16, SK-MEL-110, A375, and squamous cell carcinoma cells A431. Malignant melanoma cells B16, SK-MEL-110, A375, squamous cell carcinoma cells A431, immortalized human keratinocytes (HaCaT), and human skin fibroblasts (HSF) were cultured in DMEM medium supplemented with 10% fetal bovine serum and a mixture of 1% penicillin (100 U / mL) and streptomycin (100 μg / mL), and incubated at 37°C in a 5% CO2 incubator. Cytotoxicity was assessed using the CCK-8 tetrazolium dye colorimetric method. The specific method is as follows: 100 μL of cell suspension was incubated at 5 × 10⁻⁶ ppm... 3 Cells were seeded at a density of 10 cells / well into each well of a 96-well plate. After overnight adhesion, the cells were washed once with PBS and treated with different doses of Boma-CATH for 24 hours. After the incubation period, the culture medium in each well was discarded, and the cells were washed once with PBS. 100 μL of the prepared solution was added to each well at a volume ratio of culture medium to CCK-8 solution of 9:1. The cells were incubated for another 1.5 hours in a cell culture incubator, and the absorbance at 450 nm was measured using a full-wavelength microplate reader. The cell growth and IC50 of the cells after Boma-CATH treatment were analyzed based on the absorbance values. 50 (Half-maximal inhibitory concentration) value, this test was repeated three times.

[0047] Experimental results are as follows Figure 1As shown, Boma-CATH can kill four types of tumor cells in a concentration-dependent manner, and is most sensitive to malignant melanoma A375 cells, with an IC50 value of 32.27 μM. Therefore, A375 cells were selected as the cells for subsequent research. The in vitro effective concentrations of Boma-CATH were 16, 32, and 64 μM (as shown in Figure A). In addition, as shown in Figure B, Boma-CATH showed low toxicity to two types of skin-associated normal cells, namely human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT), at the highest effective concentration of 64 μM, with a survival rate still above 85%.

[0048] Experimental results show that the host defense peptide Boma-CATH from the giant toad exhibits strong anti-skin tumor activity, including against malignant melanoma and squamous cell carcinoma, while showing minimal toxicity to normal skin cells, suggesting that Boma-CATH can selectively target tumor cells (see details). Figure 1 ).

[0049] 2. The host defense peptide Boma-CATH from the giant toad can significantly inhibit tumor growth.

[0050] A nude mouse xenograft model was constructed using A375 cells, and then... Figure 1 Following the procedure shown in section A, the A375 cell line in logarithmic growth phase was cultured in fresh medium the day before injection to ensure the cells were in good condition at the time of injection. After trypsin digestion, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS to adjust the cell concentration to 2 × 10⁷ cells / mL. 100 μL of cell suspension was subcutaneously injected into the right side of each 4-week-old female BALB / c nude mouse. Six days after cell injection, the tumor had grown to 100 mm. 3 In the above scenario, nude mice were randomly divided into a model group and a drug treatment group. To exclude cases where tumors did not form, two extra mice were added to each group, resulting in eight mice per group. During the treatment, the mice were injected intratumorally with the peptide every two days. The model group received an equal volume of PBS instead. The tumor volume and weight were recorded. This treatment was administered for two weeks, with eight doses given. Two days after the last dose, tumor tissue and organs such as the heart, liver, spleen, lungs, and kidneys were harvested from the mice. A portion was placed in liquid nitrogen, and the other portion was placed in 4% tissue fixative for subsequent experiments. The tumors were also photographed.

[0051] Experimental results are as follows Figure 2As shown, compared with the model group, the tumors in the 20 mg / kg Boma-CATH treatment group were significantly smaller (Figure B) and the tumor weight was also significantly reduced (Figure C). Furthermore, the growth curves of mouse tumor volume during the administration period also showed that the tumor volume in the treatment group was significantly smaller than that in the model group (Figure D). Meanwhile, there was no significant difference in body weight between the model group and the treatment group (Figure E), indicating that 20 mg / kg Boma-CATH administration did not affect the normal growth of mice.

[0052] Experimental results show that the host defense peptide Boma-CATH from the giant toad can significantly inhibit tumor growth and has a strong anti-tumor effect (see details). Figure 2 ).

[0053]

Claims

1. A host defense peptide, Boma-CATH, from the giant toad, characterized in that, This host defense peptide is a polypeptide encoded by the defense peptide gene of the Chinese amphibian endemic species, the giant toad, with a molecular weight of 3124.38 Daltons and an isoelectric point of 5.

2. Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the host defense peptide Boma-CATH from the giant toad according to claim 1 in the preparation of drugs for treating skin tumors, characterized in that, The skin tumor is either melanoma or squamous cell carcinoma of the skin.

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